Polish Journal of Microbiology

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POLSKIE TOWARZYSTWO MIKROBIOLOGÓW THE POLISH SOCIETY OF MICROBIOLOGISTS

Conference Papers

Polish Journal of Microbiology Suppl.

formerly

Acta Microbiologica Polonica

2004 POLSKIE TOWARZYSTWO MIKROBIOLOGÓW

EDITORS K.I. Wolska (Editor in Chief) A. Kraczkiewicz-Dowjat, A. Skorupska, L. Sedlaczek, E. Strzelczyk E.K. Jagusztyn-Krynicka (Scientific Secretary)

EDITORIAL BOARD President: Zdzis³aw Markiewicz (Warsaw, Poland) Ryszard Chróst (Warsaw, Poland), Miros³aw Kañtoch (Warsaw, Poland), Donovan Kelly (Warwick, UK), Tadeusz Lachowicz (Wroc³aw, Poland), Wanda Ma³ek (Lublin, Poland), Andrzej Piekarowicz (Warsaw, Poland), Anna Podhajska (Gdañsk, Poland), Gerhard Pulverer (Cologne, Germany), Geoffrey Schild (Potters, Bar, UK), Torkel Wadström (Lund, Sweden), Jadwiga Wild (Madison, USA), Miros³awa W³odarczyk (Warsaw, Poland)

EDITORIAL OFFICE Miecznikowa 1, 02-096 Warsaw, Poland tel. 48 (22) 55 41 302, Tuesday and Thursday from 10 A.M. – till 2 P.M. only fax 48 (22) 55 41 402 e-mail [email protected] biol.uw.edu.pl

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Front cover: Bacillus subtilis Hly entering Int 407 epithelial cells (courtesy of Jaros³aw Wiœniewski, M.Sc. and Magdalena Sobolewska Ph.D)

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2ND INTERNATIONAL CONFERENCE 22–23TH OCTOBER 2004

INTRACELLULAR PARASITISM: BIOLOGY AND PATHOGENESIS

EUROPEAN CENTRE OF EXCELLENCE CEMERA WARSAW UNIVERSITY, FACULTY OF BIOLOGY www.cemera.pl

Polish Journal of Microbiology formerly Acta Microbiologica Polonica

2004, Vol. 53, Suppl.

CONTENTS CONFERENCE PAPERS (bacteriological and parasitological in alphabetical order) New approaches to development of mucosal vaccine against enteric bacterial pathogens; preventing campylobacteriosis JAGUSZTYN-KRYNICKA E.K., WYSZYÑSKA A., RACZKO A.

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Molecular aspects of Listeria monocytogenes infection KRAWCZYK-BALSKA A., BIELECKI J.

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Optical lectin based biosensor as tool for bacteria identification MASAROVA J., SZWAJCER DEY E., DANIELSSON B.

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Analysis of the peptidoglycan hydrolases of Listeria monocytogenes: multiple enzymes with multiple functions POPOWSKA M.

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Polymerizer-mediated intracellular movement WIŒNIEWSKI J.M., BIELECKI J.

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New methods of pathogenic bacteria elimination WOLSKA K.I., KRACZKIEWICZ-DOWJAT A., GRUDNIAK A.M., SAJKOWSKA A., WIKTOROWICZ T.

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Molecular modifications of host cells by Toxoplasma gondii D£UGOÑSKA H.

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Babesia sp.: emerging intracellular parasites in Europe GRAY J.S.

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Zoonotic reservoir of Babesia microti in Poland KARBOWIAK G.

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Apicomplexan parasites: environmental contamination and transmission SIÑSKI E., BEHNKE J.M.

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The modulation of transferrin receptors level on mouse macrophages and fibroblasts by Toxoplasma gondii DZIADEK B., DYTNERSKA-DZITKO K., D£UGOÑSKA H.

Redakcja Polish Journal of Microbiology (formerly Acta Microbiologica Polonica) zawiadamia, ¿e realny Impact Factor w roku 2003 osi¹gn¹³ wartoœæ 0,286 Krystyna I. Wolska – Redaktor naczelny

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Polish Journal of Microbiology 2004, Vol. 53, Suppl., 7–15

New Approaches to Development of Mucosal Vaccine Against Enteric Bacterial Pathogens; Preventing Campylobacteriosis EL¯BIETA K. JAGUSZTYN-KRYNICKA, AGNIESZKA WYSZYÑSKA, ANNA RACZKO

Department of Bacterial Genetics, Institute of Microbiology, Warsaw University, Miecznikowa 1 str, 02-096, Warsaw, Poland Abstract Although vaccination, after having been more than 200 years in medical practice, has proven to be the most effective and the cheapest way to prevent infectious diseases, they remain still the main cause of human premature deaths. As many pathogens enter the human body through the mucosal surfaces, the mucosal way of immunization is considered to be the most promising strategy to decrease the number of human infections. Moreover, the oral delivery system eliminates the necessity of injection what is extremely important for pediatric immunization programs. However, most of recently constructed subunit vaccines based on purified bacterial/viral antigens are rather poorly immunogenic. This review presents some novel ways to enhance and modulate host immune responses by combining antigens with specific adjuvants or by employing specific delivery systems. We also discuss some recent technologies, based on mining the genomic sequences of bacterial pathogens, which accelarate and improve identification of new candidates for vaccine construction. As an example, we focus on the progress in the development of vaccine against Campylobacter spp. Campylobacter jejuni is now recognized as a leading cause of bacterial enteritis in human. K e y w o r d s: mucosal vaccine, specific adjuvants, antigen carriers, campylobacteriosis

Introduction Antibiotics and vaccines are one of the most important medical achievements of the 20 th century. Their introduction into public medical practice resulted in a sharp decrease in infectious diseases morbidity and mortality, and gave rise to a common belief that infectious diseases can be controlled and prevented. The enormous progress recorded in the area was particularly marked in developed countries doubtlessly due to mass immunisation programs. However, the smallpox is the only infectious disease that has been completely eradicated. Vaccine-preventable diseases are still the number one cause of premature death in the world. According to World Health Organisation data over 17 million people die every year as a result of infectious diseases. A recent literature review indicates the existence of 1415 species of infectious agents pathogenic for human (viruses, bacteria, parasitic helminths, protozoa, prions) (Taylor et al., 2001). Many of them (12%) are regarded by epidemiologists as emerging and re-emerging pathogens. Most of human emerging pathogens are zoonotic. Apart from wild and domestic animals also human population and our environment are a source of new organisms pathogenic for humans. Many physiological and social factors, such as genetic variability of the pathogens (mutations, horizontal gene transfer), ecological changes in human population, progress in medical technologies, and modification of the host-pathogen relationship promote such a huge number of emerging pathogens (Woolhouse, 2002). This fact combined with the emergence of many antibiotic-resistant pathogens makes it necessary to look for new strategies to treat bacterial infections. The fundamental issue now is how to improve existing vaccines or how to create new ones. The experience of the last century proves that vaccines are the major beneficial factor in medicine of infectious diseases. Our ambition is to introduce new specimens whom would be safe, effective, cheap and easy to deliver. Attempts should be also made to shorten the time of vaccine/drug discovery. Now it takes about 10 years to create new vaccine or new antibacterial drug and it is widely accepted that the time is too long.

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The modern era of genomics technologies promises to speed our understanding of the not completely understood field of bacterial pathogenicity. New strategies have been developed for rapid microbes’ identification, for new antibacterial drug design and for selecting genes encoding protective antigens. This review outlines the current trends regarding mucosal vaccination against bacterial enteropathogens. It also presents new achievements concerning anty-Campylobacter immunization 1. Mucosal vaccination The vast majority of bacterial and viral infections originate at mucosal membranes that line interior part of the body. Colonization of these surfaces is the first step for many infectious diseases. For some pathogens mucosal surfaces are their destination point in the host body whereas for others colonization of the mucus membrane is the starting point for spreading in the mammalian organism. For example, among enteric bacteria, Helicobacter pylori and Vibrio cholerea are extracellular microorganisms existing in the lumen of the gastrointestinal track whereas Shigella, Salmonella or Yersenia cross the epithelium through M or epithelial cells causing localized or systemic infection. In most cases, the most effective approach to obtain protection against enteric pathogens is the induction of high level of s-IgA. The mucosal route of vaccination, which offers the possibility to induce locally produced and secreted sIgA antibodies, in addition to systemic IgG antibodies may be an effective and particularly convenient way to meet the requirement. Some attenuated strains of enteric bacteria are already approved vaccine against enteric bacteria (attenuated strains of Salmonella enterica sv. Typhi and Vibrio cholerae) (Dietrich et al., 2003). Despite their efficacy, there is a concern over potential side effects. Some of currently licensed antiviral mucosal vaccines failed and were withdrawn from the market due to serious adverse reactions. On the other hand, orally administrated subunit vaccines composed of purified antigenic components of the microorganisms or inactivated whole bacterial cells are frequently not strong immunogens. Varieties of strategies have been developed to enhance the level of the local immunity induced by subunit vaccines. Among the most commonly studied is the use of bacterial cells as carriers for heterologous antigens, biodegradable microparticles, different formulations of liposomes or the use of specific oral mucosal adjuvants. Also DNA vaccines have been currently developed for administration on mucosal surfaces (McCluskie and Davis, 1999). 1.1. Identification of new potential vaccine candidates The choice of the antigens is a critical point for effective live subunit vaccine construction. Several new technologies have been recently developed to identify genes required for survival of the pathogen in mammalian host. Most of the strategies allow selection of many genes active during infection in just one experiment. Among them, the most powerful are IVET technology and STM mutagenesis. The first one – positive selection method based on using reporter genes – was originally described to look for Salmonella genes expressed in vivo. From then on, this strategy has been modified several times and adjusted to study many pathogenic bacterial species and what is more important to allow identification of genes expressed on low level during infection. STM is a negative selection assay based on comparative hybridization and applying specific tag-labelled transposons. The method has been used to fish out many genes needed to establish infection by a variety of pathogens. This strategy which is complementary to IVET, although simple and extremely useful, posses also some disadvantages such as loosing all genes essential for surviving in vitro (for review see: Chiang et al., 1999). A current approach to the identification of new potential candidates for subunit vaccine constructions is reinforced by a recent progress in bacterial genome sequencing. Since the first complete bacterial genome nucleotide sequence was reported in 1995, more than 100 genome nucleotide sequences have been annotated and published and about 200 bacterial genome sequence projects are under way. Unquestionably, any genome nucleotide sequence of bacterial pathogen encodes for many strong protective antigens and the challenge is to find and characterise them. Several types of computational tools have been recently developed to screen bacterial genomes for vaccine candidates. They mainly focus on searching for new virulence factors or membranelocated proteins. Using available data from completely and incompletely sequenced bacterial chromosomes Pallen et al. (2001) identified over twenty putative new ADP-ribosylotransferases. Also data-mining of pneumococcal genome identified many surface-exposed proteins (Wizemann et al., 2001). The results of the bioinformatic studies are the starting point for further analysis and need experimental verification. DNA

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microarray methods, also based on sequencing achievements, offer an alternative way for identification of genes transcribed under different conditions as well as for studying host response to the infection. The new strategy of identification of the potential protective antigens based on in silico analysis of bacterial genomes, termed “reverse vaccinology”, for the fist time was used to search for novel protective antigens in the genome of Neisseria meningitidis B serogroup. Out of 570 genes identified as potential vaccine candidates 350 were successfully cloned and expressed in E. coli. Finally, after protein purification and immunogenicity and protective efficacy analysis, 22 out of 85 surface-exposed proteins were classified for further analysis. The technology has been currently extended to others pathogens of medical importance (Streptoccocus pneumoniae, Porphyromonas gingivalis, Staphylococcus aureus, Chlamydia pneumoniae and Bacillus anthracis). This new concept – mining of the bacterial genome in silico – has already had a marked impact on vaccine discovery research, mainly due to its rapidity. As every technology it has also several limitations, including the fact that not all protective surface-located antigens can be find by bioinformatic tools. The result of the screening is dependent on the selected criteria. In addition, the second step of reverse vaccinology – cloning of many genes and mainly immunological testing of many proteins- is a time-consuming, rate-limiting step (for review see: Mora et al., 2003). 1.2. Novel promising adjuvants 1.2.1. DNA containing CpG motifs

The first line of immunological action against pathogens is the innate immune system. The innate immune system recognises pathogen-associated molecular patterns (PAMPs) using Toll- like receptors (TLR) and is able to discriminate different microbial components. TLR receptors belong to a large family of PRR receptors (pattern recognition receptors). As far ten classes of mammalian TLRs have been described which distinguish different conserved bacterial structures For example, TLR4 receptors recognize bacterial lipopolisacharides, TLR2 proteins reacted with lipoproteins whereas TLR5 are stimulated by bacterial flagellins. TLR signalling pathways initiated by stimulation of these transmembrane proteins by PAMNs share many similarities with IL-1R signalling and resulted in induction of many cytokine productions. TLR9 receptors recognized bacterial DNA containing unmethylated CpG motifs. The DNA with unmethylated CpG acts directly or indirectly on different kinds of immune cells, such as B cells, dendritic cells, macrophages and monocytes as well. TLR9s, in contrast to others classes of TLRs, are not located on the immune cell surface but are rather directed into cytoplasmic compartments where they are stimulated by intracellularly present bacterial DNA. It was demonstrated that, synthetic oligonucleotides containing CpG motifs (CpG ODN) in a particular sequence context mimic the structure of bacterial DNA and augment immune responses to many antigens. They stimulate both, innate and adaptive immune system. The treatment of infectious diseases with CpG is a relatively new scientific area. However, since the end of XX century when the knowledge concerning CpG motif was put into practice, several bacterial and viral human infections have been experimentally treated in this way (for recent reviews see: Underhill and Ozinsky, 2002; Wagner, 2002; Dittmer and Olbrich, 2003; Klinman et al., 2004). 1.2.2. Enterotoxins and their derivatives (LT and CT)

Other strategy to develop effective mucosal vaccine deliver via oral or nasal immunization is to employ the heat-labile Vibrio cholerea toxin (CT) and closely related heat-labile enterotoxin of E. coli (LT) as mucosal adjuvant. Both toxins are composed of five B subunits responsible for binding to eucaryotic surface exposed receptors (GM1 ganglioside) and one enzymatically active A subunit. A subunit consists of two parts: N-terminal fragment termed A1 which displays ADP-rybosylating activity and COOH terminus termed A2 which binds A subunit with pentamer formed of five identical B subunits. It was documented that CT and LT are the most potent immunogens as far described. In addition they also act as immunoadjuvants towards co-administrated unrelated antigens. The data was obtained from many independent experiments for a variety of microorganisms, as well as their products. The experiments were carried out using animal models and also in human voluntaries trials. The mechanism of mucosal adjuvantcity of CT and LT, which is, to date, not well understood, seems to be extremely complex and involves interactions of several populations of cells, mainly different kinds of APC (antigen presenting cells) (for review see: Freytag and Clements, 1999; Pizza et al., 2001). There is a controversy about the augmentation of Th1 or Th2-dependent immune response by oral administration of LT and CT. Some reports indicate that CT

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induces strongly polarized Th2 response whereas LT stimulate mixed Th1 and Th2 response (Marinaro et al., 1995; Freytag and Clements, 1999; Petrovska et al., 2003). The high toxicity of both proteins attributed to ADP-ribosyltransferase activity of A1 subunits precludes their use as adjuvants for human vaccine formulations. Two alternatives have been proposed to overcome the problem. The first one suggests the use of genetically modified toxin constructed by site-specific mutagenesis. As far, more than 50 different site directed mutants of both toxins have been obtained and used to study direct correlation between structure/ function of both proteins and their immunomodulatory action. The most profoundly analyzed mutants were those in enzyme active site (Douce et al., 1998; Pizza et al., 2001). The second approach consists of employing native or recombinant B subunits of the proteins which can be co-administrated with unrelated antigens or alternatively genetically or chemically fused to them. However, in both instances (genetically modified toxin or recombinant B subunit) the effectiveness of modified adjuvants has been shown, to be weaker than those of intact toxins. Mucosal immunogenicity has been shown to be retained by the B subunits of both LT and CT. Based on this, the licensed oral anti- V. cholerea vaccine is composed of CTB co-administrated with killed whole V. cholerea cells. The correct pentameric structure of the protein has been supposed to be crucial for its immunogenicity. Further, the cellular localization of the LT-B influenced the level of its immunogenicity. There are apparently contradictory reports in the literature on the adjuvant effects of CTB and LTB. The reason for the observed discrepancies is not completely understood. This situation is due, in part, to the variables in the model systems used. It is believed that several factors, such as type of antigen, dose, and route of immunization, method of coupling (or lack of coupling) can influence the results of the experiments. The fact that some commercially originated preparations of the B subunits are contaminated by minute amounts of intact toxins should be always taken into account. Some investigators claimed that recombinant B subunits obtained from a nontoxigenic host have no adjuvant activity. On the other hand, others presented results clearly indicating that r-LTB and r-CTB had enhanced immune responses towards intranasally and orally administered antigens (Wu and Russell, 1998; Weltzin et al., 2000). In addition some reports documented distinct differences in immunomodulatory actions of CT and LT, which could resulted from dissimilar binding abilities of CT and LT (Peterson et al., 1999; Millar et al., 2001). Thus, the problem of the CTB or LTB potential adjuvant effect still remains unsolved and still is the critical issue in respect to mucosal immunization. Recently published data proved strong immunomodulatory effect displayed by genetically modify enterotoxins after intranasal coadministration with different antigens (Jakobsen et al., 1999; Jiang et al., 2003; Periwal et al., 2003). However, at the same time some serious neurological adverse reactions were observed as consequence of immunization with LT as an adjuvant. The significance of both these findings for human LT/CT application seems to have been determined (Green and Baker, 2002). 1.3. Bacterial cells as foreign antigen carriers (attenuated pathogenic bacteria, lactic acid bacteria) Live attenuated strains of bacterial enteropathogens (Salmonella, Vibrio, Shigella and Listeria spp.) able to stimulate both, innate and adaptive immune responses, are attractive candidates for the development of mucosal multivalent vaccines. Although, many bacterial and viral antigens have been cloned and expressed in attenuated bacteria, only a few have been tested in clinical trials (Kochi et al., 2003). Attenuated, genetically defined, Salmonella strains are particularly promising among bacterial live vectors for at least two reasons. Firstly, because Salmonella strains able to immunise via mucosal surface induce the broad spectrum of immune responses. Secondly, the genetic manipulation of this genus is well understood. Several attenuated Salmonella strains were constructed for using as live vectors to deliver heterologous antigens of various viral and bacterial pathogens. The most profoundly studied strains are those obtained by deleting genes whose products are involved in key biosynthetic pathways (aro mutants) or regulatory genes crp-cya; phoP-phoQ). The efficacy of genetically modified Salmonella enterica sv. Typhimurium vaccine strains was demonstrated using different animal models. The key problem to overcome in order to obtain effective vaccine based on bacterial strains used as carriers is ensuring an adequate level of the heterologous antigen to allow maximal accumulation of the antigenic protein/s without impairing live vector ability to survive and replicate in host tissues. To solve the problem, genes for foreign antigens are cloned into plasmids of different replication systems. Additionally, foreign genes can be expressed from own promoters or placed under the control of the strong, constitutive host regulatory sequences or alternatively put under the control of environmentally regulated promoters. The subcellular location of the recombinant antigen influences its immunogenicity. Then several systems have been developed

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to assure proper assembly of the foreign antigen. Heterologous antigens produced by Salmonella vaccine strains can be secreted through cytoplasmic membrane and produced as periplasmic, secreted or outermembrane anchored proteins. Recent data emerging from studies of the host-pathogen interaction encouraged several investigators to use E. coli Hly export apparatus or type III secretion system to target recombinant antigens into appropriate cell compartment (Autenrieth and Schmidt, 2000; Gentschev et al., 2002). Additionally, attenuated Salmonella strains can be employed as a carrier for DNA vaccines as well as a system targeting cytokines directly to the host antigen presenting cells (for reviews see: Bumann et al., 2000; Garmory et al., 2002). However, in the case of using genetically engineered, attenuated pathogenic microorganisms the attention should be paid to genetic stability, host-and food-independence of the vector’s attenuated phenotype as well as on the impact of vector priming on the immunogenicity of foreign antigens. Using the probiotics [LAB (lactic acid bacteria) strains with GRAS- (Generally Recognized As Safe) status] which are autochthonous microorganisms, instead of attenuated pathogenic strains to deliver foreign antigens will allow overcoming these concerns. Moreover, this LAB-based approach seems to be very promising because most strains from Lactobacillus and Lactococcus genera are acid resistant and survive in the stomach. Many genetic tools required for foreign gene cloning and manipulation of the protein locations have been developed recently (for reviews see: Pouwels et al., 1998; Steidler, 2003). 2. Preventing campylobacteriosis Food poisoning and diarrhoeal diseases in Europe as well as in USA continues to be a serious health care problem and Campylobacter spp, gram-negative microorganisms, are one of the leading causes of bacterial gastroenteritis in human worldwide. The clinical spectrum of enteric disease due to Campylobacter infection ranges from generally mild non-inflammatory diarrhoea to severe inflammatory diarrhoea with faecal blood and leukocytes. The former is the most common clinical manifestation of the disease observed in patients from developing countries, while the latter is typical for the patients living in industrialised regions of the world. C. jejuni is also considered to be, after E. coli ETEC strains, the second most common cause of traveller’s diarrhoea (Altekruse et al., 1999; Skirrow and Blaser, 2000; Coker et al., 2002). In addition to acute gastrointestinal disease, infection with C. jejuni has been shown to be associated with GBS (GuillanBarre syndrome), a neurological disease that may lead to respiratory muscle compromise and death. It was documented that about 30% of GBS cases is preceded by C. jejuni infection (Nachamkin et al., 1998). Additionally, systemic infections do occur specially in patients at the extremes of age or those who are immunocompromised such as HIV-infected individuals. Although the mortality associated with Campylobacter infections is relatively low and no specific treatment is required for the great majority of patients with Campylobacter infection it constitutes a serious problem because of high number of cases and neurological symptoms which are a consequences of Campylobacter infection as well as because of high social and economic costs of disease. As the average lifetime of Europeans has been increasing consistently, one can expect more serious complications of Campylobacter infections particularly in cases involving old patients. Campylobacter bacteraemia has a high mortality rate mainly due to increase in resistance to two commonly used antibiotics (fluoroquinolones and macrolides) among Campylobacter isolates (Aarestrup and Wegener, 1999; Engberg et al., 2001). 2.1. Attempts to construct human anty- Campylobacter vaccine C. jejuni was first isolated from human diarrhoeal stools in 1972 by a filtration technique. Despite more than thirty years of investigations, the molecular mechanisms involved in Campylobacter virulence and pathogenesis are far from being completely understood. Relatively little is also known about immune responses during Campylobacter infection. Epidemiological and human challenge studies proved that protective immunity develops after infection; thus the prevention of Campylobacter disease seems to be feasible. It is suggested that the human vaccine will be used not for global immunisation but to immunise the selected high-risk populations such as children living in the developing countries where the disease is endemic, medical personnel or individuals travelling to highly endemic areas. Several approaches to develop an oral human vaccine have been undertaken. The investigations mainly concentrate on the evaluation of the efficacy of killed whole-cell vaccine administrated with mucosal adjuvant or prepared utilizing NST (Nutriment Signal Transduction) technology (Scott et al., 1997). However, the application of the whole

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cells (killed or attenuated) becomes questionable in the light of tremendous antigenic and genetic diversity of the Campylobacter species, the lack of complete understanding of the mechanism of Campylobacterassociated polyneuropathy (GBS) and the fact that Campylobacter spp. are naturally transformable (Duim et al., 2000; Dorrell et al., 2001). Publishing of the nucleotide sequence of the Campylobacter jejuni NCTC 11168 genome greatly facilitates analysis of the mechanisms responsible for genetic diversity and colonization process (Parkhill et al., 2000; Wren et al., 2001; Gaynor et al., 2004). 2.2. Chicken anty-Campylobacter vaccine In the developed countries campylobacteriosis is, a food born disease. Outbreaks of Campylobacter enteritis are frequently traced to contaminated milk or water, whereas the most common cause of sporadic cases is eating of undercooked poultry meat. The contaminated chickens are, by far, the principal vehicles of infection. The epidemiology of C. jejuni in broiler flocks is still unclear. Generally, birds become infected at about 3 weeks of age The sources and routes of transmission of the microorganism to the broilers on the farms remain undetermined. Recently obtained data have indicated several sources of infection, including water, wild birds and farm’s personnel. Young chickens (0– 3 weeks) are protected against infection by maternally derived specific antibodies (Sahin et al., 2003). Although, the reported level of Campylobacter organisms in the chicken intestine, especially in the ceca, varies between 10 5 –1010 per g of cecael contents, the massive colonisation does not induce any signs of the disease. A large amount of C. jejuni in the bird faeces causes further cross-contamination of Campylobacter-negative chicken carcasses in the processing plants. As a result, Campylobacter contaminates 50–80% of the raw chicken carcasses, depending on the geographical region where the study was conducted out and the method used. This fact, in combination with the relatively low human infection dose can explain why eating undercooked poultry causes majority of sporadic cases of the campylobacteriosis (for review see: Corry and Atabay, 2001). Efforts to reduce the level of contamination by a variety of intervention programs such as improvement of the biosecurity in the hatchery, a competetive exclusion technology or using chlorinated water gave, as far, variable results when introduce at the farm level and in most cases have been unsuccessful (Newell and Wagenaar, 2000). An alternative, more realistic approach for the control of Campylobacter contamination is active immunisation of the birds. To date, there is limited data on chicken immune system functioning. Furthermore, the relationship between the host and microorganism is comensal, so the elimination of Campylobacter from bird intestinal tract is not an easy task. In recent years some attempts have been undertaken to develop an effective chicken vaccine against Campylobacter. The immunogenicity and efficacy of several vaccine regimens have been evaluated in a chicken model. Rice et al. (1997) demonstrated some but not significant reduction of Campylobacter colonization of chicks orally vaccinated with formalin-killed whole bacterial cells including E. coli heat-labile toxin when compared to non-vaccinated control. In contrast, Baqar et al. (1995a; 1995b) immunized nonhuman primates and mice using the same vaccine prototype and observed stimulation of the immune response by LT. A different approach involves a subunit vaccine, which requires the choice of an appropriate protective antigen and a way to deliver it to the host immune system. Recent data documented that multiple, immunogenic Campylobacter proteins are post-translationaly modified by glycosylation. In addition, glycosylation process affects ability of the pathogen to colonize chicken intestinal track (Karlyshev et al., 2004). Genetic and structural analysis of the O- and N-linked glycosylation systems of Campylobacter as well as cloning and expression of Campylobacter pgl gene cluster in E. coli facilitates selection of proper antigen for immunization (Wacker et al., 2002; Szymanski et al., 2003). So far only flagellin has been evaluated in the chicken model (Khoury and Meinersmann, 1995; Lee et al., 1999). Variability of the surface-exposed domains of the FlaA and its O-linked glycosylation complicate the use of flagellin for vaccination. Live and genetically engineered bacterial strains are considered to be most effective as vaccines against many enteropathogens. To be used as chicken vaccine a Campylobacter strain needs not only to be attenuated for humans but also to be immunogenic for birds. This means that it needs to persist long enough in the birds’ gut-associated tissues to induce the protective immune responses. Despite many efforts such a strain has not yet been developed. Ziprin et al. (1999; 2001) have shown that genetic knockout of four C. jejuni genes (ciaB, dnaJ, pldA and cadF), which code for proteins involved in different stage of pathogenesis renders strains incapable of colonizing the chicken intestinal tract, although they are able to colonize the crop (Ziprin et al., 2002). The post-genomic era results in new instruments for analyzing virulence-related mechanisms. Several new Campylobacter virulence factors have been identified by bioinformatics tools. One of them is new IM-located thiol-oxidoreductases of C. jejuni encoded by gene denoted dsbI. Campylobacter strains lacking DsbI activity will be examined as potential vaccine candidate (Raczko et al., 2004).

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The poultry are a major source of human Salmonella as well as Campylobacter infections. Since the attenuated Salmonella strains are highly effective chicken anti- Salmonella vaccine than one may suppose that avirulent Salmonella producing Campylobacter antigens may be attractive option for chicken dual vaccine (Curtiss and Hassan, 1996). Several C. jejuni genes coding immunodominant proteins have been identified. Three of them have been cloned, sequenced, characterized and expressed. in vaccine Salmonella strains. CjaA (Cj0982c – 30 kDa) and CjaC (Cj0734c – 28 kDa) proteins exhibit relevant overall homology to several prokaryotic solute-binding proteins (family 3) components of the ABC transport system (Pawelec et al., 1997; Pawelec et al., 1998). CjaD (Cj0113 – 18 kDa) protein exhibits homology to PAL (peptidoglican-associated lipoprotein) of gram-negative bacteria. Campylobacter genes cjaA, cjaC and cjaD were cloned into Asd+ cloning vector and introduced into avirulent S. enterica sv. Typhimurium Dasd Dcrp Dcya c3987. It was documented that chicken orally immunized with avirulent Salmonella strain expressing Campylobacter antigen developed serum IgG and mucosal IgA responses against Campylobacter membrane proteins. Moreover, this strategy greatly reduced the ability of heterologous wild-type strain to colonize the bird cecum (Wyszynska et al., 2004). In order to augment the efficacy of vaccine prototype several fusions of two Campylobacter genes (cjaA and cjaD) with etxB, which encodes B subunit of E. coli LT toxin, have been constructed (Wyszynska et al., 2002). Recombinant plasmids expressing hybrid proteins introduced into avirulent Salmonella enterica sv. Typhimurium strain will be utilized to study the LTB adjuvant effect on the immunogenicity of the C. jejuni co-administrated antigens on two animal models (chicken vs mice). Conclusion The post-genomic era, started in 1995, brings many new technologies useful for studying bacterial pathogenesis, specifically the host-pathogen interaction processes. Recent strategies, such as reversed vaccinology or DNA miccroarrays, doubtlessly decrease the time and cost required for identification of bacterial antigens. However, to put the knowledge into medical practice all new proteins have to be tested in animal models to prove their efficacy and safety. Moreover, determination of gene function, being sometimes hard or even impossible to accomplish, ensure for reasonable choice of proper antigen. Another limitation of the vaccinology development derives from the lack of understanding of mammalian immune system functioning. The recent discovery of TLR receptors, the better understanding of APCs (antigen presenting cells) role in prevention of the infectious diseases as well as cross-talk between innate and adaptive immune systems are the milestones of the vaccinology. The gained knowledge facilitates modulation of the immune responses by using appropriate delivery system or/and adjuvant, which are crucial to differentiate immune response into Th1 or Th2 type. Then, the recent achievements in bacterial genomics and proteomics as well as in human immunology and genomics radically changed vaccine development. Novel approaches towards vaccination should result in modern effective and easy to deliver vaccines against emerging or re-emerging pathogens. Also, there are many “old” infectious diseases for which conventional ways to discover vaccines failed. However, taken into account knowledge about mechanisms responsible for genetic diversity of microorganisms and recent vaccinology failures (oral vaccine against rotavirus diarrhea and nasal vaccine against influenza) one has to be more realistic. The future will show us to what extent the optimism is justified. Literature A a r e s t r u p F.M. and H.C. W e g e n e r. 1999. 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Safety and immunogenicity of a prototype oral whole-cell killed Campylobacter vaccine administered with a mucosal adjuvant in non-human primates. Vaccine 13: 22–28. B u m a n n D., C. H u e c k, T. A e b i s c h e r and T.F. M e y e r. 2000. Recombinant live Salmonella spp. for human vaccination against heterologous pathogens. FEMS Immunol. Med. Microbiol. 27: 357–364. C h i a n g S.L., J.J. M e k a l a n o s and D.W. H o l d e n. 1999. In vivo genetic analysis of bacterial virulence. Annu. Rev. Microbiol. 53: 129–154.

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Mucosal adjuvant effect of cholera toxin in mice results from induction of T helper 2 (Th2) cells and IL-4. J. Immunol. 155: 4621–4629. M c C l u s k i e M.J. and H.L. D a v i s. 1999. Mucosal immunization with DNA vaccines. Microbes Infect. 1: 685–698. M i l l a r D.G., T.R. H i r s t and D.P. S n i d e r. 2001. Escherichia coli heat-labile enterotoxin B subunit is a more potent mucosal adjuvant than its vlosely related homologue, the B subunit of cholera toxin. Infect. Immun. 69: 3476–3482. M o r a M., D. V e g g i, L. S a n t i n i, M. P i z z a and R. R a p p u o l i. 2003. Reverse vaccinology. Drug Discov. Today 8: 459–464. N a c h a m k i n I., B.M. A l l o s and T. H o. 1998. Campylobacter species and Guillain-Barre syndrome. Clin. Microbiol. Rev. 11: 555–567. N e w e l l D.G. and J.A. W a g e n a a r. 2000. Poultry infections and their control at the farm level, p. 497–509. In: I. Nachamkin and M.J. Blaser, Poultry infections and their control at the farm level. 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Polish Journal of Microbiology 2004, Vol. 53, Suppl., 17–22

Molecular Aspects of Listeria monocytogenes Infection AGATA KRAWCZYK-BALSKA and JACEK BIELECKI

Department of Applied Microbiology, Institute of Microbiology, Warsaw University, Miecznikowa 1, 02-096 Warsaw, Poland Abstract Listeria monocytogenes, a food-borne intracellular animal and human pathogen, interacts with infected host cells both prior to entry and during the intracellular phase of infection. This review is focused on the role of secreted proteins, including listeriolysin O and two distinct phospholipases C, in modulating the signal transduction of infected cells. K e y w o r d s: Listeria monocytogenes, intracellular signaling, listeriolysin O, phospholipases C

1. Introduction Listeria monocytogenes (Lm) is a facultative intracellular bacterial pathogen widely distributed in the environment that encounters a variety of cell types after being consumed in contaminated food, first in its passage through the digestive tract, then the liver, the circulatory system, and the central nervous system, often with fatal consequences (Schuchat et al., 1997; Vázquez-Boland et al., 2001). In the host organism Lm rapidly infects the liver and spleen. In the liver most of the bacteria are killed upon initial encounter with Kupffer cells, resident macrophages in the liver, or with immigrant macrophages. Since some Lm can survive this encounter, neutrophils play an important role in resolving these infections (Portnoy et al., 1994; Sheehan et al., 1994). The proportion of surviving bacteria will vary depending on the immunological system state of the host. In studies with isolated macrophages and macrophage-derived cell-lines, the surviving bacteria escape from the primary phagocytic vacuole, grow and divide in the cytoplasm, recruit and polymerize host cell actin, which provides the driving force for movement through the cytoplasm and into nearby cells by means of filopodia-like projections (Tilney and Portnoy, 1989). In these neighboring cells bacteria escape from double-membrane secondary phagosomes and repeat the cycle of growth, recruitment of actin and cell-to-cell spread. Lm secretes listeriolysin O (LLO) and two distinct phospholipases C (PLC), one specific for phosphatidylinositol (PI) and glycosyl-PI (GPI) – anchored proteins (PI-PLC) (LeimeisterWächter et al., 1991; Mengaud et al., 1991; Camilli et al., 1991), and a second broad-range phospholipase (PC-PLC) capable of hydrolyzing a wide variety of mammalian phospholipids, including sphingomyelin (Geoffroy et al., 1991). These three proteins are encoded by genes located in a central virulence gene cluster, and their expression is positively regulated by the transcriptional activator PrfA (Kuhn et al., 1999). A number of studies have provided evidence for the participation of both PLCs in addition to LLO in the induction of host intracellular signaling. In this review we briefly describe the properties of these proteins and their roles in various interactions with different type of infected host cells. Host cell signaling induced by internalins has been reviewed recently (Cossart, 2001; Cossart et al., 2000), and will not be discussed here. 2. Host cell responses to presence of LLO and PLCs Evidence is accumulating that LLO and PLCs are multifunctional virulence factors with many important roles in the host-parasite interaction other than phagosomal membrane disruption. Exogenous and endogenous exposure to LLO and, in many cases, PLCs which play an accessory role, may induce a number of host cell responses such as cell proliferation and focus formation in transfected fibroblasts (Demuth et al., 1994), activation of the Raf-Mek-mitogen-activated protein (MAP) kinase pathway in epithelial cells

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(Tang et al., 1996; Tang et al., 1994; Weiglein et al., 1997), mucus exocytosis induction in intestinal cells (Coconnier et al., 1998), modulation of internalization via calcium signaling (Wadsworth and Goldfine, 1999), cytokine expression in macrophages (Nishibori et al., 1996), degranulation and leukotriene formation in neutrophils (Sibelius et al., 1999), apoptosis in dendritic cells (Guzman et al., 1996), phosphoinositide metabolism, lipid mediator generation (Sibelius et al., 1996b), NF-kB activation (Kayal et al., 1999), expression of cell adhesion molecules in infected endothelial cells (Krüll et al., 1997), and expression of Fas ligand on T lymphocytes (Zenewicz et al., 2004). More details of these aspects of activity of LLO and PLCs can be found below. 3. Molecular mechanisms of intracellular signaling induced by LLO and PLCs 3.1. Calcium signaling

Ca2+ is a very important second messenger in a great variety of eukaryotic cell signaling processes (exocytosis, contraction, metabolism, gene transcription, fertilization and proliferation) and is an important regulator of actin microfilaments (Berridge et al., 2000). In case of infection of macrophage-like J774 cells with wild-type Lm three elevations of intracellular calcium (Ca2+) was observed. The first begins within 1 min after addition of a washed suspension of bacteria in PBS to a monolayer of J774 cells. After a brief decline to near basal levels, Ca2+ increases again at 5–7 min post-infection (p.i.), declines and then increases for a prolonged period starting at 15 min p.i. LLO mutant produces none of these calcium fluxes. A PI-PLC mutant produced a relatively weak signal beginning at 15 min that lasted for about 15 min, and a PC-PLC mutant produced the first signal in Ca2+, but not the others. The first and second elevations of Ca2+ occur when very few wild-type bacteria have been internalized, and these calcium signals are seen in cells containing no bound or internalized bacteria (Wadsworth and Goldfine, 1999). Thus, it appears that calcium signaling can be induced by secreted factors. Pretreatment of host cells with the receptor-operated calcium channel blocker SK&F96365 or by removal of extracellular calcium by chelation with EGTA prevented all three Ca2+ elevations. Pretreatment with thapsigargin, an inhibitor of calcium release from intracellular stores, prevented only the second and third elevations. Together, these results indicated that the first calcium elevation resulted from influx of extracellular calcium through a channel and that the latter two elevations reflected release of calcium from intracellular stores, which required the initial calcium influx. Correlation between calcium signaling pattern and adhesion, internalization of bacteria was observed. The entry of wild-type Lm into J774 cells is slower than the entry of the LLO and PI-PLC mutants. Like heat-killed bacteria, these mutants are taken up rapidly so that from 50 to 70% of bacteria associated with the host cells are internalized within 1 min after infection compared to 10–15% of the wild type. Preventing the elevation of Ca2+ with either SK&F96365 or thapsigargin increased the rate of entry, but not to that observed with the LLO and PI-PLC mutants. Treatment with either inhibitor also resulted in greatly decreased efficiency of escape of wild-type Lm from the primary phagocytic vacuole (Wadsworth and Goldfine, 1999). Calcium signaling was also observed during infection of epithelial cell line Hep-2 with Lm (Dramsi and Cossart, 2003). In this case wild type Lm, but not nonpathogenic species L. innocua or Lm mutant strain defective in listeriolysin O production, was able to induce Ca2+ fluxes. In opposition to macrophage-like cells, increase of Ca2+ observed rapidly after infection of epithelial cells, facilitates Lm entry into these cells. Pretreatment of host cells with calcium channel antagonists or chelation of extracellular calcium markedly reduced Lm entry. In contrast, chelation of cytosolic Ca2+ or blockade of Ca2+ release from intracellular stores did not affect invasion. These results suggest that Lm-induced mobilization of extracellular Ca2+ by LLO and activation of downstream Ca2+ – dependent signaling are required for efficient cell invasion (Dramsi and Cossart, 2003). Long-lasting oscillations of intracellular Ca2+ level caused by LLO applied extracellularly were also demonstrated in case of human embryonic kidney cells (Repp et al., 2002). This was presumably resulted from a pulsed influx of extracellular Ca 2+ through pores that are formed by LLO in the plasma membrane. Unfortunately, it was not tested how changes in Ca2+ affect infection of kidney cells. 3.2. Induction of host phospholipases

The ability of wild-type Lm to induce calcium fluxes in J774 macrophage-like cells and the finding that the second brief elevation and the third prolonged elevation were inhibited by factors, which deplete calcium stores, indicated that these calcium fluxes resulted from the release of Ca2+ from intracellular stores

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(Wadsworth and Goldfine, 1999). An important mediator of Ca2+ release from the endoplasmic reticulum is inositol-1, 4, 5-P 3 (IP3), a product of hydrolytic activity of eukaryotic calcium-activated PLC on PI-4, 5-P2 (PIP2). Distinct elevation of IP2 and IP3 in murine bone marrow-derived macrophages infected with wildtype Lm was shown, but not with an LLO– mutant. Infection of J774 cells prelabeled with [3H]-inositol with wild-type Lm resulted in elevation of [3H]-IP within 10 min, followed by more rapid release at 20–30 min. Much less [3H]-IP formation was observed upon infection with mutants deficient in either LLO or PI-PLC, but loss of PC-PLC did not affect IP formation. It appears that PI-PLC with the aid of LLO rapidly hydrolyzes host PI, resulting in release of diacylglycerol (DAG) and IP (Goldfine et al., 2000). Since the Lm PI-PLC does not hydrolyze PIP2, release of IP3, unlike release of IP, is an indicator of host PLC activation (Goldfine and Knob, 1992; Griffith and Ryan, 1999). IP 3 elevation occurred within 10 min of infection of J774 cells with wild-type Lm, but not with an LLO mutant. IP3 elevation was somewhat reduced with a PI-PLC mutant. Earlier studies with human umbilical vein endothelial cells (HUVEC), which had not internalized Lm during the course of these experiments, showed that a combination of LLO and Lm PI-PLC secreted by recombinant Listeria innocua strains, resulted in the formation of products of phosphoinositide hydrolysis resulting from activation of host PLC isoforms (Sibelius et al., 1996a). DAG was also observed to accumulate rapidly. It appears that host PLC isoforms were activated since a similar response was elicited by LLO alone (Sibelius et al., 1996b). It was proposed a model for the delivery system for PI-PLC in which LLO enable PI-PLC translocation into cytosol of infected cells and, which is analogous to the type III secretion system of gram-negative bacteria (Sibelius et al., 1996a; Madden et al., 2001). In this model pores formed by LLO permit access of PI-PLC to PI in the inner leaflet of the host cell plasma membrane, resulting in the formation of IP, and a separate induction of host phospholipases. From studies with J774 cells, it appears likely that LLO induces a potent phosphoinositide response, in the absence of sustained calcium elevation, since the double PI-PLC, PC-PLC deletion mutant of Lm, which produced no intracellular Ca2+ elevation (Wadsworth and Goldfine, 1999), evoked the release of IP2 and IP3, but an LLO– strain did not (Goldfine et al., 2000). In both HUVEC and human neutrophils Lm and L. innocua strains expressing LLO induced the release of platelet activating factor and products of arachidonate metabolism, prostaglandin I2 or leukotrienes, which indicates activation of host phospholipase A 2 (Sibelius et al., 1996a; Sibelius et al., 1996b; Sibelius et al., 1999). In HUVEC, a combination of LLO and PI-PLC were required for maximal responses, but in neutrophils, the response was mainly attributed to LLO. 3.3. Mobilization of isoforms of host protein kinase C

Since DAG and calcium are known activators of PKC, it was postulated that hydrolysis of PI by bacterial PI-PLC leads to the production of DAG, and thus activation of PKC isoforms in infected host cells (Wadsworth and Goldfine, 1999). The four major isoforms of PKC found in the J774 murine macrophagelike cells are ", $I, $II and * (Smith et al., 1997). Of these, ", $I, $II are “classical” isoforms activated by intracellular calcium and/or DAG, while PKC * is a “novel” isoform which is activated by DAG, but is Ca2+ – independent (Ron and Kazanietz, 1999). PKC * has been implicated in phosphorylation and opening of Ca2+ channels in eukaryotic cells (Levin et al., 1997). It was observed that PKC * moves to a peripheral location within 30 s of addition of a washed suspension of wild-type Lm or the PC-PLC mutant to J774 cells, but not after addition of either LLO or PI-PLC deletion mutants. Studies performed using calcium channel blocker show that this translocation is calcium-independent. On the other hand, it was inhibited by 3 h pretreatment with phorbol myristate acetate (PMA), which downregulates PKCs. Pretreatment with PMA or rottlerin, an inhibitor of PKC *, abolished the first calcium signal, consistent with the role of PKC * in activating calcium channels (Wadsworth and Goldfine, 2002). Further studies revealed rapid translocation of PKC $I and $II to early endosomes after infection of J774 cells with wild-type Lm or the PC-PLC mutant. The translocation of PKC $II was seen from 30 s to 3 min p.i., while that of PKC $I occurred between 1 and 4 min p.i. No PKC $I or $II translocation was observed after infection with a LLO mutant. PKC $I, but not PKC $II, translocation to early endosomes occurred after infection with a PI-PLC mutant. PKC translocation like the first calcium elevation occurred in cells that had not internalized Lm (Wadsworth and Goldfine, 2002). Translocation of PKC $II to early endosomes is calcium- and presumably DAG-dependent since it was blocked by treatment with the calcium channel blocker and required bacterial PI-PLC. Translocation of PKC $I, in contrast, did not appear to require elevated intracellular Ca2+, since it was seen upon infection with a double phospholipase mutant (plcA, plcB), which produced no calcium signal in J774 cells. DAG was presumably produced upon infection with the double phospholipase mutant by activation of host phosphoinositide-specific PLC (Wadsworth and Goldfine, 1999; Wadsworth and Goldfine, 2002). These

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results lead to a proposed signaling pathway in macrophage-like cells in which LLO and PI-PLC cooperate in cleavage of host PI, resulting in translocation of PKC " to the cell membrane. PKC d mobilization leads to Ca2+ influx, which in combination with DAG, results in translocation of PKC bII to early endosomes. Lm mutants that do not secrete phospholipases, but do secrete LLO, can activate PKC $I through activation of host phospholipases, which also produce DAG. As noted above, inhibition of calcium signaling resulted in increased early association of wild-type Lm with J774 cells and increased uptake of associated bacteria. PI-PLC or LLO mutants associate with J774 cells and enter more rapidly than the wild type, and drugs that block calcium signaling had minimal effects on uptake of these mutants (Wadsworth and Goldfine, 1999). Hispidin, which inhibits PKC $I selectively, also increased the association of wild-type Lm with J774 cells. The percent of wild-type bacteria internalized was also increased. It appears that PKC signaling modulates uptake of Lm into these macrophage-like cells. As was the case for inhibitors of calcium signaling, hispidin also greatly decreased the efficiency of escape of both wild-type Lm and PI-PLC mutant from the primary vacuole (Wadsworth and Goldfine, 2002). 3.4. Induction of host phospholipase D (PLD) activity

Activation of eukaryotic PLD is often obtained with agonists that activate PLC isoforms, and in some systems activation of PLD occurs downstream of PLC (Singer et al., 1997). Infection of J774 cells with wild-type Lm and with mutants lacking either or both of the Lm PLCs resulted in activation of host PLD. Of the strains tested, only that lacking LLO failed to activate PLD. This increase in PLD activity occurred 15 min p.i. and, therefore, after translocation of PKC bI, bII, and d. It is either coincident with or following activation of host polyphosphoinositide-specific PLC (Goldfine et al., 2000). The activation of PLD in J774 cells coincides with the entry of wild-type Lm into host cells and the beginning of vacuolar maturation (Wadsworth and Goldfine, 1999). Inhibition of PLD by 2,3-diphosphoglycerate was accompanied by decreased efficiency of escape from the primary vacuole (Goldfine et al., 2000). Since the loss of PI-PLC by mutation also results in diminished efficiency of escape, but does not affect PLD activation, PI-PLC probably contributes to the escape process through another pathway. There is a tight coupling between phagocytosis of Mycobacterium tuberculosis as well as opsonized zymosan particles and PLD activation in human macrophages (Kusner et al., 1996). PLD activation and the product of its activity, phosphatidic acid, are thought to play important roles in intracellular vesicle transport, but the mechanisms have not been established (Exton, 1997). 3.5. Activation of NF-6B

NF-6B is an important transcription factor that mediates the response to infection through regulation of genes involved in the immune response (Siebenlist et al., 1994). In its inactive form NF-6B is found in the cytoplasm bound to inhibitor proteins. Stimulation of cells by various extracellular agents such as viruses, oxidants, inflammatory cytokines and lipopolysaccharide leads to phosphorylation cascade and subsequent degradation of inhibitor proteins. Thus NF-6B is released from the complex and translocates into the nucleus, where it regulates the expression of many targets including proinflammatory cytokines. Lm infection has been shown to activate NF-6B in a number of different cell types. In P388D1 murine macrophages a biphasic activation of NF-6B was observed. Transient NF-6B activation was induced by adhesion of Lm to P388D1 cells, which also occurred upon addition of nonvirulent L. innocua or by addition of listerial lipoteichoic acid to these cells (Hauf et al., 1997; Hauf et al., 1994). The second phase of NF-6B activation was persistent and appeared to require a signal induced during escape of Lm from the phagosome or during intracellular replication, as NF-6B activation was significantly lower when cells were infected with phospholipase (plcA or plcB) or ActA mutants (Marquis et al., 1997; Hauf et al., 1997; Marquis and Hager, 2000). In the Caco-2 epithelial cell line, Lm infection induced a monophasic activation of NF-6B connected to lipoteichoic acid activation occurred prior to entry of the bacteria. This activation occurred prior to entry of the bacteria since addition of agents blocking bacterial invasion, failed to inhibit NF-6B activation (Hauf et al., 1999). Infection of HUVEC results in translocation of NF-6B to the nucleus and upregulation of adhesion molecules such as E-selectin, VCAM-1 and ICAM-1 and LLO is required for full NF-6B activation (Drevets, 1997; Schwarzer et al., 1998; Kayal et al., 1999). There is unclear that bacterial phospholipase expression is required for NF-6B activation since such evidence correlated with the ability to produce ceramide was reported in one studies (Schwarzer et al., 1998), but not in the other study that examined this requirement (Kayal et al., 1999).

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4. Conclusions It appears that Lm manipulates intracellular signaling in different type of host cells at all steps of infection including the initial phase, in which bacteria interact either through secreted proteins or by contact; after internalization, when bacteria reside in an endocytic vacuole; upon release into the cytosol; and during cell-to-cell spread, when Lm resides briefly in the double-membrane secondary vacuole. LLO and PLCs play a crucial role in modulating the signal transduction of infected cells providing the optimal level of invasion and intracellular growth, and thus these exotoxins are multifunctional virulence factors with many important roles in the host-parasite interaction other than phagosomal membrane disruption. Literature B e r r i d g e M.J., P. L i p p and M.D. B o o t m a n. 2000. The versatility and universality of calcium signalling. Nat. Rev. Mol. Cell Biol. 1: 11–21. C a m i l l i A., H. G o l d f i n e and D.A. P o r t n o y. 1991. 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Listeria monocytogenes potently induces up-regulation of endothelial adhesion molecules and neutrophil adhesion to cultured human endothelial cells. J. Immunol. 159: 1970–1976. K u h n M., W. G o e b e l, E.T. R y s e r and E.H. M a r t h. 1999. Listeria, listeriosis and food safety, p. 97–130. Marcel Dekker, New York. K u s n e r D.J., C.F. H a l l and L.S. S c h l e s i n g e r. 1996. Activation of phospholipase D is tightly coupled to the phagocytosis of Mycobacterium tuberculosis or opsonized zymosan by human macrophages. J. Exp. M. 184: 585–595. L e i m e i s t e r - W ä c h t e r M., E. D o m a n n and T. C h a k r a b o r t y. 1991. Detection of a gene encoding a phosphatidylinositol specific phospholipase C that is co-ordinately expressed with listeriolysin in Listeria monocytogenes. Mol. Microbiol. 5: 361–366. L e v i n R., A. B r a i m a n and Z. P r i e l. 1997. Protein kinase C induced calcium influx and sustained enhancement of ciliary beating by extracellular ATP. 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Polish Journal of Microbiology 2004, Vol. 53, Suppl., 23–27

Optical Lectin Based Biosensor as Tool for Bacteria Identification JANA MASAROVA, ESTERA SZWAJCER DEY and BENGT DANIELSSON

Pure and Applied Biochemistry, LTH, Lund University, S-221 00, Sweden Abstract Biosensor techniques are based on biospecific interaction between the biological parts of biosensor with the analyte. In biosensor construction, antibodies are usually used for the detection of analytes such as microorganism, because of very strong and highly specific interaction. The disadvantages of this assay are a long time needed for antibody isolation and purification as well as difficult regeneration of biosensor chip. The use of lectins instead of antibodies could solve these problems because a several hundred lectins are commercially available and their stability in standard buffers is better compared to monoclonal antibodies. While antibody can only be used to detect that antigen it was designed for, lectin as low affinity molecule may bind several different pathogens. Using the discriminative effect of an artificial neural network the application of a lectin array will compensate for the lower specificity. Microbial surfaces bear many of the sugar residues capable of interacting with lectins. The ability of lectins to react with microbial glycoconjugates means that it is possible to employ them as probes and sorbents for whole cells, mutants and numerous cellular constituents and metabolites, and it makes them useful tools for identification or typing of bacteria. Lectins are attractive reagents for the clinical diagnostic laboratory because of their diverse specificity, commercial availability, a wide range of molecular weights, and their stability in standard buffers. The construction of lectin biosensor could be an advantage method for detection of pathogenic bacteria. K e y w o r d s: biosensors, lectins application, bacteria identification

Introduction Foodborne illness has increased dramatically throughout the world. It is estimated that there are a few hundred million cases of foodborne illness each year resulting in several thousands of deaths. Pathogenic bacteria in foods are the cause of 90% of this reported illness. Council of Agricultural Science and Technology in 1998 released eighteen recommendations for producer organizations, governmental employees, scientists, and public interest groups. One of the important tasks for research is to develop rapid, accurate detection methods for foodborne pathogens and toxins. Conventional methods for microorganism identification are sensitive, but they require too long time for detection. Typically, a small sample of the analyzed food is homogenized, incubated and pathogens are identified after at least one to three days by stains, biochemical tests and/or serological reaction (De Boer and Beumer, 1999; Artault et al., 2001). Some new methods for detection of microorganisms have been developed recently including chromatography, spectroscopy, immunomagnetic-electrochemiluminiscence, immunomagnetic separation with flow cytometry, impedance monitoring, nucleic acid hybridization and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. PCR-based methods are also much used for sensitive detection (Toze, 1999), but require careful sample preparation and is more time-consuming than the proposed technique. Beside these methods, biosensor techniques play an important role since they are based on biospecific interaction between the biological parts of the biosensor with the analyte (microorganisms). In biosensor construction, antibodies are usually used for the detection of microorganisms because of very strong and highly specific interaction. The disadvantage of this assay format is the demand for antibodies against the antigen, which takes more than 3 months to obtain (including immunization, purification and shipment). Even if purified antibodies against desired antigen are obtained, cross-reactivity may occur, resulting in lower specificity. For regeneration of the biosensor chip very strong agents are needed because of strong antibody-antigen binding.

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Masarova J. et al.

Antibody recognize whole O-antigen a-Man b-Gal b-GlcNAc

Lectins recognize individual mono- and/or oligosaccharides

Fig. 1. Comparison of the binding specificity between O-antigen and lectin or antibody

The use of lectins instead of antibodies could solve these problems, since several hundred lectins with different saccharide specificities are commercially available. Lectins compared to monoclonal antibodies have a better stability, but on the other hand in some cases lower specificity. At first sight lower sensitivity is a disadvantage, but a high affinity antibody binds only the antigen it was designed for and can only be used to detect that antigen, while a low affinity molecule (lectin) may bind several different ligands (pathogens). Using the discriminative effect of an artificial neural network the application of a lectin array will compensate for the lower specificity. Figure 1 shows difference between lectin and antibody recognizing of O-antigen structure. Lectins Lectins are sugar-binding proteins of non-immune origin that agglutinate cells and/or precipitate complex carbohydrates. They contain at least two sugar-binding sites and they are capable of binding glycoconjugates even in presence of various detergents. The specificity of lectins is usually defined by the mono- or oligo-saccharides that are best at inhibiting the agglutination or precipitation the lectin causes. The precise physiological role of lectins in nature is still unknown but they have proved to be very valuable in a wide variety of applications in vitro. They have been isolated from various sources: bacteria, plant, animal, virus, and fungi and they are present in cells and biological fluids (Duverger et al., 2003). They are involved in a wide variety of biological functions including bacterial growth, recognition, adhesion, cancer metastasis, bacterial and viral infections (Lis and Sharon, 1998; Varki, 1993; Ashraf and Khan, 2003) Bacterial lectins play an important role in the initial stages of infection by mediating the interaction of pathogens with host cell surface glycoconjugates. By virtue of their ability to mediate adhesion to host tissues, the lectins can be considered as bacterial virulence factors (Listeria references+ Streptomyces). Thus, lectin-deficient mutants of the pathogens are often unable to initiate infection (Sharon and Ofek, 2001). Plant lectins are extensively used in purification, detection and structural characterization of glycoconjugates, investigation of cell-surface architecture, blood typing and identification and differentiation of various procaryotic and eucaryotic cells and as epidemiologic as well as taxonomic markers (Lis and Sharon, 1998). The main drawbacks of employed approaches were in the need for fluorescent-labelled or enzyme-labelled lectin preparations (Graham et al., 1984; Singh and Doyle, 1993) and in many cases the use of non-immobilized lectins. Direct aggregation of suspended microorganisms is one of the important applications of lectins in microbiology. Almost all microorganisms express surface-exposed carbohydrates which are potential lectin-reactive site. The lectin receptors of microorganisms are listed in Table I (Calderon et al., 1998). Table I Lectin receptors of microorganisms (Calderon et al., 1998) Fungi

Arabinans, capsules, cell wall glucan, chitin, galactans, mannans, secreted proteins

Gram-negative bacteria

Capsules, cytoplasmic membranes, lipopolysaccharides, lipooligosaccharides, outer membranes, peptidoglycan, surface array glycoproteins

Gram-positive bacteria

Capsules, group-specific polysaccharides, lipotechoic acids, peptidoglycans, surface arrays, teichoic acids, teichuronic acids

Protozoa

Galactomannans, glycoproteins, glycolipids, lipophosphoglycans, phosphoglycans

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25

Free, non-immobilized lectins were applied in most tests for identification of bacteria mainly in seventies and eighties years of last century (Reeder and Estedt, 1971; Hamada et al., 1977; DeLucca, 1984; Davidson et al., 1982; McSweegan and Pistole, 1982) but also in recent years (Hynes et al., 1999; Aabenhus et al., 2002; Hynes et al., 2002; Annuk et al., 2001). While the earlier works were focused on qualitative demonstration of bacteria agglutination by individual lectin or panel of lectins, the recent works deal about differentiation and phenotyping of bacterial isolates based on agglutination assays. Immobilized lectins were successfully used for isolation of bacteria (Listeria monocytogenes, Salmonella sp., Staphylococcus aureus, E. coli) from milk and ground beef. Lectin-derivatized surfaces were also used to capture and concentrate microorganisms and even enveloped viruses with subsequent detection by MALDI mass spectrometry. Biosensors Biosensors use a combination of biological receptor compounds (antibody, enzyme, nucleic acid, lectin, etc.) and the physical or physico-chemical transducer directing, in most cases, real-time observation of a specific biological events (e.g. antibody-antigen, lectin-carbohydrate interaction) (Leonard et al., 2003). Biosensors may be divided into four basic groups, depending on the method of signal transduction: optical, mass, electrochemical, and thermal biosensors (Goepel, 1991; Seyhi, 1994). Optical biosensors are very attractive because they allow direct label-free and real-time detection of bacteria. They are frequently used for medical purposes and they are very promising for assessment of many diseases enhancing quality of people’s life. Surface plasmon resonance (SPR) is a powerful optical biosensing technique for non-label bioaffinity interaction analysis (antigen-antibody, lectin-saccharide, and receptor-hormone). It is phenomenon that occurs during optical illumination of a metal surface and it can be harnessed for biomolecular interaction analysis (Liedberg et al., 1995). SPR allows to monitor the process of interaction in real-time and in continuous mode providing a highly automated device enabling routine analysis without need for trained personnel. Six companies currently manufacture a variety of biosensor hardware based on SPR technique: Biacore AB (Uppsala, Sweden), Affinity Sensors (Franklin, MA), Windsor Scientific Limited (Berks, UK), BioTul AG (Munich, Germany), Nippon Laser and Electronics Lab (Hokaido, Japan), and Texas Instrument (Dallas, TX). Biacore AB released the first commercial instrument in 1990 and approximately 90% of commercial biosensor publication cites the Biacore instrument (Rich and Myszka, 2000). Biacore 3000 instrument has a few advantages compared with previous models: ● It allows measure interaction between analyte and four different ligands at the same time; ● Lower sample consumption due to smaller flow cell volume; ● Online data subtraction; ● Micro-sampler recovery. Although lectins have been commonly used for characterization and determination of tumor-associated markers, synthetic carbohydrates, synthetic glycopeptides, and microbial carbohydrates using SPR technique (Mecklenburg et al., 2002, Mislovicova et al., 2002, Holmskov et al., 1996; Okazaki et al., 1995), only one report has been published in which lectin (Con A) immobilized on SPR chip was used to capture cells (erythrocytes) (Quinn and O’Kennedy, 2001). SPR has not yet been reported for monitoring of the interaction between lectin and microorganism for diagnostic purposes. In our work we probed possibility to utilize lectins for bacteria identification. Eight lectins with various specificities (Table II) were covalently attached on Biacore™ sensor chips CM5 via their amine groups and analyte containing only endotoxin or whole bacteria cells was injected onto the surface. The lectins listed in Table II were used for differentiation of bacteria based on lectin-endotoxin (lipopolysacharide; LPS) interaction. Only endotoxins (Salmonella toucra O48, Salmonella typhimurium, Escherichia coli O104, Escherichia coli C600, Klebsiella oxytocyta 666, and Citrobacter youngae O2) as well as whole bacteria (Citrobacter freundi, Salmonella typhimurium, Escherichia coli O157, and nonpathogenic Escherichia coli) were applied. Biacore 3000 was employed for determination of lectin-LPS interaction. A contribution of non-specific interaction between lipid part of endotoxin and lectins was occurred and it was reduced by addition of detergent. The obtained lectin binding patterns were well correlated with theoretical patterns derived from carbohydrate structure of O-antigens. The binding pattern of bacteria Salmonella typhimurium corresponded with one obtained by lectin-lipopolysaccharide interaction (Masarova et al., 2004a). The experiment with whole cells showed the possibility to distinguish bacteria

26

Masarova J. et al. Table II Lectin panel used in biosensor preparation Lectin

Abbrev.

Carbohydrate specificity

Mr

Canavalia ensiformis

Con A

"-Man, "-Glc

104 000

Maackia amurensis

MAA

"-SA

140 000

Ulex europeaus I.

UEA I.

"-Fuc

63 000

ECA

"/$-GalNAc "/$-Gal

54 000

Sambucus nigra

SNA

"-SA-"-Gal

150 000

Dolichos biflorus

DBA

"-GalNAc

120 000

Triticum vulgaris

WGA

$-GlcNAc

36 000

Galanthus nivalis

GNA

"-Man

52 000

Erythrina cristagalli

with cross-reactivity in immunoassays (Citrobacter freundi, Escherichia coli O157:H7) by using of lectin panel (Masarova et al., 2004b). Such method allows creating a “bacteria library” based on their interaction with lectins. The obtained results signified the construction of lectin biosensor would be an advantage method for detection of pathogenic bacteria. In the case of gram-negative pathogen even cell free lipopolysaccharide can be monitored as indicator of pathogenic infection. Acknowledgement. This work was supported by European Commission (Contract No. QLK1-CT-2002-51664)

Literature A a b e n h u s R., S.O. H y n e s, H. P e r m i n, A.P. M o r a n and L.P. A n d e r s e n. 2002. Lectin typing of Campylobacter concisus. J. Clin. Microbiol. 40: 715. A n n u k H., S.O. H y n e s, S. H i r m o, M. M i k e l s a a r and T. W a d s t r o m. 2001. Characterization and differentiation of lactobacilli by lectin typing. J. Med. Microbiol. 50: 1069. A r t a u l t S., J.L. B l i n d, J. D e l a v a l, Y. D u r e u i l and N. G a i l l a r d. 2001. Detecting Listeria monocytogenes in food. Int. Food Hyg. 12: 23. A s h r a f M.T. and R.H. K h a n. 2003. Mitogenic lectins. Med. Sci. Monit. 9: 265. C a l d e r o n A.M., G. B u c k and R.J. D o y l e. 1998. Lectin-microorganisms complexes. Lectins, Biology, Biochemistry, Clinical Biochemistry 12; (http://plab.ku.dk/tccbh/Lectins12/Calderon/paper.htm). D a v i d s o n S.K., K.F. K e l l e r and R.J. D o y l e. 1982. Differentiation of coagulase-positive and coagulase-negative staphylococci by lectins and plant agglutinins. J. Clin. Microbiol. 15: 547. D e B o e r E. and R.R. B e u m e r. 1999. Methodology for detection and typing of foodborne microorganisms. Int. J. Food Microbiol. 50: 119. D e L u c c a A.J. 1984. Lectin grouping of Bacillus thuringiensis serovars. Can. J. Microbiol. 19: 48. D u v e r g e r E., N. F r i s o n, A.C. R o c h e and M. M o n s i g n y. 2003. Carbohydrate-lectin interaction assessed by surface plasmon resonance. Biochimie 85: 167. G o e p e l W. 1991. Chemical sensing, molecular electronics and nanotechnology: interface technologies down to the molecular scale. Sens. Actuat. B 4: 7. G r a h a m K., K.F. K e l l e r, J. E z z e l l and R.J. D o y l e. 1984. Enzyme-linked lectinosorbent assay (ELLA) for detecting Bacillus anthracis. Eur. J. Clin. Microbiol. 3: 210 H a m a d a S., K. G i l l and H.D. S l a d e. 1977. Binding of lectins to Streptococcus mutans cells and type-specific polysaccharides, and effect on adherence. Infect. Immunol. 18: 708. H o l m s k o v U., P.B. F i s c h e r, A. R o t h m a n n and P. H o j r u p. 1996. Affinity and kinetic analysis of the bovine plasma C-type lectin collectin-43 (CL-43) interacting with mannan. FEBS Letters 393: 314. H y n e s S.O., N. B r o u t e t, E.S. G r o u p, T. W a d s t r o m, M. M i k e l s a a r, P.W. O’ T o o l e, J. T e l f o r d, L. E n g s t r a n d, S. K a m i y a, A.F. M e n t i s and A.P. M o r a n. 2002. Phenotypic variation of Helicobacter pylori isolates from geographically distinct regions detected by lectin typing. J. Clin. Microbiol. 40: 227. H y n e s S.O., S. H i r m o, T. W a d s t r o m and A.P. M o r a n. 1999. Differentiation of Helicobacter pylori isolates based on lectin binding of cell extracts in an agglutination assay. J. Clin. Microbiol. 37: 1994. L e o n a r d P., S. H e a r t y, J. B r e n n a n, L. D u n n e, J. Q u i n n, T. C h a k r a b o r t y and R.O. K e n n e d y. 2003. Advances in biosensors for detection of pathogens in food and water. Enzyme Microb. Tech. 32: 3. L i e d b e r g B., C. N y l a n d e r and I. L u n d s t r o m. 1995. Biosensing with surface plasmon resonance – how it all started. Biosens. Bioelectron. 10:1. L i s H. and N. S h a r o n. 1998. Lectins: carbohydrate specific protein that mediate cellular recognition. Chem. Rev. 98: 637.

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M a s a r o v a J., E.S. D e y and B. D a n i e l s s o n. 2004a. Lectins as prospective tools for bacteria identification using Biacore 3000. Biacore Nordic User Days, June 10–11, 2004, Uppsala, Sweden. M a s a r o v a J., W.A. A l - S o u d, E.S. D e y, T. W a d s t r o m and B. D a n i e l s s o n. 2004b. Lectin biosensor for bacteria detection. CSL/JIFSAN Symposium “Novel Application of Analytical Methods in Food Safety”, 30 June-2 July, 2004, York, United Kingdom. M c S w e e g a n F. and T.G. P i s t o l e. 1982. Interaction of the lectin limulin with capsular polysaccharides from Neisseria meningitidis and Escherichia coli. Biochem. Biophys. Res. Commun. 106: 1390. M e c k l e n b u r g M., J. S v i t e l, F. W i n q u i s t, J. G a n g, K. O r n s t e i n, E. D e y, X. B i n, E. H e d b o r g, R. N o r r b y, H. A r w i n, I. L u n d s t r o m and B. D a n i e l s s o n. 2002. Differentiation of human serum samples by surface plasmon resonance monitoring of the integral glycoprotein interaction with a lectin panel. Anal. Chim. Acta 459: 25. M i s l o v i c o v a D., J. M a s a r o v a, J. S v i t e l, R. M e n d i c h i, L. S o l t e s, P. G e m e i n e r and B. D a n i e l s s o n. 2002. Neoglycoconjugates of mannan with bovine serum albumin and their interaction with concanavalin A. Bioconjug. Chem. 13: 136 O k a z a k i I., Y. H a s e g a w a, Y. S h i n o h a r a and T. K a m a s a k i. 1995. Determination of the interactions between lectins and glycoproteins by surface plasmon resonance. J. Mol. Recog. 8: 95. Q u i n n J.G. and R. O’ K e n n e d y. 2001. Detection of whole cell:antibody interaction using BIAcore’s SPR technology. BIAJ. 1: 22. R e e d e r W.J. and R.D. E s t e d t. 1971. Study of the interaction of concanavalin A with staphylocccal teichoic acids. J. Immunol. 196: 334. R i c h R.L. and D.G. M y s z k a. 2000. Advances in surface plasmon resonance biosensor analysis. Curr. Opin. Biotechnol. 11: 54. Seyhi R.S. 1994. Transducer aspects of biosensors. Biosens. Bioelectron. 9: 243. S h a r o n N. and I. O f e k. 2001. Safe as mother’s milk: Carbohydrates as future anti-adhesion drugs for bacterial diseases. Glycoconjugate J. 17: 659. S i n g h J.S. and R.J. D o y l e. 1993. Salt-enhanced enzyme-linked lectinosorbent assay (SELLA). J. Microbiol. Meth. 17: 61 T o z e S. 1999. PCR and the detection of microbial pathogens in water and wastewater. Water Res. 33: 3545. V a r k i A. 1993. Biological roles of oligosaccharides: all theories are correct. Glycobiology 3: 97.

Polish Journal of Microbiology 2004, Vol. 53, Suppl., 29–34

Analysis of the Peptidoglycan Hydrolases of Listeria monocytogenes: Multiple Enzymes with Multiple Functions MAGDALENA POPOWSKA*

Department of Bacterial Physiology, Institute of Microbiology, Warsaw University Miecznikowa 1, 02-096 Warsaw, Poland Abstract Listeria monocytogenes is a ubiquitous gram-positive, rod-shaped, widespread in nature, facultative intracellular human and animal pathogen that causes infections collectively termed listeriosis. L. monocytogenes EGD encodes a total of 133 surface proteins, the abundance of which, as well as the variety of anchoring systems, probably reflects the ability of this bacterium to survive in diverse environments and to interact with many kinds of eukaryotic cells. The group of surface proteins also includes proteins with murein hydrolase activity-autolysins. To date, five L. monocytogenes autolysins have been identified: p60, P45, Ami, MurA and Auto. These enzymes are involved in numerous cellular processes including cell growth, cell wall turnover, peptidoglycan maturation, cell division and separation, formation of flagella, sporulation, chemotaxis and biofilm formation, genetic competence, protein secretion, the lytic action of some antibiotics and pathogenicity. We have recently identified a putative sixth listerial peptidoglycandegrading enzyme, which has surprisingly been identified as FlaA, a flagellar protein of L. monocytogenes. K e y w o r d s: Listeria monocytogenes, virulence, cell wall, peptidoglycan hydrolases

Introduction Listeria monocytogenes is an important food-borne opportunistic pathogen with high mortality rates, which can range from 20 to 60% in adults, especially in the case of infections of the central nervous system, or from 54 to 90% for neonates (Hof et al., 1997). The groups at risk for listeriosis are pregnant women and neonates, the elderly (up to 65 years old) and immunocompromised or debilitated adults with underlying diseases (Vazquez-Boland et al., 2001). The bacterium is widespread in nature: waters, soil, rotting parts of plants, animal feces and wastewaters. It has also been isolated from 5% of fecal samples from healthy humans (Farber, 1991) and detected in many food products (Schlech, 2000) and recently has been reported present in various waters, including surface and groundwater in mountainous regions (e.g. Gugnani, 1999; Schaffter and Parriaux, 2002). L. monocytogenes is a model organism in studies on the pathogenesis of intracellular parasites. It is able to penetrate, multiply and propagate in various types of eukaryotic cells and is also able to overcome the three main barriers encountered in the host: the intestinal barrier, the blood-brain barrier and the placenta (Vazquez-Boland et al., 2001). Once inside the host, this bacterium has the capacity to invade phagocytic and non-phagocytic cells, to replicate intracellularly, and spread directly from cell to cell, thereby escaping the humoral immune response. Each step of the infection process is dependent upon the production of virulence factors (Portnoy et al., 1992): the internalins InlA and InlB for entry, listeriolysin O, two phospholipases C – a phospholipase specific for phosphatidylinositol PlcA and phospholipase C specific for phosphosfatidylcholine PlcB for escape from the primary and secondary vacuoles, protein ActA for intra and intracellular movements and a metaloprotease witch is involved in the processing of PlcB into its mature and active form. The genes coding for 6 of these factors are located in the bacterial chromosome next to each other in a virulence gene cluster locus (pathogenicity island) and are regulated by the transcription activator PrfA (Chakraborty et al., 2000; Vazquez-Boland et al., 2001). The inlAB operon is * Corresponding author: e-mail: [email protected]

30

Popowska M.

located elsewhere (Lecuit et al., 2001). Moreover, such other determinants as protein p60 (Park et al., 2000), amidase Ami (Milohanic et al., 2001), autolysin Auto (Cabanes et al., 2004), catalase, superoxide dismutase, siderophores and protein LmaA are also required for full pathogenic activity of the bacterium (Vazquez-Boland et al., 2001). Bacterial peptidoglycan hydrolases The cell wall of gram-positive bacteria is host to a wide variety of molecules and serves a multitude of functions, most of which are critical to the viability of the cell. The major structural component of all types of bacterial walls is murein (peptidoglycan), which is composed of glycan chains in which alternating N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid are $-1,4 bound, cross-linked via peptide bridges. Peptide side chains, which are attached to the carboxyl group of muramic acid residues, are primarily composed of five amino acids (L-Ala-D-Glu-X-D-Ala-D-Ala, where X stands either for L-Lys or diaminopimelic acid). The general structure organization of the glycan chains is relatively constant, though certain modifications are known (Navarre and Schneewind, 1999). Other polymers that are associated with murein in

Fig.1. Structure of the disaccharide-pentapeptide monomer of L. monocytogenes murein. An example of each type of bond attacked by glucosaminidase (G), muramidase (T), amidase (A), endopeptidase (E), LD-carboxypeptidase (LD-C) and DD- carboxypeptidase (DD-C).

31

Minireview

different bacteria are teichoic acids, lipoteichoic acids, polysaccharides and numerous proteins. There are five major mechanisms for displaying protein at the surface of gram-positive bacteria (Popowska and Markiewicz, 2004a). Each mechanism is characterized by specific structural features that can be identified in the sequence of the proteins and are involved in their specific properties. The major types of surface protein are: LPXTG proteins-covalently linked to the cell wall, non-covalent interactions with the cell wall; choline binding proteins, GW proteins, membrane anchored proteins and the other group lipoproteins. Different kinds of bounds in murein are cleaved by autolysins that can be classified as N-acetylmuramidases, N-acetylglucosaminidases, N-acetylmuramyl-L-alanine amidases, endopeptidases, and transglycosylases (Höltje, 1995) (Fig. 1.). Autolysins of Listeria monocytogenes To date, five L. monocytogenes autolysins have been identified: p60 (CwhA, Iap), P45, Ami, MurA and Auto (Table I). Analysis of the L. monocytogenes genome reveals the presence of eleven proteins with a peptidoglycan hydrolysis domain, thus six (at least) are still unidentified. The extracellular protein p60 (gene iap) possesses a murein hydrolase activity required for a late step in cell division (Wuenscher et al., 1993; Pilgrim et al., 2003). The p60 protein has also been shown to play a role in virulence but is not regulated by PrfA (Kohler et al., 1991). Spontaneously occurring L. monocytogenes mutants (RIII mutants) with reduced p60 levels show rough colony morphology and form long chains of 10–20 cells separated by double septa; the cells fail to separate after cell division (Kuhn and Goebel, 1989). RIII mutant cells also show reduced ability to invade non-professional phagocytic 3T6 mouse fibroblasts (Bubert et al., 1992) and a recent report suggested that p60 is directly involved in binding intestinal Caco-2 cells (Park et al., 2000). The expression of p60 is controlled at the post-transcriptional level (Wuenscher et al., 1993). It was initially thought that this protein was essential for cell viability because iap mutations were always lethal. However, a viable mutant harboring a transposon inserted within iap has been isolated (Wiœniewski and Bielecki, 1999; Pilgrim et al., 2003), indicating that other proteins may be able to compensate for the loss of p60 activity. P60 is a modular protein containing two LysM domains, a bacterial Src homology 3 (SH3) domain and a carboxy-terminal NLPC/P60 domain. The LysM domain (40 residues long) is involved in degradation of the bacterial cell wall. This domain might have a general murein-binding function. Bacterial SH3 domains (60–70 residues) are homologous to eukaryotic SH3 domains and were first characterized in p60 proteins from different Listeria species but functions of this domain is as yet unknown (Ponting et al., 1999). The NLPC/p60 domain is responsible for peptidoglycan lytic activity (Cabanes et al., 2002). One of the hypotheses assumed the possibility that p60 and MurA induce altruistic autolysis that contributes to the SecA2-dependent release of cytosolic bacterial proteins. However, the p60 autolysin is not required for secretion of any heterologous L. monocytogenes proteins. Thus, these autolysins may have other roles in promoting bacterial pathogenesis (Lenz et al., 2003). P60 is predicted to digest the peptide bond linking the D-iso-glutamine and meso-diaminopimelic acid moieties of the peptide side-chain in L. monocytogenes peptidoglycan. Conversely, MurA shares homology with enzymes that cleave the N-acetylmuramide-Nacetylglucosamine linkage. SecA2-dependent secretion would be expected to coordinate peptidoglycan digestion by these two autolytic activities, the minimal product of which would be N-acetylglucosaminyl$1-4-N-acetylmuramyl-L-alanyl-D-isoglutamine (GMDP). In vivo, host lysozymes and glucosaminidases Tabela I Peptidoglycan hydrolases of Listeria monocytogenes Proteins

Gen

p60

iap

MW*

Description

Function

50.3

peptidoglycan lytic activity

cell separation and virulence

P45

spl

42.7

peptidoglycan lytic activity

?

Ami

ami

102.3

amidase

role in motility

Auto

aut

64.1

amidase

autolysis and virulence

MurA

murA

63.6

muramidase

cell separation and autolysis

FlaA

flaA

30.4

peptidoglycan lytic activity

structural component of flagella

MW* – calculated molecular mass (in kDa)

32

Popowska M.

may also process p60-cleaved peptidoglycan to generate GMDP or muramyl-dipeptide (MDP). GMDP and MDP activate host cell signaling through the Nod2 protein (Girardin et al., 2003; Inohara et al., 2003), a cytosolic leucine-rich-repeat protein of macrophages. Cleavage of the bond between D-iGlu and mDAP by p60 may interfere with proinflammatory signals (Girardin et al., 2003; Chamaillard et al., 2003). P45 (spl), similar to protein p60 (55% similarity and 38% identity) exhibiting peptidoglycan lytic activity (NLPC/p60 domain). This protein was detected in the culture supernatant and at cell surface of L. monocytogenes (Schubert et al., 2000). The surface protein Ami (102 kDa) with N-terminal amidase domain and C-terminals eight GW modules that interact with lipoteichoic acids on the bacterial surface (Cabanes et al., 2002). The C-terminal amino acid sequence shows homology to the same region of InlB L. monocytogenes, while N-terminal domain shows homology to Atl, the major autolysin of Staphylococcus aureus (Foster, 1995). Interestingly, the other six of surface protein L. monocytogenes containing GW module, like Ami, contain an amidase domain. Ami has a subtle role in motility, observed as a reduction in swarming (McLaughlan and Foster, 1997; McLaughlan and Foster, 1998) and is involved in adhesion to eukaryotic cells (Milohanic et al., 2000; 2001). The activity of the pure amidase against L. monocytogenes peptidoglycan in vitro is only 73% of that against Bacillus subtilis vegetative cell walls in spite of the similar primary structure of both substrates. This could reflect the presence of unusual modifications in the peptidoglycan of L. monocytogenes (Kamisango et al., 1982). However, we have shown that this does not seem to be the case, except for amidation of free diaminopimelic acid residues (K³oszewska et al., in preparation). It is therefore more likely that the activity of the autolytic enzymes of L. monocytogenes is very tightly regulated and that their activity is to some extent inhibited even after the death of the cells. The surface associated autolysin Auto (aut) of L. monocytogenes, with a predicted molecular mass of 64 kDa, like Ami, containing N-terminal amidase domain and C-terminal cell wall-anchoring domain up of four GW modules was described quite recently (Cabanes et al., 2002). The aut gene is expressed independently of the virulence gene regulator PrfA and encoding surface protein with an autolytic activity. Although, microscopic analysis of the aut mutant did not reveal any defect in cell separation. The aut gene is absent from the genome non-pathogenic species L. innocua. It is not to be wondered at Auto is required for entry of L. monocytogenes into cultured non-phagocytic eukaryotic cells and virulence in vivo (Cabanes et al., 2004). Reduced virulence of the aut mutant after oral and intravenous inoculation suggests that aut is critical for several steps of the listeriosis. Cabanes clearly identified Auto as the first L. monocytogenes autolysin absent from L. innocua directly implicated in virulence process, however, the exact function of Auto remains to be elucidated. The surface location of Auto could lead to the binding to a mammalian receptor directly or through a structure that binds to a eukaryotic receptor like as InlB (Braun et al., 2000). Auto as an autolysin could thus appear as a new factor required for a successful infection in controlling the general surface architecture exposed to the host by L. monocytogenes and/or the composition of the surface products released by the bacteria, implicating directly autolysins in pathogenicity. A novel cell wall hydrolase MurA is a 66 kDa cell surface protein which displays two characteristic features: an N-terminal muramidase domain with homology to muramidases from several gram-positive bacterial species and four copies of a cell wall-anchoring LysM repeat motif present within its C-terminal domain (Carroll et al., 2003). The deduced molecular mass of MurA is 63,571 Da. The cleavage of a signal peptide would result in mature secreted protein of 57,949 Da. The apparent molecular mass of MurA is larger than would be expected from its primary amino acid sequence, i.e., 66 versus 58 kDa, for the mature protein. Protein database searches showed that the MurA protein of L. monocytogenes and L. innocua are highly conserved, with 84% identities at the amino acid level. BLAST searches with the amidase_4 motif region of MurA revealed additional ORFs in L. monocytogenes EGDe genome (lmo1076 – Auto, lmo1215, lmo1216, lmo2203 and lmo2591) that display homology to this domain. The murA gene is preceded by a divergently transcribed putative transcriptional regulator. These cell wall hydrolase is involved in cell separation (mutant murA– grew as long chains) and autolysis of L. monocytogenes (the deletion mutant is more resistant to both prolonged stationary-phase autolysis and Triton X-100 induced autolysis). A putative novel murein degrading enzyme from L. monocytogenes, which would be the sixth identified murein-degrading activity, was recently discovered by us in the course of a zymographic analysis of surface-associated autolysins of the bacterium released using 4 M LiCl (Popowska and Markiewicz, 2004b). To our surprise the enzyme was identified as the previously described flagellar protein FlaA (Dons et al., 1992). The amino acid sequence of the autolysin showed 100% identity with that of FlaA. Our preliminary observations will be followed up in subsequent studies aimed, amongst others, at identifying the bond in murein cleaved by the enzyme.

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Conclusions Autolysins enzymes, defined as murein hydrolases, are ubiquitous among both gram-positive and gramnegative bacteria. However, the exact roles of these enzymes cell growth and division has not yet been fully elucidated. It is obvious that autolysins, which are involved in various biological functions including cell growth, cell wall turnover, peptidoglycan maturation, cell division and separation, formation of flagella, sporulation, chemotaxis and biofilm formation, genetic competence, protein secretion, the lytic action of some antibiotics and pathogenicity, should be under strict spatial and temporal control. Though it is generally considered that the activity of autolysins is regulated post-translation level, examples are known of regulation at the level of transcription. The gene bvg product in Bordetella pertussis (Tuomanen et al., 1990) inhibits the expression of virulence determinants in this bacterium. Avirulent cells easily undergo autolysis, which points to derepression of hydrolase activity by the product of this gene. In Staphylococcus aureus identified of rta gene, coding a RAT protein, witch appears to be a negative regulator of autolysin genes including lytM and lytN (Ingavale et al., 2003). Sequence analysis indicated that Rat is homologous to the SarA protein families that modulated virulence determinants (Cheung and Zhang, 2002). Mutation in rat resulted in decreased expression of known autolytic regulators lytRS, lrgAB and arlRS. The RAT binds to the lytRS and arlRS promoters, thus confirming Rat as a DNA-binding protein to these known repressors of autolytic activity. It is known that SarA regulates lytRS and lrgAB (Fournier et al., 2001) but zymographic analysis of the lysates of rat and sarA mutants suggest that rat and sarA may act on different target genes in autolysis (Ingavale et al., 2003). It is also known that on the one hand these enzymes make the growth of the cell wall possible, but on the other can lead to the death of a cell in the process of autolysis. Cell wall hydrolases are thought to be involved in autolysis of the bacterial cell, and this phenomenon is usually observed after inhibition of further synthesis of peptidoglycan either nutritionally or by the addition of an antibiotic or treatment with certain non-specific chemicals (Shockman and Höltje, 1994). For these reasons the enzymes are considered very significant in therapy involving the use of antibiotics. This direct correlation with pathogenicity further reinforces the importance of understanding bacterial autolysis. Recent findings suggest that autolysis is not just an unfavourable side effect of the enzymes that control bacterial cell wall synthesis but that it provides some advantage to the organism that is necessary for its survival (Carroll et al., 2003). The peptidoglycan hydrolase profile of L. monocytogenes is complex and consists of multiple bandsautolysins (McLaughlan and Foster, 1997), including members of the P60 family such as p60 ( iap), P45 (spl) and Lmo0394 (Cabanes et al., 2002) and members of the amidase protein family such as Ami or Auto and Lmo1215, Lmo1216, Lmo1521, Lmo2203, Lmo2591 as well muramidase MurA. All these enzymes appear to have alternative functions in the organism. The presence of multiple autolysins complicates the process of determining the roles of each autolysin in the organism. Literature B r a u n L. and P. C o s s a r t. 2000.Interactions between Listeria monocytogenes and host mammalian cells. Micro. Infect. 2: 803–811. B u b e r t A, M. K u h n, W. G o e b e l and S. K o h l e r. 1992. Structural and functional properties of the p60 proteins from different Listeria species. J. Bacteriol. 174: 8166–71. C a b a n e s D., P. D e h o u x, O. D u s s u r g e t, L. F r a n g e u l and P. C o s s a r t. 2002. Surface proteins and the pathogenic potential of Listeria monocytogenes. Trends in Microbiol. 5: 238–245. C a b a n e s D., P. D e h o u x, O. D u s s u r g e t, L. F r a n g e u l and P. C o s s a r t. 2004. Auto, a surface associated autolysin of Listeria monocytogenes required for entry into eukaryotic cells and virulence. Mol. Microbiol. 51: 1601–14. C a r r o l l S.A, T. H a i n, U. T e c h n o w, A. D a r j i, P. P a s h a l i d i s, S.W. J o s e p h and T. C h a k r a b o r t y. 2003. Identification and characterization of a peptidoglycan hydrolase, MurA, of Listeria monocytogenes, a muramidase needed for cell separation. J. Bacteriol. 185: 6801–8. C h a k r a b o r t y T., T. H a i n and E. D o m a n n. 2000. Genome organization and the evolution of the virulence gene locus in Listeria species. Int. J. Med. Microbiol. 290: 167–174. C h a m a i l l a r d M., M. H a s h i m o t o, Y. H o r i e, J. M a s u m o t o, S. Q i u, L. S a a b, Y. O g u r a, A. K a w a s a k i, K. F u k a s e, S. K u s u m o t o, M.A. V a l v a n o, S.J. F o s t e r, T.W. M a k, G. N u n e z and N. I n o h a r a. 2003. An essential role for NOD1 in host recognition of bacterial peptidoglycan containing diaminopimelic acid. Nat. Immunol. 4: 702–7. C h e u n g A.L. and G. Z h a n g. 2002. Global regulation of virulence determinants in Staphylococcus aureus by the SarA protein family. Front. Biosci. 7: 1825–42. D o n s L., O.F. R a s m u s s e n and J.E. O l s e n. 1992. Cloning and characterization of a gene encoding flagellin of Listeria monocytogenes. Mol. Microbiol. 6: 2919–2929. F a r b e r J.M. 1991. Listeria monocytogenes, a food-borne pathogen. Microbiol. Rev. 55: 476–511.

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Foster S.J. 1995. Molecular characterization and functional analysis of the major autolysin of Staphylococcus aureus 8325/4. J. Bacteriol. 177: 5723–5. F o u r n i e r B., A. K l i e r and G. R a p o p o r t. 2001. The two-component system ArlS-ArlR is a regulator of virulence gene expression in Staphylococcus aureus. Mol. Microbiol. 41: 247–261. G i r a r d i n S.E., I.G. B o n e c a, L.A. C a r n e i r o, A. A n t i g n a c, M. J e h a n n o, J. V i a l a, K. T e d i n, M.K. T a h a, A. L a b i g n e, U. Z a h r i n g e r, A.J. C o y l e, P.S. D i S t e f a n o, J. B e r t i n, P.J. S a n s o n e t t i and D.J. P h i l p o t t. 2003. Nod1 detects a unique muropeptide from gram-negative bacterial peptidoglycan. Science 6: 1584–7. G u g n a n i H.C. 1999. Some emerging food and water borne pathogens. J. Commun. Dis. 31: 65–72. H o f H., T. N i c h t e r l e i n and M. K r e t s c h m a r. 1997. Management of listeriosis. Clin. Microbiol. Rev. 10: 345–357. H ö l t j e JV. 1995. From growth to autolysis: the murein hydrolases in Escherichia coli. Arch Microbiol. 164: 243–54. I n g a v a l e S.S., W. Va n W a m e l and A.L. C h e u n g. 2003. Characterization of RAT, an autolysis regulator in Staphylococcus aureus. Mol. Microbiol. 48: 1451–1466. I n o h a r a N., Y. O g u r a, A. F o n t a l b a, O. G u t i e r r e z, F. P o n s, J. C r e s p o, K. F u k a s e, S. I n a m u r a, S. K u s u m o t o, M. H a s h i m o t o, S.J. F o s t e r, A.P. M o r a n, J.L. F e r n a n d e z - L u n a and G. N u n e z. 2003. Host recognition of bacterial muramyl dipeptide mediated through NOD2. Implications for Crohn’s disease. J. Biol. Chem. 21: 5509–12. K a m i s a n g o K., I. S a i k i, Y. T a n i o, H. O k u m u r a, Y. A r a k i, I. S e k i k a w a, I. A z u m a and Y. Y a m a m u r a. 1982. Structures and biological activities of peptidoglycans of Listeria monocytogenes and Propionibacterium acnes. J. Biochem. 92: 23–33. K o h l e r S., A. B u b e r t, M. Vo g e l and W. G o e b e l. 1991. Expression of the iap gene coding for protein p60 of Listeria monocytogenes is controlled on the posttranscriptional level. J. Bacteriol. 173: 4668–74. K u h n M. and W. G o e b e l. 1989. Identification of an extracellular protein of Listeria monocytogenes posibly involved in intracellular uptake by mammalian cells. Infect. Immun. 57: 55–61. L e c u i t M., S. V a n d o r m a e l - P o u r n i n, J. L e f o r t, M. H u e r r e, P. G o u n o n, C. D u p u y, C. B a b i n e t and P. C o s s a r t. 2001. A transgenic model for listeriosis: role of internalin in crossing the intestinal barrier. Science 292: 1722–1725. L e n z L.L., S. M o h a m m a d i, A. G e i s s l e r and D.A. P o r t n o y. 2003. SecA2-dependent secretion of autolytic enzymes promotes Listeria monocytogenes pathogenesis. Proc. Natl. Acad. Sci. USA 14: 12432–7. M c L a u g h l a n A.M. and S.J. F o s t e r. 1998. Molecular characterization of an autolytic amidase of Listeria monocytogenes EGD. Microbiol. 144: 1359–1367. M c L a u g h l a n A.M. and S.J. F o s t e r. 1997. Characterisation of the peptidoglycan hydrolases of Listeria monocytogenes EGD. FEMS Microbiol. Lett. 1: 149–54. M i l o h a n i c E., B. P r o n, P. B e r c h e and J.L. G a i l l a r d. 2000. Identification of new loci involved in adhesion of Listeria monocytogenes to eukaryotic cells. European Listeria Genome Consortium. Microbiol. 146: 731–9. M i l o h a n i c E., R. J o n q u i e r e s, P. C o o s s a r t, P. B e r c h e, J.L. G a i l l a r d. 2001. The autolysin Ami contributes to the adhesion of Listeria monocytogenes to eukaryotic cells via its cell wall anchor. Mol. Microbiol. 39: 1212–1224. N a v a r r e W.W. and O. S c h n e e w i n d. 1999. Surface proteins of gram-positive bacteria and mechanisms of their targeting to the cell wall envelope. Microbiol. Mol. Biol. Rev. 63: 174–229. P a r k J.H., Y.S. L e e, Y.K. L i m, S.H. K w o n, C.U. L e e and B.S. Y o o n. 2000. Specific binding of recombinant Listeria monocytogenes p60 protein to Caco-2 cells. FEMS Microbiol. Lett. 186: 35–40. P i l g r i m V., A. K o l b - M a u r e r, L. G e n t s c h e v, W. G o e b e l and M. K u h n. 2003. Deletion of the gene encoding p60 in Listeria monocytogenes leads to abnormal cell division and los of actin-based motility. Infect. Immun. 71: 3473–3484. P o n t i n g C.P., L. A r a v i n d, J. S c h u l t z, P. B o r k and EV. K o o n i n. 1999. Eukaryotic signalling domain homologues in archaea and bacteria. Ancient ancestry and horizontal gene transfer. J Mol. Biol. 18: 729–45. P o p o w s k a M. and Z. M a r k i e w i c z. 2004a. Classes and functions of Listeria monocytogenes surface proteins. Pol. J. Microbiol. 53: 75–88. P o p o w s k a M. and Z. M a r k i e w i c z. 2004b. Murein-hydrolyzing activity of flagellin FlaA of Listeria monocytogenes. Pol. J. Microbiol. 53: P o r t n o y D.A., T. C h a k r a b o r t y, W. G o e b e l and P. C o s s a r t. 1992. Molecular determinants of Listeria monocytogenes pathogenesis. Infect. Immun. 60: 1263–1267. S c h a f f t e r N. and A. P a r r i a u x. 2002. Pathogenic-bacterial water contamination in mountainous catchments. Water Res. 36: 131–139. S c h l e c h W.F. 2000. Foodborne listeriosis. Clin. Infect. Dis. 31: 770–5. S c h u b e r t K., A.M. B i c h l m a i e r, E. M a g e r, K. W o l f f, G. R u h l a n d and F. F i e d l e r. 2000. P45, an extracellular 45 kDa protein of Listeria monocytogenes with similarity to protein p60 and exhibiting peptidoglycan lytic activity. Arch. Microbiol. 173: 21–28. S h o c k m a n G.D. and J.V. H ö l t j e. 1994. Microbial peptidoglycan (murein) hydrolases. Ghuysen J.M., Hakenbeck R. Bacterial Cell Wall 131. T u o m a n e n E., U. S c h w a r z and S. S a n d e. 1990. The vir locus affects the response of Bordetella pertussis to antibiotics: phenotypic tolerance and control of autolysis. J. Inf. Dis. 162, 560. V a z q u e z - B o l a n d J.A., M. K u h n, P. B e r c h e, T. C h a k r a b o r t y, G. D o m i n g u e z - B e r n a l, W. G o e b e l, B. G o n z a l e s - Z o r n, J. W e h l a n d and J. K r e f t. 2001. Listeria pathogenesis and molecular virulence determinants. Clin. Microbiol. Rev. 14: 584–640. W i œ n i e w s k i J.M. and J.E. B i e l e c k i. 1999. Intracellular growth of Listeria monocytogenes insertional mutant deprived of protein p60. Acta Microbiol. Pol. 48: 317–329. W u e n s c h e r M.D., S. K o h l e r, A. B u b e r t, U. G e r i k e and W. G o e b e l. 1993. The iap gene of Listeria monocytogenes is essential for cell viability, and its gene product, p60, has bacteriolytic activity. J. Bacteriol. 175: 3491–501.

Polish Journal of Microbiology 2004, Vol. 53, Suppl., 35–38

Polymerizer-Mediated Intracellular Movement JAROS£AW M. WIŒNIEWSKI and JACEK BIELECKI

Department of Applied Microbiology, Institute of Microbiology, Warsaw University, Poland Abstract Bacterial movement inside the cytoplasm is a major virulence factor in that it is necessary for efficient colonization of the infected tissues. Molecules from both the host and the pathogen present possible sites of pharmacologic intervention. Because locomoting Listeria and Shigella mimic the activated state of the leading edge of nonmuscle cells, these pathogens are powerful tools for dissecting the molecular machinery of actin-based motility. Analysis of the movement linked to cytoskeleton may lead to: (I) improved understanding of the mechanisms of disease transmission, including carriers and carrier states, pathogen movements, environmental factors and pharmacokinetics of the uptake and residues of vaccines and other biologics, and drugs in cultivated organisms; (II) new therapeutic developments, since it identifies the molecular targets involved in the pathogenicity of Listeria and Shigella and vaccinia intracellular enveloped virus. Recent knowledge about the intracellular movement in cytoplasm may lead to a better understanding of the processes governing actin dynamics within the cell and disease spread. K e y w o r d s: intracellular pathogens, parasite spread. movement in cytoplasm

Intracellular movement Many microorganisms can exist in an intracellular infectious phase that protects them from the effector functions of the antibody system. These agents infect host cells and live enclosed by the host membrane, in the endosome or free in cytoplasm. Movement is necessary for these parasites to spread within the host animal, it is necessary for their ability to enter host cells, escape from the host cell, and find a new cell. Intracellular pathogens are well known to co-opt cellular machinery to accomplish their life cycle. Many intracellular parasites have special, genetically-encoded mechanisms to get into host cells that are nonphagocytic. Intracellular pathogens such as Yersinia, Listeria, Salmonella, Shigella and Legionella possess a complex machinery for cellular invasion and intracellular survival. These systems involve various types of virulence factors. Intracellular parasites have evolved diverse mechanisms to enhance their survival and replication within eukaryotic host cells (Moulder, 1985). These mechanisms largely involve adaptations for survival in distinct intracellular compartments that permit the parasites to avoid lysosomal killing. Parasites can infect different host cells, and once inside the cell, some of them escape from the endocytic vacuole as soon as within an hour of infection. The interactions between bacterial intracellular parasites and host cells may be divided conceptually into three stages. The first interaction to be considered results in the adherence of bacteria on the surface of the cells. The adherence of invading bacteria is a first major step in the pathogenic process. The second stagen of bacteria/host interaction consists of signal transduction events that result in cytoskeletal rearrangements (Tang et al., 1996, Tang et al., 1998) and the third stage is bacterial internalization and intracellular movement (Bliska and Falkow, 1993). Polymerizer-mediated movement L. monocytogenes is an intracellular pathogen that survives by passing from phagosomes into cytoplasm. Listeria can dramatically stimulate host-cell actin assembly in a directional manner, which serves to rapidly propel the bacteria through the cytoplasm, allowing the organisms to move to peripheral membranes and

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spread to uninfected cells. It secretes a hemolytic protein, listeriolysin O (LLO), which mediates bacterial passage into cytoplasm. Measurements of phagosomal pH during infection with L. monocytogenes showed that LLO-mediated perforation of phagosomes occurs optimally at pH 6.0, and requires an acidic environment for perforation. It has been shown that only a small percentage of an L. monocytogenes population that enters macrophage cells undergoes intracellular growth, while the rest of the bacterial population is killed (Chastellier and Berche, 1994; Raybourne and Bunning, 1994). Chastellier and Berche showed that intacellular multiplication in fact resulted from rapid evasion of a very small number of bacteria (14%) from the phagosome compartment during this early stage to prior phagosome-lysosome fusion (Chastellier and Berche, 1994). By treating cells with DAMP, a weak base that accumulates in the acidic compartment of the cell the authors showed that all phagosomes, whether they contained intact or damaged bacteria, were acidified. Acidification seems to be required for bacterial escape from the phagosome compartment. Listeriolysin is optimally active at an acidified pH (Portnoy et al., 1992). The intracellular actin-based movement was reported for L. monocytogenes by Tilney and Portnoy (1989). The bacteria use proteins inside the host cell to form rocketlike tails. They then ride these tails to the cell wall, deform the wall and invade neighboring cells. L. monocytogenes induce polarized actin assembly at the surface to gain propulsive force in infected cells. Upon entering the host cell’s cytoplasm, the pathogen L. monocytogenes can subvert the normal contractile system of the host cell; subsequent assembly of polar actin-filament structures is likely to provide the force for rapid intracellular bacterial movement and its cell-to-cell spread. The Listeria surface protein ActA, which is accumulated over the posterior bacterial body during movement in host cells, is crucial for actin-based motility. The growth of barbed ends, restricted to the bacterial surface is initiated by the bacterial protein ActA (Domann et al., 1992; Kocks et al., 1992; Kocks et al., 1993). Directed Listeria movement occurs when a bacterial surface protein, ActA, and cytoplasmic actin-binding proteins distribute to one pole of the bacterium. The first step in actin filament formation is called nucleation. In this low probability event, three actin monomers are thought to combine simultaneously, forming a thermodynamically unstable trimeric nucleus. Once a trimer is formed, the nucleus most frequently dissociates back into monomers; however, the nucleus occasionally survives long enough to permit subsequent binding of additional actin molecules. Monomer addition is more rapid at the plus end. Once actin filaments reach a steady-state length, ADP-actin monomers are released from the minus ends of the filaments at the same rate as new ATP-actin monomers are added to the plus ends. In the cell, two additional issues are important: (a) actin monomer addition occurs mostly, if not exclusively, at the plus-end; and (b) this process probably produces a pool of ADP-actin from which actin-ATP must be regenerated by exchange (not direct phosphoryl transfer) with ATP in the cytoplasm. ATP-actin has a much higher affinity for the ends of actin filaments than does ADP-actin, and ATP-actin is the primary monomeric species of actin that adds to filament ends in the cell. The Arp2/3 complex from the cytoplasm, using ActA as a scaffold, constitutes the minimal requirement for the nucleation of actin polymerization, which continues at the interface between the quickly growing (barbed) actin end and the bacterial surface. Insertion of subunits at the barbed end of cross-linked filaments generates compression forces used for propulsion. Within the host cell, actin filament assembly is exquisitely well regulated through the action of a number of actin-regulatory proteins, including actin filament capping and severing proteins, actin monomer sequestering proteins, actin bundling proteins and actin cross-linking proteins. One host cell component likely to play a central role in Listeria actin-based motility is profilin. This protein binds to actin monomers in a one to one complex, alters the conformation of the actin monomer, and accelerates the exchange of ATP with actin-bound ADP. Because ATP-actin has a higher affinity for the ends of actin filaments, catalysis of nucleotide exchange should enhance actin-filament assembly. Profilin is likely to be most highly concentrated wherever new actin filaments assemble. Profilin is the only actin regulatory protein that binds polyproline, and the host cell protein vasodilator-stimulated phosphoprotein (VASP) has taken advantage of this unique characteristic to concentrate profilin in specific regions of the cell where new actin filaments assemble. The VASP monomer contains four potential profilin binding sites each containing a series of proline residues. VASP exists as a tetramer in the host cell; therefore each VASP molecule could attract up to 16 profilin molecules. Finally, a third actin-regulatory protein of importance in Listeria intracellular movement is called alpha-actinin. This host cell cytoskeletal protein binds to the sides of actin filaments, linking them into bundles. Bundling of actin filaments creates the more rigid co-linear filament network required to form structures such as actin stress fibers and Listeria actin tails. The growing actin filaments become crosslinked into the now familiar “comet tail”, which acts to anchor the structure so that addition of more actin monomers to the filaments at the gap between the bacterium and the filaments now pushes the bacterium forward. ActA has multiple functional domains and

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interacts with several host factors, the Arp2/3 complex, Drosophila Enabled (Ena)/VASP family proteins and PtdIns(4,5)P2 (Chakraborty et al., 1995; Chakraborty, 1999; Gertler et al., 1996, Welch et al., 1997). The N-terminal domain of ActA (residues 30 to 263) can not only interact with the Arp2/3 complex but can also stimulate its actin nucleation activity (Pistor et al., 2000; Zalevsky et al., 2000). The scientists found that Listeria do not move continuously on the molecular level, but instead move in a steplike fashion. Kuo and McGrath (2000) used high-resolution laser tracking to follow the trailing ends of Listeria moving in the lamellae of COS7 cells, and found that pauses during motility occur frequently and that episodes of step-like motion often show pauses spaced at about 5.4 nm. Each step may correspond to the addition of individual protein building blocks to the tail. Surprising information about Listeria movement has been provided by Buchwalow et al. (1997) who reported that the comet tails involved in Listeria movement have a tubulin-like component. Shigella causes bacillary dysentery, a disease provoking severe bloody and mucous diarrhea. When the pathogen reaches the colon, bacteria translocate through the epithelial barrier by way of the M cells that overlay the solitary lymphoid nodules (Sansonetti et al., 1991; Sansonetti et al., 1996). The movement of intracellular Shigella was first reported by Ogawa et al. (1968). The virG (also called icsA) gene on the large plasmid of S. flexneri is required for cell-to-cell spreading and actin-based motility in mammalian cells (Makino et al., 1986; Bernardini et al., 1989). Shigella internalization involves cytoskeletal rearrangements that lead to dramatic changes in the plasma membrane at the point of direct contact between the bacterium and the host cell. To propel itself in infected cells, the pathogen S. flexneri subverts the Cdc42-controlled machinery responsible for actin assembly during filopodia formation. Bacterial protein IcsA binds N-WASP and activates it in a Cdc42-like fashion. Dramatic stimulation of actin assembly is linked to the formation of a ternary IcsA-N-WASP-Arp2/3 complex, which nucleates actin polymerization. VASP is not involved in Shigella movement, and the function of profilin does not require its binding to proline-rich regions. This process promotes a random intracellular movement of the bacteria and leads to the infection of adjacent cells by the formation of protrusions. This movement, which involves the nucleation, polymerization, and subsequent polarization of actin, is referred to as the Ics phenotype (intra/intercellular spread) (Egile et al., 1999). Spotted fever group Rickettsia, and the vaccinia virus also induce polarized actin assembly at the surface to gain propulsive force in infected cells. The viral particles are too large to move from their replication site near the center of the cell to the cell periphery by passive diffusion. The enveloped form of vaccinia virus, called intracellular enveloped virus (IEV), also induces formation of an actin comet tail in infected cells (Cudmore et al., 1995). Unlike Listeria, vaccinia forms actin comet tails only at the periphery of infected cells, where the virus can interact with the plasma membrane. The mechanism of the actin tail formation of IEV resembles that of Shigella VirG more than that of Listeria ActA with respect to the involvement of N-WASP. However, vaccinia virus movement occurs depending on protein tyrosine phosphorylation of one of the surface proteins, called A36R (Frischknecht et al., 1999). The tyrosinephosphorylated A36R links to N-WASP but does so indirectly via binding to adapter proteins such as Nck and WIP. Unlike Shigella actin-based motility, the activation of N-WASP is independent of Cdc42 (Moreau et al., 2000). R. ricketsii actin filament tails are more stable than those induced by L. monocytogenes and contain some of the cellular cytoskeletal-associated components found in the comet tails of Listeria and Shigella, but not Arp2:3 and N-WASP (Heinzen et al., 1999; Gouin et al., 1999). These structural and compositional differences suggest that the mechanism of actin recruitment and polymerization by Ricketsiia is unique compared with Shigella and Listeria. Literature B e r n a r d i n i M.L., J. M o u n i e r, H. d’ H a u t e v i l l e, M. C o q u i s - R o n d o n and P. J. S a n s o n e t t i. 1989. Identification of icsA, a plasmid locus of Shigella flexneri that governs bacterial intra- and intercellular spread through interaction with F-actin. Proc. Natl. Acad. Sci. USA 86: 3867–3871. B l i s k a J.B. and S. F a l k o w. 1993. The role of host tyrosine phosphorylation in bacterial pathogenesis. Trends Genet. 9: 85–89. B u c h w a l o w I.B., M. E m o t o, M. B r i c h and S.H. K a u f m a n n. 1997. Involvement of tubulin and inhibitory G proteins in the interaction of Listeria monocytogenes with mouse hepatocytes. Infect. Immun. 65: 1095–1097. C h a s t e l l i e r d e C. and P. B e r c h e. 1994. Fate of Listeria monocytogenes in murine macrophages: evidence for simultaneous killing and survival of intracellular bacteria. Infect. Immun. 62: 543–553. C h a k r a b o r t y T., F. E b e l, E. D o m a n n, K. N i e b u h r, B. G e r s t e l, S. P i s t o r, C.J. T e m m - G r o v e, B.M. J o c k u s c h, M. R e i n h a r d, U. W a l t e r and J. W e h l a n d. 1995. A focal adhesion factor directly linking intracellularly motile Listeria monocytogenes and Listeria ivanovii to the actin-based cytoskeleton of mammalian cells. EMBO J. 14: 1314–1321.

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C h a k r a b o r t y T. 1999. Molecular and cell biological aspects of infection by Listeria monocytogenes. Immunobiology 201: 155–63. C u d m o r e S., P. C o s s a r t, G. G r i f f i t h s and M. W a y. 1995. Actin-based motility of vaccinia virus. Nature 378: 636–638. D o m a n n E., J. W e h l a n d, M. R o h d e, S. P i s t o r, M. H a r t l, W. G o e b e l, M. L e i m e i s t e r - W a c h t e r, M. W u e n s c h e r and T. C h a k r a b o r t y. 1992. A novel bacterial gene in Listeria monocytogenes required for host cell microfilament interaction with homology to the proline-rich region of vinculin. EMBO J. 11: 1981–1990. E g i l e C., T.P. L o i s e l, V. L a u r e n t, R. L i, D. P a n t a l o n i, P.J. S a n s o n e t t i, M.F. C a r l i e r. 1999. Activation of the CDC42 effector N-WASP by the Shigella flexneri IcsA protein promotes actin nucleation by Arp2/3 complex and bacterial actin-based motility. Cell Biol. 146: 1319–1332. F r i s c h k n e c h t F., V. M o r e a u, S. R o t t g e r, S. G o n f l o n i, I. R e c k m a n n, G. S u p e r t i - F u r g a and M. W a y. 1999. Actin-based motility of vaccinia virus mimics receptor tyrosine kinase signalling. Nature 401: 926–929. G e r t l e r F.B., K. N i e b u h r, M. R e i n h a r d, J. W e h l a n d and P. S o r i a n o. 1996. Mena, a relative of VASP and Drosophila Enabled, is implicated in the control of microfilament dynamics. Cell 87: 227–239. G o u i n E., H. G a n t e l e t, C. E g i l e, I. L a s a, H. O h a y o n, V. V i l l i e r s, P. G o u n o n, P.J. S a n s o n e t t i and P. C o s s a r t. 1999. A comparative study of the actin-based motilities of the pathogenic bacteria Listeria monocytogenes, Shigella flexneri and Rickettsia conorii. J. Cell Sci. 112: 1697–1708. H e i n z e n R.A., S.S. G r i e s h a b e r, L.S. V a n K i r k and C.J. D e v i n. 1999. Dynamics of actin-based movement by Rickettsia rickettsii in vero cells. Infect Immun 67: 4201–4207. K o c k s C., E. G o u i n, M. T a b o u r e t, P. B e r c h e, H. O h a y o n and P. C o s s a r t. 1992. Listeria monocytogenesinduced actin assembly requires the actA gene product, a surface protein. Cell 68: 521–531. K o c k s C., R. H e l l i o, P. G o u n o n, H. O h a y o n and P. C o s s a r t. 1993. Polarized distribution of Listeria monocytogenes surface protein ActA at the site of directional actin assembly. J. Cell Sci. 105: 699–710. K u o S.C. and J.L. M c G r a t h. 2000. Steps and fluctuations of Listeria monocytogenes during actin-based motility. Nature 407: 1026–1029. M a k i n o S., C. S a s a k a w a, T. K a m a t a and M. Y o s h i k a w a. 1986. A genetic determinant required for continuous reinfection of adjacent cells on a large plasmid in Shigella flexneri 2a. Cell 46: 551–555. M o r e a u V., F. F r i s c h k n e c h t, I. R e c k m a n n, R. V i n c e n t e l l i, G. R a b u t, D. S t e w a r t and M. W a y. 2000. A complex of N-WASP and WIP integrates signalling cascades that lead to actin polymerization. Nat. Cell Biol. 2: 441–448. M o u l d e r J.W. 1985. Comparative biology of intracellular parasitism. Microbiol Rev. 49: 298–337. O g a w a H., A. N a k a m u r a and R. N a k a y a. 1968. Cinemicrographic study of tissue cell cultures infected with Shigella flexneri. Jpn. J. Med. Sci. Biol. 21: 259–273. P i s t o r S., L. G r ö b e, A.S. S e c h i, E. D o m a n n, B. G e r s t e l, L.M. M a c h e s k y, T. C h a k r a b o r t y and J. W e h l a n d. 2000. Mutations of arginine residues within the 146-KKRRK-150 motif of the ActA protein of Listeria monocytogenes abolish intracellular motility by interfering with the recruitment of the Arp2/3 complex. J. Cell Sci. 113: 3277–3287. P o r t n o y D.A., R.K. T w e t e n, M. K e h o e and J. B i e l e c k i. 1992. Capacity of listeriolysin O, streptolysin O, and perfringolysin O to mediate growth of Bacillus subtilis within mammalian cells. Infect. Immun. 60: 2710–2717. R a y b o u r n e R.B. and V.K. B u n n i n g. 1994. Bacterium-host cell interactions at the cellular level: fluorescent labelling of bacteria and analysis of short-term bacterium-phagocyte interaction by flow cytometry. Infect Immun. 62: 665–672. S a n s o n e t t i P.J., J. A r o n d e l, A. F o n t a i n e, H. d’ H a u t e v i l l e and M.L. B e r n a r d i n i. 1991. ompB (osmo-regulation) and icsA (cell-to-cell spread) mutants of Shigella flexneri: vaccine candidates and probes to study the pathogenesis of shigellosis. Vaccine 9: 416–422. S a n s o n e t t i P.J., J. A r o n d e l, J.R. C a n t e y, M.C. P r é v o s t and M. H u e r r e. 1996. Infection of rabbit Peyer’s patches by Shigella flexneri: effect of adhesive or invasive bacterial phenotypes on follicle-associated epithelium. Infect. Immun. 64: 2752–2764. T a n g P., I. R o s e n s h i n e, P. C o s s a r t and B.B. F i n l a y. 1996. Listeriolysin O activates mitogen-activated protein kinase in eucaryotic cells. Infect. Immun. 64: 2359–2361. T a n g P., C.L. S u t h e r l a n d, M.R. G o l d and B.B. F i n l a y. 1998. Listeria monocytogenes invasion of epithelial cells requires the MEK-1/ERK-2 mitogen-activated protein kinase pathway. Infect. Immum. 66: 1106–1112. T i l n e y L.G. and D.A. P o r t n o y. 1989. Actin filaments and the growth, movement, and spread of the intracellular bacterial parasite Listeria monocytogenes. J. Cell Biol. 109: 1597–1608. W e l c h M.D., A. I w a m a t s u and T.J. M i t c h i s o n. 1997. Actin polymerization is induced by Arp2/3 protein complex at the surface of Listeria monocytogenes. Nature 385: 265–269. Z a l e v s k y J., I.I. G r i g o r o v a and R.D. M u l l i n s. 2000. Activation of the Arp2/3 complex by the Listeria ActA protein: ActA binds two actin monomers and three subunits of the Arp2/3 complex. J. Biol. Chem. 276: 3468–3475.

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New Methods of Pathogenic Bacteria Elimination KRYSTYNA I. WOLSKA, ANNA KRACZKIEWICZ-DOWJAT, ANNA M. GRUDNIAK, ANNA SAJKOWSKA and TATIANA WIKTOROWICZ

Department of Bacterial Genetics, Institute of Microbiology, University of Warsaw, Miecznikowa 1, 02-096 Warsaw, Poland Abstract The growing bacterial resistance to antibiotics calls for the elaboration of new pathogens elimination strategies. Some of these methods are based on the conjugative transfer of recombinant plasmids able to eliminate pathogenic recipients by plasmid run-away replication or by killing activity of plasmid-encoded bacteriocins. Using live bacteria as donors of plasmid vectors carrying killing determinants requires meeting many safety restrictions in order to eliminate potential biohazard. K e y w o r d s: bio-therapeutic, recombinant plasmids, conjugation

Introduction Enormous amounts of anti-microbial chemical agents, mainly antibiotics, are produced and used to treat human and animal infections and to promote the growth of animals and plants (Levy, 1998). This overusage, sometimes misusage of antibiotics is correlated with the growing emergence of bacterial resistance to multiple drugs whose dissemination is greatly facilitated by the association of resistance encoding determinants with the mobile genetic elements, e.g. integrons, transposons and plasmids (Mazel and Davies, 1999). The growing bacterial resistance to antibiotics causes that they are no longer effective and many people die from bacterial infections that previously were easily treated with antibiotics. It should be mentioned here that some anti-microbial agents themselves promote the evolution of resistance. Tetracycline, for example, stimulates the transmission of transposons coding for tetracycline resistance (Miller and Sulavik, 1996). Moreover anti-microbial agents that often cause DNA damage or decrease in translation fidelity directly enhance the frequency of mutations and thus can potentially influence bacterial susceptibility to antibiotics (Heinemann, 1999). Another problem hampering safe antibiotic usage in therapy is that they are frequently broad spectrum, indiscriminately killing “good” and pathogenic bacteria. Many approaches have been taken to find better antimicrobial agents and this task is executed by modification and improvement of traditional antibiotics and development of fundamentally new chemical antibiotics by modern genomic-identified, target-based discovery (Walsh, 2003). This includes, for example, antagonists of bacterial communication (such as quorum sensing) to turn off the expression of virulence factors. A better understanding of bacterial molecular biology will eventually allow us to discover new efficient “smart” antibiotics which will not cause resistance spread (Heinemann, 2001). These drugs probably might have a more limiting therapeutic spectra and will be more expensive and therefore many big pharmaceutic companies have greatly curtailed their antibacterial research (Projan, 2003). The alarming data presented above indicate a need for alternative anti-microbial agents, mainly biotherapeutic agents. This role can be played by antibacterial peptides, bacteriophages and mobile elements, mainly plasmids which can be delivered in a natural way to unwanted bacteria resulting in their death. Bacteroiphages and bacteriophage encoding enzymes have been explored and appear to be effective in the treatment of certain pathogenic E. coli and S. pneumoniae strains (Chibani-Chennoufi et al., 2004; Jado et al., 2003). Bacteriophages as bio-therapeutic agents are being used in the USA, Poland, Georgia (for review see Carlton, 1999). The major limitations of this therapy are narrow host-specificity and relatively quick development of bacteria resistant to phage infection. The very new strategy of pathogenic

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bacteria elimination based on recombinant plasmids transferred to pathogenic recipient, preferentially by conjugation is the scope of presented minireview. Horizontal DNA transfer in bacteria Horizontal DNA transfer between various bacterial species and even genera is often observed in the environment. Transfer of DNA to recipient bacteria can be executed by one of the tree mechanisms – conjugation, transformation and transduction among which the first one has the main impact on bacterial variability, diversity and evolution (Davidson, 1999; Wolska, 2003). Conjugation, first described by Lederberg and Tatum (1946) denotes the transfer of DNA from cell to cell by direct contact. It requires a specific type of DNA replication in which one strand of replicated molecule is retained in the donor and the second is transferred to the recipient in 5’→ 3’ direction (Lanka and Wilkins, 1995). Conjugation functions in gram-negative bacteria are usually plasmid encoded however horizontal transfer of conjugative transposons or genomic islands has also been reported (Schubert et al., 2004). Plasmids are small DNA molecules, mainly circular, which are stably maintained in bacterial population. Plasmids replicate extra-chromosomally and a lot of them can transfer their DNA by conjugation. Some plasmids can integrate themselves into the chromosome and are then able to promote transfer of chromosomal markers. Conjugative transfer are quite efficient in the environment. Briefly, the DNA sequences that control the extra-chromosomal replication of plasmids are called vegetative origins (oriV) and rep genes, those controlling conjugative transfer are the origins of transfer (oriT) and tra genes. Gram-negative bacterial conjugative systems demand the presence of special structure – pilus – on the donor cell surface. These systems are grouped together into the type IV secretion system (Christie, 2001) composed of multi component transporters adapted to functions as diverse as conjugative DNA transfer or the delivery of effector proteins into eukaryotic target cells in pathogenesis (Seubert et al., 2003). No pili are present in gram-positive conjugation systems in which cell-to-cell contact is facilitated by pheromones (Clewell, 1993). Conjugative transfer was described in a variety of ecosystems among which human and animal bodies are of major importance in relation to the spread of antibiotic resistance. Conjugative transfer of plasmids encoding multiple drug resistance was reported for coliform bacteria living in human and animal intestine tracts (Balis et al., 1996) and for bacteria of human and bovine origins in a farm environment (Oppregaard et al., 2001). Transfer of plasmids was also observed in insects e.g. in digestive tract of cutworm Peridroma saucia (Armstrong et al. 1990) and lepidopterous larvae Galleria mellonella and Spodoptera littoralis (Jarret and Stephenson, 1990). Horizontal DNA transfer events in the natural environments by transformation and transduction have also been reported (Nielsen et al., 1997; Jiang and Paul, 1998) however they are not as common as conjugation, especially in animal ecosystems and therefore do not play the crucial role in the antibiotic resistance spread. Recombinant plasmid as the elimination agents Research design. Recently, studies have been intensified on the construction of recombinant palsmids encoding anti-bacterial functions and optimization of their conjugative transfer in order to create the new possibility of pathogenic bacteria killing after plasmid delivery from benign host to pathogenic recipient. Two main killing strategies are explored, e.g. killing by unabated (run-away) replication and killing by expression of lethal functions. Plasmids are present within host cell in characteristic copy number controlled in part by repressor-like molecules (Helinski et al., 1996; Filutowicz and Rakowski, 1998). Therefore, mutations that disrupt the repressor function cause plasmids over-replication leading to an increase in their copy number (Blasina et al., 1996). These copy-up mutations result in run-away replication due to the loss of copy-control mechanisms, what in turn stops the replication of bacterial chromosomes because of titration of multi-protein complexes required for DNA synthesis. Thus run-away replication can result in cell death. The method of killing demands the use of self-transmissible, preferentially broad-host-range plasmids able to replicate in diverse assortment of bacteria (Sakai and Komano, 1996). Special precautions should be taken to avoid further spread of newly delivered plasmids to other pathogenic/nonpathogenic recipients. To ensure that,

Minireview

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Fig. 1. Approach to killing of recipient cell.

Three methods of plasmid-based killing of bacteria are shown: A) killing by run-away replication, B) killing by activation of bacteriocin (lack of its neutralization), C) killing by derepression of gene encoding bacteriocin.  – repressor of oriV, o – repressor of bacteriocin transcription, – bacteriocin, – antidote.

plasmid minireplicons devoid of tra genes are used. Transfer of minireplicons to the recipients demands cloning of tra region in chromosome of donor cells. The use of runaway plasmid replication has a great advantage – resistance to over-replication based killing is unlikely to occur and spread horizontally. Another mechanism of killing is based on plasmid-encoded bacteriocin activity. Cells producing bacteriocin produce also a bacteriocin-specific antidote, typically a peptide or RNA (Engelberg-Kulka and Glaser, 1999). As a result of transfer of the gene encoding bacteriocin to antidote-deprived recipient cell death can occur. In another approach a donor, but not recipient, might be rendered insensitive to bacteriocin by using a tightly regulated promoter-operator system preventing repressor synthesis in donor cells. The approaches described above are diagrammed in Fig. 1. Killing of unwanted recipient bacteria demands very efficient conjugative transfer of plasmids so the conditions should be worked out to ensure the maximal frequency of this process. As biofilms provide ideal

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niches for conjugation the proposed approaches are especially suitable to use against biofilm-forming pathogens (Ghigo, 2001; Hausner and Wuertz, 1999). It should be mentioned here that biofilms of certain pathogens are thought to be the major obstacle for conventional antibiotics, preventing them from reaching and killing pathogenic bacteria (Mah and O’Toole, 2001). Reduction of biohazard. Special attention should be drawn to reduce biohazard connected with using live bacteria for delivering killing agents. Environmentally safe bacteria must be used as donors, such as E. coli F18 or various Lactobacillus and Lactococcus strains. Application of live bacteria is nothing new in therapy and agriculture. For example, the use of live attenuated bacterial vaccine strains allows the targeted delivery of macromolecules to mammalian cells and tissues and recently the ability of attenuated strains of Salmonella, Shigella and Yersinia spp., as well as Listeria monocytogenes and invasive E. coli, to deliver eukaryotic expression plasmids into mammalian cells in vivo and in vitro has been determined (Loessner and Weiss, 2004). Pseudomonas fluorescens and Erwinia herhicola are used to control the fire blight. These strains compete for nutrients with the pathogenic E. amylovora thus inhibiting its growth (Johnson and Stockwell, 2000). Another strategy recommends application of non-growing donors cells such as minicells and maxicells instead of living cells. Minicells lack chromosomal DNA but may contain plasmids, they neither divide nor grow (Frazer and Curtiss, 1975). Maxicells are obtained from a strain of E. coli that carries mutations in the key DNA repair pathways, recA, uvrA and phr (Sancar et al., 1979). This strain dies upon exposure to low doses of UV but plasmid molecules can replicate and plasmid-directed transcription and translation can occur efficiently. It is also very important, as mentioned before, to use plasmids which are mobilizable by conjugative machinery but not self-transmissible. Moreover antibiotic resistance markers for selection should be avoided. Of special advantage is the use of conditionally suicidal donors and/or plasmids systems, for example conditionally replicated plasmids which can replicate in the donor but are not able to replicate in the recipient (not in run-away replication strategy). It should be also mentioned that even for plasmids transferred by conjugation, the host range can be modulated by restriction-modification systems, ubiquitous in bacteria (Roberts and Macelis, 1996). 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Polish Journal of Microbiology 2004, Vol. 53, Suppl., 45–54

Molecular Modifications of Host Cells by Toxoplasma gondii HENRYKA D£UGOÑSKA

Department of Immunoparasitology, Institute of Microbiology and Immunology, University of £ódŸ, ul. Banacha 12/16, 90-237 £ódŸ, Poland, e-mail: [email protected] Abstract Toxoplasma gondii, the etiological agent of toxoplasmosis, is an Apicomplexa obligate intracellular protozoan parasite, which is able to infect any nucleated cell of numerous endothermic vertebrates. The combined abilities to actively penetrate host cells and perfectly control the fate of the parasite-containing vacuole (parasitophorus vacuole, PV) contribute to the remarkable global success of Toxoplasma as an intracellular parasite. Very broad host range and the relative ease of growth both in cell cultures in vitro and in vivo suggest that the parasite is able to manipulate the host cell apoptotic machinery. The article describes different aspects of host-parasite interplay focusing on molecular modifications of infected host cells. K e y w o r d s: Toxoplasma gondii, host-parasite interplay, host cells modifications

Host cells as microontohabitat for Toxoplasma gondii – life in parasitophorus vacuole Toxoplasma gondii invades both phagocytic and nonphagocytic cells by an active multistep and carefully orchestrated process including attachment, penetration and internalization. During invasion, the host cell is essentially passive and no change is detected in membrane ruffling, the actin cytoskeleton or phosphorylation of host cell proteins (Dobrowolski and Sibley, 1997). Table I Phagocytosis and invasion (penetration) of host cells by T. gondii (according to Dobrowolski and Sibley, 1997) Process

Phagocytosis

Invasion

Duration

2 – 5 min

15 – 40 sec

Host cells: type actin condensation membrane ruffling tyrosine phosphorylation

macrophages, granulocytes yes yes yes

all nucleated cells no no no

Toxoplasma exhibits several highly characteristic forms of motility: circular gliding, twirling and helical gliding and only the latter leads to cell invasion, the parasite gets screwed into host cell like cork-screw (Sibley, 2003). The invasion is initiated by contact between the apex of T. gondii and the host cell surface, involving both many host receptors (proteoglycans: heparin and heparan sulphate, $-integrins) and parasite ligands: surface antigens (SAG), surface antigen-related sequences (SRS), microneme proteins (MIC) and laminin (Furtado et al., 1992; Jacquet et al., 2001; Manger et al., 1998; Ortega-Baria and Boothroyd, 1999). While T. gondii uses sulphated glycosaminoglycans such as heparin and heparan sulphate as target molecules for binding to their host cells, tightly related apicomplexan parasite N. caninum prefentially interacts with chondroitin suphate residues (Naguleswaran et al., 2002).

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D³ugoñska H. Attachment

Internalization

Penetration helical gliding

dense granules rhoptries micronemes

host cell membrane

host cell

ER

M

M

TVMN

Fig. 1. The invasion of host cell by Toxoplasma gondii. Parasite’s (▼) and host’s (■) cell components involved in attachment, PVM – parasitophorus vacuole membrane, M – mitochondria, ER – endoplasmic reticulum with ribosomes, TVMN – tubulovesicular membranous network (modified from Bonhomme et al., 1999)

During invasion, the contents of three separate secretory organelles of T. gondii are discharged sequentially (Carruthers and Sibley, 1997; Dubremetz, 1998), Fig. 1. First, small apical vesicles called micronemes release a family of adhesive proteins, involved in recognition of and attachment to the host cells. The second wave of exocytosis occurs from rhoptries whose content is responsible for forming (biogenesis) of nascent parasitophorus vacuole (PV). Rhoptry proteins are commonly located on the host cytoplasmic side of PVM (parasitophorus vacuole membrane), suggesting their role in host-parasite biochemical communications. The cytoplasmic face of PVM is lined with continuous layer of host mitochondria (M) and endoplasmic reticulum (ER). The latter has no ribosomes on the side oriented to PVM. T. gondii tachyzoites have evolved a surface-associated proteolytic machinery which extensively modifies their secretory proteins (microneme and rhoptry origin), contributing to the adhesive properties of the parasite (Naguleswaran et. al., 2003). Finally, dense granule proteins (GRA1-GRA8) are released to fully formed vacuole and the secretion is continued during T. gondii replication. They participate in modifying of the vacuole and can be dispatched to various targets: PVM, reticular network in PV, vacuolar space and even host membrane and cytosol (Bonhomme et al., 1999). Consistent with the formation of a specialized compartment-parasitophorus vacuole, the invasion of T. gondii into hosts cell leads to exclusion of the transferrin receptors and rab5 as markers of early endosomes (Mordue and Sibley, 1997). Furthermore, within PVM have been not detected late endosomal markers: rab7 and cation-independent mannose 6-phosphate receptor (CI-M6PR), lysosomal marker LAMP1 (lysosome-associated membrane protein 1) and proton pump (Joiner et al., 1990; Mordue and Sibley, 1997; Mordue et al., 1999). Entering of the PV by parasite is associated with the increase of K+ level and sPLA2 II (phospholipase 2) activity in host cytosol and the host membrane becomes hyperpolarized (Bonhomme et al., 1999). Following entry, Toxoplasma rapidly divides by binary fission, forming sequential pairs of daughter cells (endodyogeny). After six to eight parasite divisions the host cell lyses and released tachyzoites infect surrounding cells and tissues. Chronic infection is associated with differentiation into bradyzoites that form cysts preferentially in central nervous system, skeletal muscle and eye. At the beginning of cyst formation, the PVM remains relatively smooth, making a few long tubular invaginations inside the cyst. Irrespective of their size and age, the tissue cysts are present within intact brain cells that provide continuing niche for parasite’s survival (Beyer et al., 2002). The infection with Toxoplasma gondii occurs usually by the accidental ingestion of cysts present in contaminated meat or oocysts excreted by cats. Chemokines released by intestinal epithelial cells recruit polymorphonuclear neutrophils, dendritic cells, macrophages and lymphocytes. The relation and influence of the parasite on diverse host cells will be presented.

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Toxoplasma gondii and various host cells Neutrophils. Several lines of evidence suggest that neutrophils are essential for survival during the first few days of infection, but their parasiticidal effect is not associated with reactive oxygen intermediates. Neutrophils were recently identified as a source of several proinflammatory cytokines: IL-12 (interleukin 12), TNF-", (tumor necrosis factor ") and chemokines: MIP-1", MIP-1$ (macrophage inflammatory protein 1", 1$) that are stored in preformed pools in secretory or possibly gelatinase granules. Recruited rapidly (2–3 h after infection) and in large numbers to the site of infection, neutrophils are ready to release cytokines and chemokines that are very important regulators of cell recruitment and activation, among them immature dendritic cells (Denkers et al., 2004, Kasper et al., 2004). Dendritic cells (DC). DC, previously thought to be only very efficient antigen-presenting cells, have been transformed recently into architects of immunity. Many studies have implicated that the interaction parasite-dendritic cell is a key element of the host response, because dendritic cells play a crucial role not only in inducing cellular immunity, but also in directing its profile – Th1 or Th2 (T helper 1 or 2 lymphocytes). The ability of DC to produce IL-12 relies on synergy of MyD88 (a Toll-like receptor adaptor molecule) and CCR5 (CC chemokine receptor 5) signaling (Aliberti and Sher, 2002; Denkers et al., 2004). Immature DC in response to CCR1 and CCR5 ligands, migrate to inflammatory sites, where they process the antigen, mature, down-regulate CCR1 and CCR5 and up-regulate CCR7, which facilitates them homing into T-cell areas of secondary lymphoid organs. It is worth noticing that, almost as a rule, invaded by toxoplasms DC either fail to respond or are functionally suppressed. Nevertheless, DC can be activated by tachyzoite extracts, secreted parasite components or through cross-priming by other infected cells (Sher et al., 2003). Aliberti and Sher (2002) have proposed two general mechanisms by which T. gondii soluble extract (STAg) could trigger IL-12 production. The first hypothesis (autocrine stimulation model) argues both the direct activation of IL-12p40 gene expression and CCR5 ligands production. The chemokines produced interact with CCR5, amplifying IL-12 production. In the alternative hypothesis (chemokine mimicry model) the second enhancing signal for IL-12 production is provided by parasite component, CCR5 ligand mimic. The authors emphasize that G-protein coupled CCR5 signaling is an enhancing factor to the weak signal from undetermined parasite triggering molecule. The most fascinating question is, why CCR5-dependent G-protein signaling is stimulatory in the case of T. gondii, but inhibitory in the case of other microbial stimuli. After exposure to STAg, DC are unresponsive 5– 7 days to further stimulation. The mechanisms of the paralysis are based on lipoxin A4, an eicosanoid, arachidonate inhibitor of acute inflammation produced by macrophages under T. gondii stimulation. Lipoxin, by binding to formyl-peptide receptor ligand -1 (FPRL-1), down-regulates CCR5 expression on DC and consequently, IL-12 production. This phenomenon, termed “DC paralysis”, might be unique for Toxoplasma, because lipoxin had no inhibitory effect on IL-12 production triggered by other microbial products, for instance LPS. DC paralysis is a likely mechanism to avoid proinflammatory cytokine production (Scott and Hunter, 2002). Interestingly, human monocyte-derived DC discriminate between alive and killed tachyzoites. Only the first ones up-regulate CD28 and CD40 on DC and initiate IL-12 synthesis, whereas a Toxoplasma lysate was a poor inducer of IL-12 (Subauste and Wessendarp, 2000). Fischer et al. (2000) found that Toxoplasma gondii triggered expansion and maturation of DC from their progenitors in central nervous system and emerged as a major source of IL-12, suggesting a role of long-term IL-12 production in protection. The long-lasting presence of activated DC in infected brain might contribute to the chronicity of the intracerebral cellular response in Toxoplasma encephalitis. The traditional concept of the brain as “immunoprivileged” organ should be modified. Macrophages. Macrophages, evolutionary ancient cells, play a key role in defense against infection. Tachyzoites of different T. gondii strains, but not bradyzoites, induce chemokine MCP-1 (macrophage chemotactic protein -1) that recruit intensively macrophages during acute infection (Brenier-Pinchart, 2002). Despite the fact that macrophages are potent effectors of innate immunity (phagocytosis, killing by reactive oxygen and nitric intermediates, limiting iron and tryptophane resources, producing proinflammatory cytokines) the cells serve as hosts for T. gondii. After macrophage penetration, the parasite inhibits acidification of the parasitophorus vacuole, actively suppresses proinflammatory cytokine synthesis by interference with intracellular signaling pathway or stimulates production of counter-regulatory cytokines. When macrophages are infected with the parasite, the cells fail to produce TNF-" and IL-12 production occurs only later, after delay of approximately 24 h. Toxoplasma infection induces rapid activation of transcription factors such as STAT1 and NF6B, but blocks their translocation from cytoplasm to nucleus for 24 hours (Butcher et al., 2001). After nuclear import blockade, macrophages begin to release IL-12, but remain actively suppressed in their ability to produce TNF-". T. gondii triggers two independent cell pathways leading to IL-12 production: the first

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D³ugoñska H.

co-dependence?

1

2 CCR5 ligand mimic cyclophilin - 18

T. gondii TLR ligand (?) TLR

CCR5

MyD88 GaI - like protein NFKB MAPK

IL - 12 Fig. 2. Production of IL-12 by Toxoplasma gondii – triggered host immune cells: MyD88- and CCR5 – dependent pathways. TLR – Toll like receptor, MyD88 – transducer molecule, NF6B – nuclear factor kappa B, MAPK – mitogen-activated protein kinase, CCR5 – CC chemokine receptor 5, IL-12 – interleukin 12 (modified from Denkers, 2003; Denkers et al., 2003)

(in macrophages, dendritic cells and neutrophils) through TLR receptor and transducer molecule MyD88 and the second – in dendritic and possibly other cells through CCR5 and G-protein (Denkers et al., 2003). The parasite is known as a strong activator of innate and acquired immunity, associated with high levels of IL-12, IFN-( and TNF-", all important in protective immunity. The view that the parasite is solely a proinflammatory pathogen is oversimplistic. Many observations clearly indicate active suppression mediated by Toxoplasma. 1. inducing IL - 10 production 2. inducing lipoxin A4

suppression of IL - 12 activity suppression of IL - 12 activity

extracellular intracellular T. gondii

PV

1. blocking NFKB nuclear import suppression of proinflammatory cytokine activities 2. blocking STAT1 nuclear import down-regulation of MHC class I and II expression 3. blocking caspase activation and cytochrome c release inhibition of apoptosis Fig. 3. Immunosuppression in toxoplasmosis – extracellular and intracellular pathways. IL-10 – interleukin 10, IL-12 – interleukin 12, NF6B – nuclear factor kappa B, STAT1 – signal transducer and activator of transcription 1, MHC – major histocompatibility complex, PV – parasitophorus vacuole (modified from Denkers, 2003)

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The ability to delay or dampen the proinflammatory cytokine synthesis allows T. gondii for early expansion and prevents the host from immunopathology due to overproduction of those cytokines. By comparing the immune response of mice infected with different Toxoplasma strains, it was observed that highly virulent strain RH elicited IFN-( hyperproduction, extensive apoptosis and marginal NO• production, whereas the infection by low-virulent strain ME49 induced low levels of IFN-(, less apoptosis, but higher levels of NO•. The ingestion of IFN-(-induced apoptotic cells triggers an alternatively activated macrophages population, that owing to their minimal NO• production could support the intracellular growth of the virulent parasites (No et al., 2004). As shown by Lüder et al. (2003a) the infection in vitro of primary bone marrowderived macrophages or monocyte/macrophage line RAW264.7 with mouse low-virulent strain down-regulated of IFN-( or LPS-induced NO• synthesis as well. The infection of human monocytes (and other cells) with T. gondii first induces a hyperpolarisation of host plasma membrane, followed by transient polarization of PVM, which decreases during parasite replication. The observed depolarization might be controlled by H + and H+/K+ ATPases as was shown by use of proper ATPases inhibitors (Bouchot et al., 2001). Lymphocytes. Initiating and sustaining strong T lymphocytes – mediated immunity is crucial in preventing the emergence of T. gondii as a serious pathogen. Parasite-specific Th1 lymphocytes release high levels of IFN-(, a pitoval protective cytokine in Toxoplasma infections, which is required to prevent cyst reactivation. Besides, both CD8+ and CD4+ lymphocytes show cytolytic activity against host cells infected with the parasite (Denkers and Gazinelli, 1998). Mast cells. The studies performed on experimental model, a wild mouse-like autochthonous rodent from South America Calomys callosus, extremely susceptible to toxoplasmosis, demonstrated that mast cells were involved in acute phase of inflammatory response. After 1 h of infection, a significant influx of mastocytes into peritoneal cavity was observed. Their morphology suggested degranulation process, triggered probably by secreted parasite antigens. Degranulation initiated a remarkable increase in neutrophils influx after 12 h post-infection (Ferreira et al., 2004) Apoptosis as a strategy of modulating T. gondii – host interactions Toxoplasma is able to induce and inhibit apoptosis using different strategies. The factors determining the direction of the opposite effects have not been determined, but might be dependent on the virulence of the parasite, host cell type and stage of its infection. Programmed cell death (i.e. apoptosis) is a complex and highly regulated process of multicellular organisms. Besides its critical roles in development, aging and homeostasis, apoptosis is an important defense mechanism against viruses, bacteria and parasites. Parasitic pathogens have evolved diverse strategies to interfere with cell host apoptosis and thereby modulating the host’s immune response, facilitating intracellular survival and promoting dissemination within the host (Lüder et. al., 2001b). There are two primary pathways of apoptosis, induced by extrinsic (death receptor activation) or intrinsic (DNA damage) stimuli. The first one triggers the caspase 8 dependent pathway, while the second activates caspase 9 pathway (mitochondrial pathway) following the release of cytochrom c into cytoplasm that promotes the formation of the apoptosome, a complex composed of cytoplasmatic protein APAF1, cytochrome c and caspase 9. Activated by proteolytic processing (trans-cleavage) within apoptosome caspase 9 acts on procaspase 3 and its activation initiates the executioner phase of apoptosis resulting in cleavage of many nuclear, plasma membrane and cytoskeletal targets (Sinai et al., 2004). The mitochondrial pathway of apoptosis is particularly interesting with regard to T. gondii, because of intimate association of parasitophorous vacuole with host mitochondria (Sinai and Joiner 2001, Goebel et al., 2001). Why does T. gondii block the apoptosis? To survive long time in its host. T. gondii is auxotrophic to critical metabolites, including purines, certain amino acids (Fox et al., 2004) and cholesterol (Coppens et al., 2000), which have to be supplied by the host cell. By blocking apoptosis, the rapidly growing parasite ensures the steady providing of metabolites for its replication. Inhibition of apoptosis seems to be also important for stage conversion from aggressive tachyzoites with high growth rate to dormant, slowly multiplying bradyzoites and establishment of chronic phase of infection (Tenter et al., 2000). Keeping the infected host cell alive is likely to be critical and this protection from apoptosis is implied from the observations in vivo that tissue cysts containing bradyzoites may survive for years or even decades. Manipulation of apoptosis could reveal in the immune response, both natural and adaptive. Apoptotic cells, sending the signal “eat me” (for example, by exposure of surface phosphatidylserine), are in vivo rapidly cleared by macrophages. Recent observations

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D³ugoñska H.

of Fox et al. (2004) suggest that arginine depletion of infected host cell can trigger bradyzoite differentiation. These findings are consistent with a model of host-parasite evolution that favoured host control of bradyzoite induction over parasite control of tachyzoite proliferation, trading off virulence for increased transmission. Interestingly, T. gondii also induces apoptosis (of T lymphocytes, macrophages), possibly initiated by Fas-FasL interaction. The results of Liesenfeld et al. (1997) indicate that peroral infection with T. gondii IFN-( leads to Fas dependent apoptosis of CD4+ and CD8+ lymphocytes in Peyer’s patches, induced by IFN-(. The Fas-FasL interaction on lymphocytes and on ocular tissues is involved in pathogenesis of acquired ocular toxoplasmosis. Intraocular inoculation of T. gondii induced inflammation associated with increase in Fas and FasL expression (Hu et al., 1999). The dual activity of Toxoplasma on host cell apoptosis might require an exclusively balanced interplay between pro- and anti-apoptotic signals of the infected host. T. gondii as an obligate intracellular parasite needs continuous supply of essential metabolites and host cell apoptosis limits parasites replication. Induction of apoptosis of certain immune cells downregulates parasite-specific immune response resulting in increased parasites survival and prevention of immunopathological processes, so induction of apoptosis in the course of infection may be beneficial for the parasite and for the host (Lüder et al., 2001b). Another aspects of T. gondii – host interplay One of the evasion mechanisms induced by T. gondii in host cells is the downregulation of MHC (major histocompatibility complex) class II expression. Protective immunity against T. gondii is mediated by specific CD8+ and CD4+ lymphocytes. The first step of priming and activation of these cells is presentation of antigenic peptides in the context of MHC class I and class II molecules. According to a classical paradigm in immunology, antigens presented by class I molecules are classified as cytosolic (endogenous) and those presented by class II molecules as extracellular (exogenous) derived ones. T. gondii, like other intracellular pathogens, has evolved different strategies to interfere with class II-restricted presentation to evade host immune response and survive intracellularly. PV containing viable parasites does not fuse with lysosomes and does not acidify, thereby avoiding degradation by proteases. In addition to mediating formation of nonfusogenic PV, T. gondii down-regulates MHC class II expression on human melanoma cells (Yang et al., 1996), primary mouse macrophages (Lüder et al., 1998) and neural antigen-presenting cells (Lüder et al., 2003b). In contrast, the expression of MHC class I molecules was not influenced by the parasite (Lüder and Seeber, 2001). Despite the interference of T. gondii with MHC class II presentation pathway, infection of immunocompetent hosts induces priming and expansion of CD4+ lymphocytes. Most T. gondii proteins which elicit CD4+ T cell responses are secretory proteins, particularly dense granule proteins and the major surface antigen SAG1 (Fischer et al., 1996; Reichmann et al., 1997; Prigione et al., 2000) which are recognized in the context of defined MHC class II molecules. Lüder and Seeber (2001) proposed that secretory proteins as well as those derived from non-viable parasites or associated with cellular debris from lysed host cell are endocytosed by APC (antigen presenting cell) and then presented, to some extent, via the conventional MHC class II pathway. However, due to the fact of PV fusion incompetence and down-regulation of MHC class II molecules, host APC infected by live parasites are not recognized by CD4+ lymphocytes thus facilitating intracellular T. gondii survival. Since surface MHC class II molecules are excluded from the nascent PVM during invasion of the host cell, alternative loading of antigenic peptides within early endosomes seems unlikely. In both humans and mice, T. gondii infection provides a potential stimulation for the generation of CD8+ effectors capable of lysing infected target cell (Denkers and Gazinelli, 1998).The problem is, how T. gondii antigens reach the MHC class I pathway in infected cells. The PVM is believed to act as a molecular sieve, allowing bi-directional passive transport of molecules up to 1300 Da. This would mean that only small peptides degradated by parasite proteases or intact proteins actively transported could gain access to host cell MHC class I pathway. Other mechanisms can be also not excluded, e.g. direct loading of host cell MHC class I molecules by released parasite antigens at the time of invasion. Recently, an alternative, non-classical route of MHC class I antigen presentation, called “cross-presentation”, has been widely accepted. Exogenous antigen can be processed extracellularly by serum or surface host cell proteases and load empty MHC class I molecules or alternatively can be internalized (endocytosis, macropinocytosis and phagocytosis), transported to intersection between MHC class I and II pathways, degradated by proteases and resulting peptides are loaded onto MHC class I molecules, in endoplasmic reticulum by TAP (transporter associated with antigen processing) or in endosomes, independent of TAP (Lüder and Seeber, 2001).

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After infection the mammalian cells acquire a novel dynamic compartment, i.e. parasitophorus vacuole, which contains live and dividing parasites. The replication of T. gondii within parasitophorus vacuole must coincide with a significant increase in membrane mass (biogenesis of nascent parasites membranes and enlargement of PVM). Confocal microscopic analyses demonstrated that host lipids were compartmentalized into parasite endomembranes or concentrated in discrete lipid bodies, distinct from the parasite secretory organelles. (Charon and Sibley, 2002). The mechanism(s) by which host-derived lipids are transferred across the PVM to the parasite remains unknown. One of a few possibilities is carrier protein-mediated transport, for example by using host low-density lipoprotein receptors. The exposure of T. gondii – infected cells to low-density lipoproteins led to their internalization in host endosomes and lysosomes and after 20 min they were transported through PVM inside vacuolar space, then surrounded by PVM. The phenomenon illustrates a new feature of the parasite to have an access to essential nutrients in lysosomes without being exposed to digestive hydrolases (Lüder et al., 2001a). Toxoplasma is also capable of lipid biosynthesis, at least fatty acid biosynthesis. Although cholesterol-enriched organelle – rhoptries discharge at the time of host cell entry and contribute to PWM formation, host but not rhoptry cholesterol is incorporated into the forming PVM, through a caveolae-independent mechanism. Depleting host cell membrane cholesterol inhibits parasite internalization by reducing rhoptry proteins release (Coppens and Joiner, 2003). The analysis of transcriptional profile of human fibroblats infected with Toxoplasma or bacterial intracellular pathogens revealed two genes that were specifically up-regulated by toxoplasms: gene for transferrin receptors and gene for MacMARCKS (responsible for integration of Ca2+ – calmodulin and protein kinasedependent signals) (Gail et al., 2001). Although nearly one-third of the world population has been exposed to Toxoplasma, the infection occurs mainly in place of precarious sanitary conditions and nutrition. Using an experimental toxoplasmosis model the genotoxicity of the parasite in vivo was evaluated in isogenic mice under dietary restriction and submitted to a treatment with sulphonamides, which are widely used for treatment of the toxoplasmosis. The results indicated that dietary restriction induced DNA damage in peripheral blood cells of infected mice, whereas the liver and brain cells were not influenced. Sulphonamide therapy did not show any genotoxicity, as detected by the comet assay (Ribeiro et al., 2004). Does Toxoplasma gondii manipulate us? The infection with T. gondii modulates not only the host immune responses in both acute and chronic phases of toxoplasmosis but also physiological and behavioural changes. In experimentally infected mice a decline in serum thyroxine (T4) was observed (Stahl and Kaneda, 1998) that is likely do to perturbation of the pulsative release of TSH from pituitary. Besides, concomitant immunization of the mice with nonrelated soluble antigens leads to predominant production of IgG2a specific antibodies, instead of IgG1 as seen in non-infected animals. Such a change could significantly influence the reactivity of hosts vaccinated with different microorganisms antigens (Nguyen et al., 1998). Animals infected with T. gondii may show a variety of neurological and behavioral symptoms, including changes in activity, learning and memory. The transmission of parasites during early pregnancy causes mental retardation (Webster, 2001). The high prevalence of T. gondii in the human population offers the opportunity of studying the influence of the parasite on human behaviour. Flegr et al. (1995) found a positive correlation between duration of latent toxoplasmosis and the intensity of superego (willingness to accept group moral standards) decrease. The studies, which have been done on individuals with schizophrenia, reported a higher percentage of seropositive in affected group and increased levels of cognitive impairment compared to age- and severitymatched individuals with schizophrenia but seronegative (Torrey and Yolken, 2003). In conclusion, latent toxoplasmosis, although frequently dismissed as asymptomatic and clinically unimportant in both humans and rodents does alter host behaviour, both in rodents, where altered responses may be proposed to benefit the parasite (enhancing transmission rate through food chain), and humans, where altered responses may arise as a side-effect with non current adaptive significance (Webster, 2001). Are we and other endothermic vertebrata puppets of Toxoplasma? Concluding remarks Stability of the host-parasite relationship during T. gondii infection demands that the parasite avoids elimination by effectors of host immunity but also demands that triggered immunity is enough to prevent

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the host from uncontrolled parasite replication and death. The fact that one third or even more of the human population in the world is chronically and asymptomatically infected with Toxoplasma indicates that the parasite and its hosts have co-adapted very well to achieve a stable balance. In addition to its clinical importance, T. gondii is recently increasingly becoming an attractive model organism not only for investigating apicomplexan parasites but also basic cellular functions of interest to broader scientific community beyond parasitologists, physicians and veterinarians (Kim and Weiss, 2004). Investigations of fundamental pathways in parasites may provide insight into organisms where direct experimentation is difficult or impossible. Besides, such studies might lead to the identification of crucial structures that can be then used to target parasites without damaging the host cells, for example pyridinylimidazoles block selectively parasite replication, probably by targeting an enzyme, p38 mitogen-activated protein kinase (MAPK) homologue in Toxoplasma gondii, but not MAPK in host cells (Wei et al., 2002). Toxoplasma gondii, an ancient parasite, enjoys its second youth? Literature A l i b e r t i J. and A. S h e r. 2002. Role of G-protein-coupled signaling in the induction and regulation of dendritic cell function by Toxoplasma gondii. Microb. Infect. 4: 991–997. B e y e r T.V., N.V. S v e z h o v a, A.I. R a d c h e n k o and N.V. S i d o r e n k o. 2002. Parasitophorus vacuole: morphofunctional diversity in different coccidian genera (a short insight into the problem). Cell Biol. Inter. 26: 861–871. B o n h o m m e A., A. B o u c h o t, N. P e z z e l l a, J. G o m e z, H. L e M o a l and J.M. P i n o n. 1999. 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Regulation and function of T-cell-mediated immunity during Toxoplasma gondii infection. Clin. Microbiol. Rev. 11: 5679–588. D e n k e r s E.Y., B.A. B u t c h e r, L. D e l R i o and S. B e n n o u n a. 2004. Neutrophils, dendritic cells and Toxoplasma. Int. J. Parasitol. 34: 411–421. D e n k e r s E.Y., L. K i m and B.A. B u t c h e r. 2003. In the belly of the beast: subversion of macrophage proinflammatory signaling cascades during Toxoplasma gondii infection. Cell. Microbiol. 5: 75–83. D o b r o w o l s k i J. and L.D. S i b l e y. 1997. The role of the cytoskeleton in host invasion by Toxoplasma gondii. Behring Inst. Mitt. 99: 90–96. D u b r e m e t z J.F. 1998. Host cell invasion by Toxoplasma gondii. Trends Microbiol. 6: 27–30. F e r r e i r a G.L.S., J.R. M i n e o, J.G. O l i v e i r a, E.A.V. F e r r o, M.A. S o u z a and A.A.D. S a n t o s. 2004. 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G o e b e l S., U. G r o s s and C.G.K. L ü d e r. 2001. Inhibition of host cell apoptosis by Toxoplasma gondii is accompanied by reduced activation of the caspase and alterations of poly(ADP-ribose) polymerase expression. J. Cell Sci. 114: 3495–3505. H u M.S., J.D. S c h w a r t z m a n, G.R. Y e a m a n, J. C o l l i n s, R. S e g u i n, I.A. K h a n and L.H. K a s p e r. 1999. Fas-FasL interaction involved in pathogenesis of ocular toxoplasmosis in mice. Infect. Immun. 67: 928–935. J a c q u e t A., L. C o u l o n, J. D e N é v e, V. D a m i n e t, M. H a u m o n t, L. G a r c i a, A. B o l l e n, M. J u r a d o and R. B i e m a n s. 2001. The surface protein SAG3 mediates the attachment of Toxoplasma gondii to cell-surface proteoglycans. Mol. Biochem. Parasitol. 116: 35–44. J o i n e r K.A., S.A. F u h r m a n, H.M. M i e t t i n e n, L.H. K a s p e r and L. M e l l m a n. 1990. Toxoplasma gondii: fusion competence of parasitophorus vacuole in Fc receptor-transfected fibroblasts. Science 249: 641–646. K a s p e r L., N. C u r r e t, S. D a r c h e, S. L u a n g s a y, F. M e n n e c h e t, L. M i n n s, N. R a c h i n e l, C. R o n e t and D. Buzoni-Gatel. 2004. Toxoplasma gondii and mucosal immunity. Int. J. Parasitol. 34: 401–409. K i m K. and L.M. W e i s s. 2004. Toxoplasma gondii: the model apicomplexan. Int. J. Parasitol. 34: 423–432. L i e s e n f e l d O., J.C. K o s e k and Y. S u z u k i. 1997. Gamma interferon induces Fas-dependent apoptosis of Peyer’s patch T cells in mice following peroral infection with Toxoplasma gondii. Infect. Immun. 65: 4682–4689. L ü d e r C.G.K., M. A l g n e r, C. Lang, N. B l e i c h e r and U. G r o s s. 2003a. Reduced expression of inducible nitric oxide synthase after infection with Toxoplasma gondii facilitates parasite replication in activated murine macrophages. Int. J. Parasitol. 33: 833–844. L ü d e r C.G.K., W. B o h n e and D. S o l d a t i. 2001a. Toxoplasmosis: a persisting challenge. Trends Parasitol. 17: 460–463. L ü d e r C.G.K., U. G r o s s and M.F. L o p e s. 2001b. Intracellular protozoan parasites and apoptosis: diverse strategies to modulate parasite – host interactions. Trends Parasitol. 17: 480–486. L ü d e r C.G., T. L a n g, B. B e u e r l e and U. G r o s s. 1998. Down-regulation of MHC class II molecules and inability to up-regulate class I molecules in murine macrophages after infection with Toxoplasma gondii Clin. Exp. Immunol. 112: 308–316. L ü d e r C.G.K., C. L a n g, M. G i r a l d o - V e l a s q u e z, M. A l g n e r, J. G e r d e s and U. G r o s s. 2003b. Toxoplasma gondii inhibits MHC class II expression in neural antigen-presenting cells by down-regulating the class II transactivator CIITA. J. Neuroimmunol. 134: 12–24. L ü d e r C.G.K. and F. S e e b e r. 2001. Toxoplasma gondii and MHC-restricted antigen presentation: on degradation, transport and modulation. Int. J. Parasitol. 31: 1355–1369. M a n g e r I.D., A.B. H e h l and J.C. B o o t h r o y d. 1998. The surface of Toxoplasma tachyzoites is dominated by a family of glycosylphosphatidylinositol-anchored antigens related to SAG1. Infect. Immun. 66: 2237–2244. M o r d u e D.G., S. H å k a n s s o n, I. N i e s m a n and L.D. S i b l e y. 1999. Toxoplasma gondii resides in a vacuole that avoids fusion with host cell endocytic and exocytic vesicular trafficking pathways. Exp. Parasitol. 92: 87–99. M o r d u e D.G. and L.D. S i b l e y. 1997. Intracellular fate of vacuoles containing Toxoplasma gondii is determined at the time of formation and depends on the mechanism of entry. J. Immunol. 159: 4452–4459. N a g u l e s w a r a n A., A. C a n n a s, N. K e l l e r, N. Vo n l a u f e n, C. B j ö r k m a n and A. H e m p h i l l. 2002. Vero cell surface proteoglycan interaction with the microneme protein NcMIC 3 mediates adhesion of Neospora caninum tachyzoites to host cells unlike that in Toxoplasma gondii. Int. J. Parasitol. 32: 695–704. N a g u l e s w a r a n A., N. M ü l l e r and A. H e m p h i l l. 2003. Neospora caninum and Toxoplasma gondii: a novel adhesion/ invasion assay reveals distinct differences in tachyzoite-host cell interactions. Exp. Parasitol. 104: 149–158. N g u y e n T.D., G. B i g a i g n o n, J. V a n B r o e c k, M. V e r c a m m e n, M. D e l m e e, M. T u r n e e r, S.F. W o l f and J.P. C o u t e l i e r. 1998. Acute and chronic phases of Toxoplasma gondii infection in mice modulate the host immune responses. Infect. Immun. 66: 2991–2995. N o W., G. R a e s, G.H. G h a s s a b e h, P. D e B a e t s e l i e r and A. B e s c h i n. 2004. Alternatively activated macrophages during parasite infections. Trends Parasitol. 20: 126–133. O r t e g a - B a r i a E. and J.C. B o o t t h r o y d. 1999. A Toxoplasma gondii activity specific for sulfated polysaccharides is involved in host cell infection. J. Biol. Chem. 274: 1267–1276. P r i g i o n e I., P. F a c c h e t t i, L. L e c o r d i e r, D. D e s l é e, S. C h i e s a and M.F. C e s b r o n - D e l a u w. 2000. T cell clones raised from chronically infected healthy humans by stimulation with Toxoplasma gondii excretory-secretory antigens cross-react with live tachyzoites: characterization of the fine antigenic specificity of the clones and implications for vaccine development. J. Immunol. 164: 3741–3748. R e i c h m a n n G., H. D l u g o n s k a and H.G. F i s c h e r. 1997. T cell receptor specificities of Toxoplasma gondii – reactive mouse CD4+ T lymphocytes and Th1 clones. Med. Microbiol. Immunol. 186: 25–30. R i b e i r o D.A., P.C.M. P e r e i r a, J.M. M a c h a d o, S.B. S i l v a, A.W.P. P e s s o a and D.M.F. S a l v a d o r i. 2004. Does toxoplasmosis cause DNA damage? An evaluation in isogenic mice under normal diet or dietary restriction. Mutation Res. 559: 169–176. S c o t t P. and C. H u n t e r. 2002. Dendritic cells and immunity to leishmaniasis and toxoplasmosis. Curr. Opin. Immunol. 14: 466–470. S h e r A., E. P e a r c e and P. K a y e. 2003. Shaping the immune response to parasites: role of dendritic cells. Curr. Opin. Immunol. 15: 421–429. S i b l e y L.D. 2003. Toxoplasma gondii: perfecting an intracellular life style. Traffic 4: 581–586. S i n a i A.P. and K.A. J o i n e r. 2001. The Toxoplasma gondii protein ROP2 mediates host organelle association with parasitophorous vacuole membrane. J. Cell Biol. 154: 95–108. S i n a i A.P., T.M. P a y n e, J.C. C a r m e n, I. H a r d i, S.J. W a t s o n and R.E. M o l e s t i n a. 2004. Mechanisms underlying the manipulation of host apoptotic pathways by Toxoplasma gondii. Int. J. Parasitol. 34: 381–391. S t a h l W. and Y. K a n e d a. 1998. Impaired thyroid function in murine toxoplasmosis. Parasitology 117: 217–222.

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S u b a u s t e C.S. and M. W e s s e n d a r p. 2000. Human dendritic cells can discriminate between viable and killed Toxoplasma gondii tachyzoites: dendritic cell activation after infection with viable parasites results in CD28 and CD40 ligand signaling that controls IL-12-dependent and independent T cell production of IFN-(. J. Immunol. 165: 1498–1505. T e n t e r A.M., A.R. H e c k e r o t h and L.M. W e i s s. 2000. Toxoplasma gondii: from animals to humans. Int. J. Parasitol. 30: 1217–1258. T o r r e y E.F. and R.H. Y o l k e n. 2003. Does Toxoplasma gondii cause some cases of schizophrenia? abstr., p. 52–53, International Congress on Schizophrenia Research 2003. W e b s t e r J. 2001. Rats, cats, people and parasites: the impact of latent toxoplasmosis on behaviour. Microb. Infect. 3: 1037–1045. W e i S., F.B. M a r c h e s, B. D a n i e l, S. S o n d a, K. H e i d e n r e i c h and T. C u r i e l. 2002. Pyridinylimidazole p. 38 mitogen activated protein kinase inhibitors block intracellular Toxoplasma gondii replication. Int. J. Parasitol. 32: 969–977. Y a n g T.H., F. A o s a i, K. N o r o s e, M. U e d a and A. Y a n o. 1996. Differential regulation of HLA-DR expression and antigen presentation in Toxoplasma gondii – infected melanoma cells by interleukin 6 and interferon gamma. Microbiol. Immunol. 40: 443–449.

Polish Journal of Microbiology 2004, Vol. 53, Suppl., 55–60

Babesia sp.: Emerging Intracellular Parasites in Europe JEREMY S. GRAY

Department of Environmental Resource Management, University College Dublin Dublin 4, Republic of Ireland Abstract The emergence of Lyme borreliosis as the most prevalent arthropod disease of humans in the temperate northern hemisphere has resulted in renewed interest in human babesiosis, transmitted by the same tick vectors. The advent of new molecular tools has made possible a reappraisal of the main parasites involved (Babesia divergens in Europe and Babesia microti in the USA). B. divergens is probably restricted to European cattle, though there are several nearly identical species. B. microti occurs as a world-wide species complex rather than as a single species, and although both phenotypic and genotypic features lend support to suggestions that zoonotic B. microti may occur in Europe, convincing medical evidence is lacking. Comparative biology should support genetic data in taxonomic studies of these parasites. K e y w o r d s: human babesiosis, B. divergens and B. microti taxonomy

Introduction Babesiosis is caused by tick-borne intraerythrocytic protozoan parasites of the genus Babesia and more than 100 species have now been recorded in mammal hosts (Telford et al., 1993). Although these parasites are best known as a cause of disease in animals, particularly in the tropics and subtropics, increasing attention is being paid to zoonotic human babesiosis. Most human cases are thought to be caused by B. microti, a rodent parasite (in the USA), and B. divergens, a bovine parasite (in Europe). However, several other species are involved, mostly of unknown host origin (Kjemtrup and Conrad, 2000; Homer et al., 2000), and it is only now, with the increasing availability and use of molecular tools, that real progress is being made in characterising zoonotic Babesia species. This review will consider B. divergens and B. microti in relation to their taxonomic identity and their possible roles in human disease in Europe. Babesia divergens Biology and pathology. B. divergens probably occurs wherever cattle and the vector, Ixodes ricinus, coexist. The precise humidity requirements of I. ricinus restrict it to areas with a moisture-saturated microhabitat at the base of permanent herbage in forest woodland, on rough hill-land and damp low-lying meadows. Since most of the continental-European habitat for I. ricinus is woodland and cattle make limited use of such habitat, the distribution of B. divergens-infected ticks shows incomplete overlap with the overall distribution of the vector. Countries with the highest incidence of the disease appear to be those where significant tick populations occur in open hill-land or meadows, for example Ireland (Gray and Harte, 1985), and where woodland frequently abuts cattle pasture, for example France (L’Hostis and Seegers, 2002). The parasite is transmitted transovarially and all active feeding-stages, larva, nymph and adult female, are infective. Only adult females can acquire the infection from the host. Ticks are important reservoirs of infection because the infection can persist, even in the absence of cattle, for more than one generation. Cattle are also reservoirs and subclinical infections may occur for more than two years. Although the Mongolian gerbil, Meriones unguiculatus, is a highly susceptible laboratory host, there is no evidence that wild rodents can become infected and serve as reservoir hosts (Zintl et al., 2003). Many cases of bovine babesiosis are relatively mild, but case fatality rates may be as high as 10% despite treatment (Gray and Harte, 1985). Severe cases present with high fever, anaemia, anorexia, depression,

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weakness, cessation of rumination and an increase in respiratory and heart rate. Parasitaemias may rise to between 30 and 45% causing extensive erythrocyte destruction. The resulting haemoglobinuria, which gives the disease the colloquial name of redwater fever, is frequently the first clinical sign detected and may manifest at parasitaemias as low as 3%. Fatal cases usually result from cardiac failure, or hepatic and renal insufficiency. Human patients show most of the symptoms of the acute bovine disease, including haemoglobinuria. All the recorded cases (> 30) have occurred in splenectomised individuals, frequently with further immunocompromising conditions, and are characterised by rapid fulmination (Zintl et al., 2003). Current recommended treatment consists of exchange transfusion (2 or 3 exchanges) followed by treatment with clindamycin (sometimes with quinine) (Gorenflot et al., 1998). Though the anti-malarial atovaquone appears to be a much more effective drug against B. divergens (Pudney and Gray, 1997), it has not yet been used in human cases. Identity of B. divergens. In recent years DNA detection methods have shown that closely related but hitherto uncharacterised parasites can cause symptoms characteristic of B. divergens infection. Two European cases, one Italian and one Austrian, were found to have been caused by a babesia (EU1) that on phylogenetic analysis of the sequenced 18S rRNA gene, clusters with B. odocoilei, a parasite of American whitetailed deer (Odocoileus virginianus). B. odocoilei has not been recorded in Europe, but another babesia, also apparently related to B. odocoilei and differing from EU1 by only one base was reported from I. ricinus in Slovenia (Duh et al., 2001) (Table I). Conversely, although B. divergens has been regarded as exclusively European, very similar parasites have been identified in three acute cases of human babesiosis in the USA. In a fatal case that occurred in Missouri, the parasite (MO1) was found to cluster with B. divergens. A small fragment (144 bases) of the 18S rRNA gene was sequenced initially and showed 100% homology with B. divergens, but the authors nevertheless concluded that this parasite was probably not identical to B. divergens. Subsequent sequencing of the whole gene supported this view, though it is clearly very closely related (Table I). The second case occurred in Kentucky and was described as “B. divergens” since the 18S rDNA sequence only differed by 3 bases (99.8% homology) (Beattie et al., 2002). A third case from Washington State (Herwaldt et al., 2004) was found to differ from B. divergens by 8 bases (99.5% homology, though comparison with the partial sequence of the Purnell strain U13670 gives a 6-base difference i.e. 99.6% homology – Table I). In this case the authors refrained from designating the parasite B. divergens. This latter research group established, by resequencing the whole 18S rRNA gene of the three original GenBank depositions (U13670, Z48751 and U07885), that all are in fact identical with respect to this gene (100% homology) (Slemenda et al., unpublished, in Herwaldt et al., 2004). The only accurate sequence for the 18S rRNA gene among these original depositions is for the Purnell strain (U16370). This finding puts the identity of some parasites isolated from human cases and described as B. divergens (e.g. from Portugal, Centeno-Lima et al., 2003; from Madeira, Olmeda et al., 1997; from Kentucky, Beattie et al., 2002) in serious doubt (Table I). A few B. divergens-like parasites from animals other than cattle and humans have been described. A parasite isolated from Scottish red deer (Cervus elaphus) was morphologically and serologically (IFA) identical to B. divergens, but did not infect splenectomised cattle and was therefore described as B. capreoli (Adam Table I Babesia divergens 18S rRNA gene homology (EMBL-EBI ClustalW analysis) of selected isolates estimated from GenBank sequences, with reference to the Purnell strain (GenBank Accession number U16370) Strain

Accession No.

Base pairs compared

% homology

Source

Author

Madeira

AF435415

309

99.7

Human

Olmeda et al., 1997

MO1, Missouri, USA

AY048113

1724

99.7

Human

Slemenda et al., 2001

Y274114

1724

99.6

Human

Herwaldt et al., 2004

Portugal

AY048113

1712

99.2

Human

Centeno-Lima et al., 2004

Reindeer, UK

AY098643

1712

99.8

Reindeer

Langton et al., 2003

Deer, Slovenia

AY572456

1724

99.6

Red, roe deer

Duh et al., unpubl.

Rabbit, Nantucket, USA

AY144688

1166

99.7

Cottontail rabbit

Goethert and Telford, 2003

Washington State, USA

EU1 Slovenia

AY553915

1727

98.1

I. ricinus

Duh et al., 2004

EU1 Austria, Italy

AY046575

1723

98.2

Human

Herwaldt et al., 2003

Purnell-2 B.divergens

AY046576

1664

100.0

bovine

Herwaldt et al., 2003

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et al., 1976). Similarly a parasite isolated from sika deer (Cervus nippon) in Ireland (Gray et al., 1990) was shown by the same criteria to be identical to B. divergens, but failed to infect splenectomised cattle or gerbils (Meriones unguiculatus), a laboratory host that is highly susceptible to bovine isolates of B. divergens (L’Hostis and Chauvin, 1999). It is tempting to conclude that these parasites are the same as a parasite sequenced from red and roe deer (Capreolus capreolus) in Slovenia (Duh et al., unpublished), which is not identical to B. divergens (Table I). A similar babesia was isolated from reindeer (Rangifer tarandus) in the UK (Langton et al., 2003) and this too was uninfective to gerbils, though indistinguishable from B. divergens morphologically and serologically. In this case the 18S rRNA gene was found to differ from the Purnell isolate GenBank sequence by 4 bases (99.8% homology); a very small difference but possibly enough, together with non-infectivity to gerbils, to differentiate it from bovine isolates of B. divergens. The sequence also shows small differences from that obtained from roe and red deer in Slovenia (Duh et al., unpublished). The EU1-like parasite detected in Slovenian ticks (Duh et al., 2001 differs from that obtained from red/roe deer and from the UK reindeer by at least 27 bases (98.4% homology), so is certainly a separate species. Lastly, an intriguing paper (Goethert and Telford, 2003) on transmission of “B. divergens” among American cottontail rabbits (Sylvilagus floridanus) describes a parasite reported by the authors as showing 100% 18S rDNA homology with the Kentucky human isolate (Beattie et al., 2002) and only 3 base differences with the bovine B. divergens Purnell strain (99.8% homology). It seems likely that this parasite can be infective for humans, but in view of even the small difference shown from the type-bovine B. divergens isolate (Purnell strain), together with lack of information on its infectivity to cattle or gerbils, it is perhaps too early to synonymise it with European bovine B. divergens. Its apparent identity with the Kentucky isolate (for which no sequence has yet been deposited in GenBank) allows the Kentucky, Washington and MO1 isolates to be compared. The Kentucky and Washington isolates are not identical (99.6% homology), but there is 100% 18S rDNA homology between the partial sequence from the cottontail rabbit isolate (and therefore the Kentucky isolate) and the MO1 isolate from Missouri. Although this finding is based on partial 18S rDNA sequences there may be two rather than five B. divergens-like species in the USA. Babesia microti Biology and pathology. Although assigned to the same genus, B. divergens and B. microti appear to be only distantly related, and B. microti may be nearer to the genus Theileria (Homer et al., 2000). B. microti has long been regarded as a parasite of small rodents but morphologically identical species have been reported from many hosts throughout the world (Goethert and Telford, 2004). Human infections caused by these parasites are much less widespread. The majority of cases occur on the north-eastern seaboard of the USA (Kjemtrup and Conrad, 2000) but the disease has also been reported from Japan (Saito-Ito et al., 2000). Infected rodents occur throughout Europe but so far no confirmed human cases have been recorded; this is addressed in more detail below. Unlike cases of B. divergens, B. microti-infection can give rise to disease in spleen-intact patients, manifesting as malaise, myalgia, anorexia and mild fever. In splenectomised and elderly patients the disease is likely to be more severe, resembling cases of B. divergens infection and characterised by haemoglobinuria, splenomegaly, hepatomegaly and jaundice (Kjemtrup and Conrad, 2000). Even in mild cases parasitaemia may persist for months or even years after treatment (Krause et al., 1998). The infection is transmitted transstadially by ticks of the I. ricinus complex and human infections are acquired in habitats where both small rodents and ticks are present, for example mixed woodland. B. microti is much less susceptible than B. divergens to antibabesials, reflecting the taxonomic differences referred to earlier. For example, atovaquone, the drug of choice (in combination with azithromycin) for B. microti, (Krause et al., 2000) was estimated to have an ED 50 more than 18 times that for B. divergens (Gray and Pudney, 1999). Co-infection with B. microti is thought to exacerbate Lyme borreliosis (caused by the spirochaete Borrelia burgdorferi sensu lato, which shares the same habitat and is transmitted by the same vectors) (Krause et al., 1996). Treatment problems perceived to arise from co-infections of B. microti and B. burgdorferi s.l. appear to be behind the recent use of the anti-malarials artemesia and mefloquine in “chronic” LB patients (Horowitz et al., 2000), despite the fact that no anti-babesial activity for these two drugs was evident in earlier laboratory studies (Marley et al., 1997). There is no firm evidence that any cases of human babesiosis have resulted from infection with European B. microti. One suggested explanation is that the main vector is the rodent-specific tick, I. trianguliceps (Homer et al., 2000; Kjemtrup and Conrad, 2000). However, at least one strain has been shown to be transmitted by

58

Gray J.S.

I. ricinus (Gray et al., 2002), and B. microti has been detected in I. ricinus specimens collected from vegetation (Duh et al., 2001; Skotarczak and Cichocka, 2001; Foppa et al., 2002; Kalman et al., 2003). Serological evidence for human infection with B. microti exists (Krampitz et al., 1986; Hunfeld et al., 2002; Foppa et al., 2002) but no isolations of parasites have been made from human patients despite speculation that B. microti infection may underlie atypical presentations of Lyme borreliosis. The establishment of B. microti as a European zoonosis is possibly only a matter of time (see next section) and indeed a recent publication (Meer-Scherrer et al., 2004) claims to report the first autochthonously acquired case in Europe. However, there are many unconvincing aspects of this case. The clinical presentation was persuasive, but immunofluorescence assay titres were very low, some positive PCR results were obtained but the product was not sequenced, the PCR was negative at a time when parasites were reportedly observed in blood smears, and objects identified in photographs as parasites appear to be platelets. The zoonotic status of B. microti in Europe must therefore remain open to question. Future attempts to identify human cases of B. microti infection in Europe should include blood transfer to susceptible laboratory hosts, such as hamsters or gerbils, and also the sequencing of any PCR products obtained. Identity of B. microti. B. microti has long been regarded, based on morphological and host characteristics, as a single species. However, molecular techniques have recently provided new insights and it is evident that B. microti consists of a genetically diverse species complex. Goethert and Telford (2004) analysed the 18S rDNA and beta-tubulin genes of isolates of B. microti-like parasites from the USA (including Alaska), Switzerland, Spain and Russia, from a variety of vertebrate hosts (humans, voles, woodmice, shrews, foxes, skunks, raccoons, and dogs) and from ticks. The 18S rRNA gene proved the most discriminatory and resulted in identification of three clades, only one of which (Clade 1) contained strains thought to be zoonotic. All of these are probably parasites of rodents, but rodent babesias are also present in Clade 3. Clade 2 appears to consist entirely of carnivore parasites. Interestingly, Clade 1 includes Swiss and Russian isolates, supporting the possibility of the occurrence of zoonotic strains in Europe. It is also notable that the confirmed zoonotic parasite GI from the USA can be transmitted by I. ricinus as easily as a German isolate (HK) (Gray et al., 2002). Although not included in the analysis of Goethert and Telford (2004), these parasites evidently both belong to Clade 1. The GI strain, used as a reference strain in Table II, is identical to the Clade 1 Nantucket strain, while HK shows a high level of homology to GI, as do the Clade 1 Swiss and Russian strains (all 99.9%). The Swiss strain and another European strain from Berlin proved to be identical to HK (Table II), further strengthening the case for the occurrence of a zoonotic form of B. microti in Europe. The Russian strain showed 99.8% 18S rDNA homology to these European strains. It is also of interest to note that despite sharing vector infectivity and a relatively high degree of genetic homology, GI and HK are of very different appearance in the erythrocytes of laboratory gerbils (Meriones unguiculatus) (Gray et al., 2002). The Japanese Kobe strain (AB032434), despite being zoonotic and of rodent origin, does not appear to belong to Clade 1 (Table II). However, a rodent strain showing 100% beta-tubulin gene homology with the zoonotic US types, including the GI strain, has recently been identified in northern Japan (Zamoto et al., 2004a). Similar genotypes to this potentially zoonotic Clade 1 parasite have also been found on mainland northeastern Eurasia (Zamoto et al., 2004b). The rodent Kobe strain is clearly distinct from another European rodent isolate, ‘Munich’ (AB071177) (98.2% 18S rDNA homology) (Table II) and the Munich isolate is also separated from Clade 3, the other group containing rodent parasites in Goethert and Telford’s study Table II Babesia microti 18S rRNA gene homology (EMBL-EBI ClustalW analysis) of selected isolates estimated from GenBank sequences, with reference to the GI strain (GenBank Accession number AF231348) Accession No

Base pairs compared

% homology

HK* Hannover

AB085191

1705

99.9

Clethrionomys

Tsuji et al., 2002

Berlin*

AF231349

1705

99.9

I. ricinus

Zahler et al., 2000

Switzerland*

AY144692

1255

99.9

I. ricinus

Goethert and Telford, 2004

Russia

AY144693

1254

99.9

Clethrionomys

Goethert and Telford, 2004

Munich

AB071177

1701

89.9

Mus musculus

Tsuji et al., 2001

Kobe (Japan)

AB032434

1705

99.5

Human, Apodemus,

Saito-Ito et al., 2000

Nantucket (USA)

AY144722

1255

100.0

Human, I. scapularis,

Goethert and Telford, 2004

Strain

* HK, Berlin and Switzerland strains show 100% 18S rDNA homology

Source

Author

Minireview

59

(98.2% 18S rDNA homology compared with the Clade 3 isolate from Maine, USA, AY144690). Goethert and Telford’s Clade-2 isolates are all parasites of carnivores, and the recently described B. microti-like parasite of dogs in Spain (Theileria annae?) (Camacho et al., 2001) also appears to belong to this group since it (AY534602) shows 100% 18S-rDNA homology to a Clade 2 parasite (AY144702) from a fox in Cape Cod, MA, USA. There is no evidence to suggest that T. annae is zoonotic. In addition to the above observations on homology between isolates in respect to the 18S rRNA gene, an unpublished analysis of other genes has shown that UK isolates, transmitted by the rodent tick I. trianguliceps rather than I. ricinus, are well separated from a group of continental European strains that includes the Berlin strain (M. Zahler, pers. comm.). Clearly, further characterisation of European strains of B. microti is necessary. B. divergens and B. microti biology in relation to genotype The 18S rRNA gene has been analysed by several researchers in order to determine the identity of isolates of B. divergens and B. microti. While this has proved to be revealing in many respects, it is difficult to determine the degree of homology required for conspecificity. For example, whereas several isolates of B. divergens-like parasites showed sufficient homology to be considered the same species, in the few cases where biological data were available obvious discrepancies arose, for example the apparent lack of infectivity of deer ‘B. divergens’ for gerbils or cattle (Langton et al., 2003). Similarly, the isolation of B. divergens-like parasites from humans in the USA initially suggested that B. divergens occurs on that continent, but there is no suggestion that bovine cases have ever occurred there. The evident 100% homology between bovine isolates with respect to the 18S rRNA gene (Slemenda et al., unpublished, in Herwaldt et al., 2004) now suggests that the Kentucky (Beattie et al., 2002), Portuguese (Centeno-Lima et al., 2003), Madeira (Olmeda et al., 1997), cottontail rabbit (Goethert and Telford, 2003) and reindeer (Langton et al., 2003) parasites are not B. divergens. There is still no reason to think that the pathogenic bovine parasite, B. divergens, is not restricted to Europe. In the case of B. microti, we now know that many strains exist and that B. microti should be regarded as a species complex (Goethert and Telford, 2004). Whether these strain differences manifest in significant biological differences remains to be seen, though it is already apparent that there is some variation in host specificity. The occurrence in Europe of strains with a high degree of 18S rDNA homology to zoonotic American strains (GI) suggests that some European strains may also be zoonotic, but there is no conclusive medical evidence to support this. Further genetic and biological characterisation of European B. microti isolates are required. Acknowledgements. Thanks are due to Pat Holman, Norman Pieniazek, Sam Telford 3 rd and Monika Zahler for helpful discussions and shared information.

Literature A d a m K.M., D.A. B l e w e t t, D.W. B r o c k l e s b y and G.A. S h a r m a n. 1976. The isolation and characterization of a Babesia from red deer (Cervus elaphus). Parasitology 73: 1–11. B e a t t i e J.F, M.L. M i c h e l s o n and P.J. H o l m a n. 2002. Acute babesiosis caused by Babesia divergens in a resident of Kentucky. N. Engl. J. Med. 347: 697–698 C a m a c h o A.T., E. P a l l a s, J.J. G e s t a l, F.J. G u i t a n, A.S. O l m e d a, H.K. G o e t h e r t and S.R T e l f o r d. 2001. Infection of dogs in north-west Spain with a Babesia microti-like agent. Vet Rec. 149: 552–555. C e n t e n o - L i m a S., V. d o R o s a r i o, R. P a r r e i r a, A.J. M a i a, A.M. F r e u d e n t h a l, A.M. N i j h o f and F. J o n g e j a n. 2003. A fatal case of human babesiosis in Portugal: molecular and phylogenetic analysis. Trop. Med. Int. Health. 8: 760–764. D u h D., M. P e t r o v e c and T. A v s i c - Z u p a n c. 2001. Diversity of babesia infecting European sheep ticks (Ixodes ricinus). J. Clin. Microbiol. 39: 3395–3397. F o p p a I.M., P.J. K r a u s e, A. S p i e l m a n, H. G o e t h e r t, L. G e r n, B. B r a n d and S.R. T e l f o r d 3rd. 2002. Entomologic and serologic evidence of zoonotic transmission of Babesia microti, eastern Switzerland. Emerg. Infect. Dis. 8: 722–726. G o e t h e r t H.K. and S.R. T e l f o r d 3rd. 2003. Enzootic transmission of Babesia divergens among cottontail rabbits on Nantucket Island, Massachusetts. Am. J. Trop. Med. Hyg. 69: 455–460. G o e t h e r t H.K. and S.R. T e l f o r d 3rd. 2004. What is Babesia microti? Parasitology 127: 301–309. G o r e n f l o t A., K. M o u b r i, E. P r e c i g o u t, B. C a r c y and T.P.M. S c h e t t e r s. 1998. Human babesiosis. Ann. Trop. Med. Parasitol. 92: 489–501.

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G r a y J.S. and L.N. H a r t e. 1985. An estimation of the prevalence and economic importance of clinical bovine babesiosis in the Republic of Ireland. Ir. Vet. J. 39: 75–78. G r a y J.S., T.M. M u r p h y, S.M. T a y l o r, D.A. B l e w e t t and R. H a r r i n g t o n. 1990. Comparative morphological and cross transmission studies with bovine and deer babesias in Ireland. Prev. Vet. Med. 9: 185–193. G r a y J.S. and M. P u d n e y. 1999. The activity of atovaquone against Babesia microti in the Mongolian gerbil, Meriones unguiculatus. J. Parasitol. 85: 723–728. G r a y J.S., L.V. v o n S t e d i n g k, M. G ü r t e l s c h m i d and M. G r a n s t r ö m. 2002. Transmission studies on Babesia microti in Ixodes ricinus ticks and gerbils. J. Clin. Microbiol. 40: 1258–1263. H e r w a l d t B.L., G. d e B r u y n, N.J. P i e n i a z e k, M. H o m e r, K.H. L o f y, S.B. S l e m e n d a, T.R. F r i t s c h e, D.H. P e r s i n g, A.P. L i m a y e. 2004. Babesia divergens-like infection, Washington State. Emerg. Infect. Dis. 10: 622–629. H o m e r M.J., I. A g u i l a r - D e l f i n, S.R. T e l f o r d 3rd, P.J. K r a u s e and D.H. P e r s i n g. 2000 Babesiosis. Clin. Microbiol. Rev. 13: 451–869. H o r o w i t z R.: Mefloquine and artemesia: A prospective trial of combination therapy in chronic babesiosis. 13th International Scientific Conference on Lyme Disease and Other Tick-Borne Disorders, March 24–26, 2000, Hartford Marriott Farmington, CT, USA 2000. (also at http://www.ilads.org/mefloquine.htm). H u n f e l d K.P., A. L a m b e r t, H. K a m p e n, S. A l b e r t, C. E p e, V. B r a d e and A.M. T e n t e r. 2002. Seroprevalence of Babesia infections in humans exposed to ticks in midwestern Germany. J. Clin. Microbiol. 40: 2431–2436. K a l m a n D., T. S t r e t e r, Z. S z e l l and L. E g y e d. 2003. Babesia microti infection of anthropophilic ticks (Ixodes ricinus) in Hungary. Ann. Trop. Med. Parasitol. 97: 317–319. K j e m t r u p A.M. and P.A. C o n r a d. 2000. Human babesiosis: an emerging tick-borne disease. Int. J. Parasitol. 30: 1323–1337. K r a m p i t z H.E., H. B u s c h m a n n and P. M ü n c h o f f. 1986. Gibt es latente Babesien-infektionen beim Menschen in Süddeutschland? Mitt. Österr. Ges. Tropenmed. Parasitol. 8: 233–243. K r a u s e P.J., T. L e p o r e, V.K. S i k a n d, J. G a d b a w J r., G. B u r k e, S.R. T e l f o r d 3 rd, P. B r a s s a r d, D. P e a r l, J. A z l a n z a d e h, D. C h r i s t i a n s o n, D. M c G r a t h and A. S p i e l m a n. 2000. Atovaquone and azithromycin for the treatment of babesiosis. N. Engl. J. Med. 343: 1454–1458. K r a u s e P.J., A. S p i e l m a n, S.R. T e l f o r d 3rd, V.K. S i k a n d, K. M c K a y, D. C h r i s t i a n s o n, R.J. P o l l a c k, P. B r a s s a r d, J. M a g e r a, R. R y a n and D.H. P e r s i n g. 1998. Persistent parasitemia after acute babesiosis. N. Engl. J. Med. 339: 160–165. K r a u s e P.J., S.R. T e l f o r d 3rd, A. S p i e l m a n, V. S i k a n d, R. R y a n, D. C h r i s t i a n s o n, G. B u r k e, P. B r a s s a r d, R. P o l l a c k, J. P e c k and D.H. P e r s i n g. 1996. Concurrent Lyme disease and babesiosis. Evidence for increased severity and duration of illness. JAMA. 275: 1657–1660. L a n g t o n C., J.S. G r a y, P.F. W a t e r s and P.J. H o l m a n. 2003. Naturally-acquired babesiosis in a reindeer (Rangifer tarandus tarandus) herd in Great Britain. Parasitol. Res. 89: 194–198. L’ H o s t i s M. and A. C h a u v i n. 1999. Babesia divergens in France: descriptive and analytical epidemiology. Parasitologia 41: 59–62. L’ H o s t i s M. and H. S e e g e r s. 2002. Tick-borne parasitic diseases in cattle: current knowledge and prospective risk analysis related to the ongoing evolution in French cattle farming systems. Vet Res. 33: 599–611. M a r l e y S.E., M.L. E b e r h a r d, F.J. S t e u r e r, W.L. E l l i s, P.B. M c G r e e v y, T.K. R u e b u s h 2 nd. 1997. Evaluation of selected antiprotozoal drugs in the Babesia microti-hamster model. Antimicrob. Agents Chemother. 41: 91–94. M e e r - S c h e r r e r L., M. A d e l s o n, E. M o r d e c h a i, B. L o t t a z and R. T i l t o n. 2004. Babesia microti infection in Europe. Curr. Microbiol. 48: 435–437. O l m e d a A.S., P.M. A r m s t r o n g, B.M. R o s e n t h a l, B. V a l l a d a r e s, A. d e l C a s t i l l o, F. d e A r m a s, M. M i g u e l e z, A. G o n z á l e z, J.A. R o d r í g u e z, A. S p i e l m a n and S.R. T e l f o r d 3rd. 1997. A subtropical case of human babesiosis. Acta Trop. 67: 229–234. P u d n e y M. and J.S. G r a y. 1997. Therapeutic efficacy of atovaquone against the bovine intraerythrocytic parasite, Babesia divergens. J. Parasitol. 83: 307–310. T e l f o r d S.R. I I I, A. G o r e n f l o t, P. B r a s s e u r and A. S p i e l m a n. 1993. Babesial infections in man and wildlife. p. 1–28. In: Parasivic Protozoa Vol 4, 2nd ed, J. P. Kreier and J. R. Baker (eds), Academic Press New York. S k o t a r c z a k B. and A. C i c h o c k a. 2001. The occurrence of DNA of Babesia microti in ticks Ixodes ricinus in the forest areas of Szczecin. Folia Biol. (Kraków) 49: 247–250. S a i t o - I t o A., M. T s u j i, Q. W e i, S. H e, T. M a t s u i, M. K o h s a k i, S. A r a i, T. K a m i y a m a, K. H i o k i and C. I s h i h a r a. 2000. Transfusion-acquired, autochthonous human babesiosis in Japan: isolation of Babesia microti-like parasites with hu-RBC-SCID mice. J. Clin. Microbiol. 38: 4511–4516. Z a m o t o A., T. M a s a y o s h i, T. K a w a b u c h i, Q. W e i, M. A s a k a w a and C. I s h i h a r a. 2004a. U.S.-type Babesia microti isolated from small wild mammals in Eastern Hokkaido, Japan. J. Vet. Med. Sci. 66: 919–926. Z a m o t o A., M. T s u j i, Q. W e i, S.H. C h o, E.H. S h i n, T.S. K i m, G.N. L e o n o v a, K. H a g i w a r a, M. A s a k a w a, H. K a r i w a, H. T a k a s h i m a and C. I s h i h a r a. 2004b. U.S.-type Babesia microti isolated from small wild mammals in Eastern Hokkaido, Japan. J. Vet. Med. Sci. 66: 919–926. Z i n t l A., G. M u l c a h y, H.E. S k e r r e t t, S.M. T a y l o r and J.S. G r a y. 2003. Babesia divergens: a bovine blood parasite of veterinary and zoonotic importance. Clin. Microbiol. Rev. 16: 622–636.

Polish Journal of Microbiology 2004, Vol. 53, Suppl., 61– 65

Zoonotic Reservoir of Babesia microti in Poland GRZEGORZ KARBOWIAK*

W. Stefañski Institute of Parasitology of Polish Academy of Sciences Twarda str. 51/55 00-818 Warsaw, Poland Abstract Babesiosis is as one of the emerging human and animal diseases transmitted by ticks. It is caused intraerythrocytic parasites of the genus Babesia. Current evidence of human babesiosis suggests that the majority of cases are involved by Babesia divergens and Babesia microti piroplasms. As zoonotic reservoir of B. microti serve small mammals – insectivores and rodents. The occurrence of this parasite in natural environment in Poland is documented for various regions, in the wide range of mammal hosts. The most important role as Babesia microti reservoir play Microtus voles. The prevalence of infection in Microtus arvalis studied in Mazurian Lakeland is 9–33%; in Microtus agrestis in Katowice agglomeration reach almost 50%, Microtus oeconomus in Bia³owie¿a 7.7–50%. The lesser role as zoonotic reservoir play Clethrionomys voles, Apodemus mice and shrews; the prevalence of infections in these mammals don’t exceed 2 %. The vectors for B. microti piroplasms in middle-European conditions are Ixodes ricinus, I. trianguliceps and Dermacentor reticulatus. There were recorded the infections of Ixodes ricinus ticks with B. microti in Szczecin and Tri-City, the rate was 6.2–13.3%. The variation in B. microti prevalence in rodents and ticks is very changeable and determined by season, the interaction with other hemoparasites, host’s age and local conditions. K e y w o r d s: babesiosis, Babesia spp. reservoires in Poland

Piroplasmosis is the dangerous tick-borne disease of human and animals, caused by protozoans of the Babesia genus. Human babesiosis has been ascribed to cause animal babesias, but current evidence suggests that the majority of cases are caused by Babesia divergens and Babesia microti. Including the first case described by Skrabalo and Deanovic in 1957 in Former Yugoslavia, over 20 cases of human babesiosis have been reported in Europe. B. divergens, cattle pathogen, was involved in a majority of human babesiosis in Europe, the cases caused by Babesia microti are seldom (Homer et al., 2000; Skotarczak and Cichocka, 2001a). Only recently, the case of imported babesiosis was reported in Poland (Humiczewska and KuŸna-Grygiel, 1997). Splenectomy is the main factor of risk which was found in 86% of the patients (Brasseur and Gorenflot, 1996). As zoonotic reservoir of Babesia microti serves small mammals; there is documented ability of 16 European species of insectivores and rodents to be host of this piroplasm (Cox, 1970; Šebek, 1975; Šebek et al., 1977; Šebek et al., 1980; Siñski, 1999). The occurrence of some Babesia in natural environment in Poland is documented from various regions in the wide range of mammal hosts. Babesia microti was found in rodents in Bia³owie¿a National Park, Katowice agglomeration and Mazurian Lakeland (Bajer et al., 1998; Karbowiak et al., 1999). Infections of ticks with Babesia microti, in many cases mixed with B. divergens and Borrelia burgdorferi spirochetes, are recognised in Szczecin (Skotarczak and Cichocka, 2001a) and Tri-City (Stañczak et al., 2004). Voles from the genus Microtus are still considered to be the main reservoir of Babesia microti in natural environment in Europe. The analysis of data collected from various research centres shows that the prevalence rate of infection in Microtus voles is much higher than in other rodents (Table I). Similar zoonotic situation is observed in Poland. The prevalence of infection are the highest in Microtus voles: in common vole Microtus arvalis studied in Mazurian Lakeland the prevalence is 9–33%; in field vole Microtus agrestis in Katowice agglomeration reach almost 50%, in root vole Microtus oeconomus in Bia³owie¿a 7.7–50%. The lesser role as zoonotic reservoir play Clethrionomys voles, Apodemus mice and shrews; the prevalence of infections in these mammals don’t exceed 2% (Table II). * Corresponding author: e-mail [email protected]

62

Karbowiak G. Table I The hosts recorded and prevalence (in %) of Babesia microti infections in Europe (apart Poland) Host’s species Microtus arvalis

Microtus agrestis

8.3

a

Localisation

Authors

Austria, Steiermark

Šebek et al. (1980)

0.7

Czech, s. Moravia

Šebek (1975)

0.6

Czech

Šebek et al. (1977)

25.5

southern England

Baker et al. 1963

4.1–22.2

England

Healing (1981)

25.2

England

Cox (1970)

38.0

Germany, Bavaria

Kramptiz and Bäumler (1978)

30.5

Austria, Steiermark

Šebek et al. (1980)

6.5

Austria, North Tyrol

Mahnert (1972)

0.5

Czech, s. Bohemia

Šebek (1975)

0.5

Czech

Šebek et al. (1977)

Microtus nivalis

2.3

Austria, North Tyrol

Mahnert (1972)

Microtus socialis

50.0

Ukraine, Askania Nova

Karbowiak et al. (2002)

Clethrionomys glareolus

Pitymys subterraneus Apodemus flavicollis Apodemus sylvaticus

9.6–13.0

England

Healing (1981)

21.3

England, Sussex

Turner (1986)

16.3

England

Cox (1970)

1.8

Austria, Steiermark

Šebek et al. (1980)

1.0

Austria, North Tyrol

Mahnert (1972)

0.4

Czech, s. Moravia

Šebek (1975)

0.3

Czech

Šebek et al. (1977)

11.1a

Austria, Steiermark

Šebek et al. (1980)

18.1a

Austria, North Tyrol

Mahnert (1972)

1.6

Austria, Steiermark

Šebek et al. (1980)

0.1

Bosnia-Herzegovina

Šebek et al. (1977)

England

Healing (1981)

8.8

England, Sussex

Turner (1986)

1.6

England

Cox (1970)

2.1–10.0

0.1

Bulgaria

Šebek et al. (1977)

1.1

Bosnia-Herzegovina

Šebek et al. (1977)

0.8

Eastern Slovakia

Karbowiak et al. (2003)

Mus musculus

0.2

Macedonia

Šebek et al. (1977)

Sorex araneus

6.8

England

Cox (1970)

1.9

Austria, North Tyrol

Mahnert (1972)

Sorex minutus

5.4

England

Cox (1970)

Neomys anomalus

1.3a

Macedonia

Šebek et al. (1977)

Apodemus agrarius

a

Prevalence

statistically insignificant

The morphological features of B. microti strain found in Poland is identical to described by other authors. The parasites were mostly of the ring-like and pear-shaped form (Fig. 1.) and were 1.5–3.0 :m in diameter. Dividing stages were 2.5–3.5 mm in diameter. The mean intensity of the erythrocyte infection was 2.5%. Usually one parasite was seen in infected erythrocyte. The regular form of four cells – “maltese cross”, characteristic for “small” Babesia species, was noticed very seldom. The infection of Microtus voles with Babesia microti resulted in a dramatically enlarged spleen (Fig. 2). This phenomenon hasn’t been observed with other common hemoparasite infections, as Trypanosoma or Hepatozoon. However, apart splenomegaly symptoms, natural Babesia infections haven’t any visible signs, so it is evident that piroplasms cause chronic avirulent infections in its natural hosts (Baker et al., 1963; Krampitz and Bäumler, 1978; Turner, 1986).

63

Minireview Table II The hosts recorded and prevalence (in %) of Babesia microti infections in small mammals in Poland Rodent species Microtus arvalis

Prevalence

Localisation

Authors

13.8

Mazurian District

a a

Siñski (1999) Bajer et al. (2001); Pawe³czyk et al. (2004)

9.0

Mazurian District

Microtus arvalis

33.3

Mazurian Districtb

Karbowiak (unpublished)

Microtus agrestis

50

Katowice

Karbowiak et al. (1999)

17.6

Bia³owie¿a

Karbowiak et al. (1999)

7.7

Bia³owie¿a

Karbowiak et al. (2002)

Bia³owie¿a

Karbowiak (unpublished)

Microtus eoconomus

50 Microtus sp. Clethrionomys glareolus

c

14.3

Mazurian Districta

Bajer et al. (1998) Karbowiak and Siñski (1996)

?

Mazurian District

0.6

Mazurian Districta

Siñski (1999)

1.0

Mazurian Districta

Bajer et al. (2001)

Apodemus flavicollis

2.1

Mazurian District

Bajer et al. (1998)

Apodemus sp.

0.7

Mazurian Districta

Siñski (1999)

Sorex minutus

20

Bia³owie¿a

Karbowiak (unpublished)

Abbreviations:

a

c

Urwita³t near Miko³ajki;

b

b

a

Kosewo Górne near Mr¹gowo; c statistically insignificant

The variation in Babesia microti prevalence in rodents and ticks is very changeable and determined by season, the interaction with other hemoparasites, host’s age, sex and local conditions (Healing, 1981; Turner 1986, Pawelczyk et al., 2004). The seasonal pattern of B. microti incidence correlates with seasonal changes in the abundance of the tick vector; the seasonal variation shows a characteristic rise in the early summer time and a minimum in January (Krampitz and Bäumler, 1978; Turner 1986). The differences of prevalence between some closely located hosts population were noted in Microtus oeconomus living in open habitats in Bia³owie¿a Primeval Forest (Karbowiak, unpublished). The differentiation of infections rate in some tick species are also showed by Skotarczak (Skotarczak and Cichocka 2001ab). However, several studies of

Fig. 1. Ring forms of Babesia microti in blood of naturally infected root-vole Microtus oeconomus in Bia³owie¿a. Scale bar 10 :m

Fig. 2. Splenomegaly induced by natural Babesia microti infection in root-vole Microtus oeconomus

64

Karbowiak G.

rodent blood parasites have used a longitudinal survey format conducted in natural environment, and this requires further investigations. The vectors for Babesia piroplasms in middle-European conditions are Ixodes ricinus and I. trianguliceps, as well as Dermacentor reticulatus (Randolph, 1995). In the case of Babesia microti the most important species is Ixodes ricinus – this is common in whole area of Poland, whereas Dermacentor reticulatus Eastern part only. Ixodes trianguliceps occurs in Eastern Europe seldom due to it has small significance. The role of D. reticulatus is not clear; there is demonstrated the ability of transfer Babesia canis piroplasms, but is nothing known about other babesias. Moreover, only Ixodes ricinus regularly attacks human, in every active developmental stages; for other tick species human isn’t attractive host (Siuda, 1993; Homer et al., 2000). Many authors accent the ability of Rhipicephalus in spreading of piroplasms, however this genus as not permanent component of polish fauna is able to play marginal role only (Siuda, 1993). Nevertheless, young stages of all these species are able to maintain the transmission cycle in population of rodents and the presence of zoonotic foci in environment. The presence of two species of Babesia piroplasms in ticks Ixodes ricinus in Poland is documented using PCR method in Szczecin area and in the forests near the Tri-City agglomeration. There were found B. microti (infection rate 6.2–13.3%) and B. divergens (3.0%) in Szczecin (Skotarczak and Cichocka, 2001ab, Skotarczak et al., 2003), and B. microti in Tri-City (infection rate of in ticks 2.3%) (Stañczak et al., 2003). These demonstrations confirm recent findings that I. ricinus can be also involved in circulation of B. microti in Europe; tick infection rates with babesiae have been calculated at 7.4% in Slovenia (Duh et al., 2001) and 3.5% in Hungary (Kálmán et al., 2003). The most common infections were found in adult females – till 14.6%, other developmental forms were lighter infected – 11.1% of nymphs were infected (Skotarczak et al., 2002). The percentage of infection is changeable, depending on the season (Skotarczak and Cichocka, 2001ab, Skotarczak et al., 2002; 2003). There are many potential reasons of differences in the epidemiology of human babesiosis between Europe and Northern America. Apart from the various virulence between European and American Babesia microti strains, there are some differences in the zoonotic foci structure. In Northern America the most competent reservoirs are white-footed mouse Peromyscus leucopus and meadow vole Microtus pennsylvanicus. Other reservoirs and vectors, as prairie vole Microtus ochrogaster (Burkot et al., 2000) have local importance. Peromyscus is the most important reservoir host, with Microtus pennsylvanicus being a minor reservoir. Field surveys estimate that up to 40–60% of these mice are infected (Homer et al., 2000; Burkot et al., 2000). Both rodent species have large geographic range and are found in a progressively greater variety of habitats. The habitat of Peromyscus leucopus is chiefly wooded areas, they are most abundant in bottom lands, less so in post oak uplands and almost completely absent from prairie lands – but these open grassland are inhabited by Microtus pennsylvanicus (Hall and Kelson, 1959). The main competent vectors for transmitting B. microti are ticks Dermacentor variabilis, Ixodes scapularis, and Ixodes dammini. These species are widespread in the Eastern and Central United States as well as Western States, in various habitats (Kjemtrup and Conrad, 2000). Young stages fees on the rodents; however, man and many wild and domestic animals are also attacked (Furman and Loomis, 1984). Such structure maintains the easy ways to transmission of B. microti infections from animal reservoir to human. In the European conditions the most competent reservoir are Microtus voles. These are the animals of the open country. Their preferred habitats are moist fields and meadows, forests edges and cropfield, rather than regular forests (Kowalski et al., 1981). Such places are inhabited by Dermacentor reticulatus ticks, not Ixodes ricinus. Ixodes ricinus chooses bush and woodland, preferably old deciduous forests, well sheltered and moist. It avoids open places (Siuda, 1993). The field observations confirm that voles living in open areas are more often infested with young adult stages of Dermacentor reticulatus than with Ixodes ricinus, in comparison with these same species living in woodland (Karbowiak, unpublished). Moreover, young and adult Dermacentor reticulatus practically don’t attack human and their significance as vector is slight. In such zoonotic foci structure there are few possibilities to transmit the Babesia piroplasms from rodent reservoir to human. Literature B a j e r A., M. B e d n a r s k a, A. P a w e ³ c z y k, E. K o n o p k a, G. K a r b o w i a k and E. S i ñ s k i. 1998. Blood parasites in a wild rodent community of Mazury Lakes District, Poland. Wiad. Parazytol. 44: 426. B a j e r A., A. P a w e ³ c z y k, J. B e h n k e, F. G i l b e r t and E. S i n s k i. 2001. Factors affecting the component community structure of haemoparasites in bank voles (Clethrionomys glareolus) from the Mazury Lake District region of Poland. Parasitology 122: 43–54

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B a k e r J.R., D. C h i t t y and E. P h i p p s. 1963. Blood parasites of wild voles, Microtus agrestis, in England. Parasitology 53: 297–301. B r a s s e u r P. and A. G o r e n f l o t. 1996. Human babesial infections in Europe. Ann. Acad. Med. Bialost. 41: 117–122. B u r k o t T.R., B.S. S c h n e i d e r, N.J. P i e n i a z e k, C.M. H a p p, J.S. R u t h e r f o r d, S.B. S l e m e n d a, E. H o f f m e i s t e r, G.O. M a u p i n and N.S. Z e i d n e r. 2000. Babesia microti and Borrelia bissettii transmission by Ixodes spinipalpis tick among prairie voles, Microtus ochrogaster, in Colorado. Parasitology 121: 595–599. C o x F.E.G. 1970. Parasitic protozoa of British wild mammals. Mammal Rev. 1: 1–28. D u h D., M. P e t r o v e c and T. A v s i c - Z u p a n c. 2001. Diversity of Babesia infecting European sheep ticks (Ixodes ricinus). J. Clin. Microbiol. 39: 3395–3397. F u r m a n D.P. and E.C. L o o m i s. 1984. The ticks of California. Bulletin of the California Insect Survey, 25. University California Press, Berkeley, Los Angeles. H a l l E.R. and K.R. K e l s o n. 1959. The mammals of North America. The Ronald Press Company, New York. H e a l i n g T.D. 1981. Infections with blood parasites in the small British rodents Apodemus sylvaticus, Clethrionomys glareolus and Microtus agrestis. Parasitology 83: 179–189. H o m e r M. J., I. A g u i l a r - D e l f i n, S.R T e l f o r d I I I, P.J. K r a u s e and D.H. P e r s i n g. 2000. Babesiosis. Clin. Microbiol. Rev. 13: 451–469. H u m i c z e w s k a M. and W. K u Ÿ n a - G r y g i e l. 1997. A case of imported human babesiosis in Poland (in Polish). Wiad. Parazytol. 43: 227–229. K á l m á n D., T. S r é t e r, Z. S z é l l and L. E g y e d. 2003. Babesia microti infection of anthropophilic ticks (Ixodes ricinus) in Hungary. Ann. Trop. Med. Parasitol. 97: 317–319. K a r b o w i a k G. and E. S i ñ s k i. 1996. The finding of Babesia microti in bank vole Clethrionomys glareolus in the district of Mazury Lakes (Poland). Acta Parasitol. 41: 50–51. K a r b o w i a k G., M. S t a n k o, L. R y c h l i k, W. N o w a k o w s k i and K. S i u d a. 1999. The new data about zoonotic reservoir of Babesia microti in small mammals in Poland. Acta Parasitol. 44: 142–144. K a r b o w i a k G., M. S t a n k o, J. F r i è o v a, I. W i t a and U. C z a p l i ñ s k a. 2003. The communities of blood parasites in field-mouse Apodemus agrarius. Wiad. Parazytol. 49: 407. K a r b o w i a k G., I. W i t a and U. C z a p l i ñ s k a. 2002. Protozoan parasites in the blood of the social vole (Microtus socialis) in Askania Nova Reserve, Ukraine, abstr. p. 131. XII Konference of Ukrainian Parasitological Society, 2002. K j e m t r u p A.M. and P.A. Conrad. 2000. Human babesiosis: an emerging tick-borne disease. Int. J. Parasitol. 30: 1323–1337. K o w a l s k i K., Z. P u c e k and A.L. R u p r e c h t. 1981. Rodents – Rodentia, p. 164–247. [In:] Pucek E. (ed.) Keys to vertebrates of Poland. Mammals. PWN, Warszawa. K r a m p i t z H.E. and W. B ä u m l e r. 1978. Occurrence, host range and seasonal prevalence of Babesia microti (França, 1912) in rodents of southern Germany (in German). Z. ParasitKde. 58: 15-33. M a h n e r t V. 1972. Grahamella and Sporozoa as blood parasites of Alpine small mammals (in German). Acta Tropica. 29: 88–100. P a w e l c z y k A., A. B a j e r, J.M. B e h n k e, F.S. G i l b e r t and E. S i n s k i. 2004. Factors affecting the component community structure of haemoparasites in common voles (Microtus arvalis) from the Mazury Lake District region of Poland. Parasitol. Res. 92: 270–284. R a n d o l p h S.E. 1995. Quantifying parameters in the transmission of Babesia microti by the tick Ixodes trianguliceps amongst voles (Clethrionomys glareolus). Parasitology 110: 287–295. Š e b e k Z. 1975. Blood parasites of small wild mammals in Czechoslovakia (in German). Folia Parasitol. (Praha). 22: 11–20. Š e b e k Z., B. R o s i c k ý and W. S i x l. 1977. The occurrence of Babesiasis affecting small terrestrial mammals and the importance of this zoonosis in Europe. Folia Parasitol. (Praha). 24: 211–228. Š e b e k Z., W. S i x l, D. S t ü n z n e r, M. Va l o v á, Z. H u b á l e k and H. T r o g e r. 1980. Blood parasites of small wild mammals in Steiermark and Burgenland (in German). Folia Parasitol. (Praha). 27: 295–301. S i ñ s k i E. 1999. Enzootic reservoir for new Ixodes ricinus-transmitted infections (in Polish). Wiad. Parazytol. 45: 135–142. S i u d a K. 1993. Ticks of Poland (Acari: Ixodida) (in Polish). Polskie Towarzystwo Parazytologiczne, Warszawa. S k o t a r c z a k B. and A. C i c h o c k a. 2001a. Isolation and amplification by polymerase chain reaction DNA of Babesia microti and Babesia divergens in ticks in Poland. Ann. Agric. Environ. Med. 8: 187–189. S k o t a r c z a k B. and A. C i c h o c k a. 2001b. The occurrence DNA of Babesia microti in ticks Ixodes ricinus in the forest areas of Szczecin. Folia Biologica (Kraków). 49: 247–250. S k o t a r c z a k B., B. W o d e c k a and A. C i c h o c k a. 2002. Coexistence DNA of Borrelia burgdorferi sensu lato and Babesia microti in Ixodes ricinus ticks from north-western Poland. Ann. Agric. Environ. Med. 9: 25–28. S k o t a r c z a k B., A. R y m a s z e w s k a, B. W o d e c k a and M. S a w c z u k. 2003. Molecular evidence of coinfection of Borrelia burgdorferi sensu lato, human granulocytic ehrlichiosis agent, and Babesia microti in ticks from northwestern Poland. J. Parasitol. 89: 194–196. S t a ñ c z a k J., R.M. G a b r e, W. K r u m i n i s - £ o z o w s k a, M. R a c e w i c z and B. K u b i c a - B i e r n a t. 2004. Ixodes ricinus as a vector of Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum and Babesia microti in urban and suburban forests. Ann. Agric. Environ. Med. 11: 109–114. Tu r n e r C.M.R. 1986. Seasonal and age distributions of Babesia, Hepatozoon, Trypanosoma and Grahamella species in Clethrionomys glareolus and Apodemus sylvaticus populations. Parasitology 93: 279–280.

Polish Journal of Microbiology 2004, Vol. 53, Suppl., 67–73

Apicomplexan Parasites: Environmental Contamination and Transmission EDWARD SIÑSKI1 and JERZY M. BEHNKE2 1 Department

of Parasitology, Institute of Zoology, Faculty of Biology, Warsaw University, Miecznikowa 1, 02-096 Warsaw, Poland 2 School of Biology, University Park, University of Nottingham, Nottingham, NG7 2RD, UK

Abstract The Apicomplexa are a diverse group of intracellular parasitic protists. The majority of species from the classes Coccidea, Haemosporea and Piroplasmea are responsible for widespread diseases of humans and domestic animals. Oocysts of these parasites can persist for long periods of time in the environment (i.e. in water, soil, on vegetation and other food resources), maintaining their infectivity even under harsh environmental conditions and therefore are important for dispersal and transmission to hosts. This review will address the biology, transmission patterns and survival in the environment of Cryptosporidium, Cyclospora and Toxoplasma species, the most common causes of human diseases. K e y w o r d s: Intracellular parasites, Cryptosporidium, Cyclospora, Toxoplasma, transmission, environmental survival

The phylum Apicomplexa contains over 4600 named species of protists, many of which are of significant medical and economic importance. Major species responsible for widespread diseases of humans and domestic animals are presented in Table I. All are unicellular and obligatory intracellular parasites occupying a variety of cells within the body of the host. Apicomplexa are characterized by lacking obvious locomotory structures and presence of an apical complex in their infective life cycle stage (zoities). The apical complex comprises polar rings and associated cortical microtubules, rhoptries, micronemes, and usually a conoid. Life cycles of the apicomplexans include both successive sexual and asexual phases of development: gamogony, the sexual phase with production of gametes and fertilisation (syngamy); sporogony, the asexual production of numerous sporozoites from the zygote within oocyst; and schizogony (merogony), a phase of asexual multiplication characterized by multiple fission (Fig. 1). The Protists of the Apicomplexa are currently divided into seven classes (Marquardt at al., 2000), including Coccidiea, which form oocysts and have direct, contaminative life cycle, Haemosporea and Piroplasmea which do not form oocysts and have an indirect life cycle with an arthropod vector. However, the essential features of the life cycles of all these classes are very similar, differing mainly in aspects relating to transmission. Table I Major apicomplexan parasites of humans and domestic animals Parasite Plasmodium spp.

Cell red blood cells

Disease

Host

malaria

humans humans

Babesia spp.

red blood cells

piroplasmosis

Cryptosporidium spp.

enterocytes

cryptosporidiosis humans

Cyclospora cayetanensis

enterocytes

cyclosporosis

Sarcocystis hominis (Isospora spp.) enterocytes

sarcocystosis

Toxoplasma gondii

macrophages and many others toxoplasmosis

humans humans humans

68

Siñski E., Behnke J.M.

Merogony

Meront Merozoite

Sporont Sporozoite

Sporogony

Gamont Micro- and macrogametes

Gamogony

Fig. 1. Life cycle of the Apicomplexa. The sporont, meront and gamont multiply by an asexual reproductive process called schizogony

Class: Coccidea In the families of Cryptosporidae and Eimeriidae the normal mode of transmission is via a resistant oocyst, including: 1. direct faecal-oral, host-to-host transmission and indirect transmission following contamination of food or water resources (e.g. Cryptosporidium sp., Cyclospora cayetanensis) 2. direct faecal-oral transmission in definitive hosts and indirect transmission by food, characteristic for extra-intestinal or tissue coccidians (e.g. Toxoplasma gondii). They have so-called isosporoid oocysts with 2 sporocysts, each of which has 4 sporozoite, similar to those of the genus Isospora. The life cycles of the tissue coccidia involve two hosts, usually a carnivore and a herbivore. The life cycle of Toxoplasma can be completed in one host, the cat, and its intermediate hosts are not mandatory. Classes: Haemosporea and Piroplasmea Indirect transmission between hosts is achieved by blood-sucking arthropod vectors. Maturation of the gamonts, fertilization, and sporogony take place in the vector, the definitive host, merogony and gamogony take place in the vertebrate host, the intermediate host (e.g. Plasmodium, Babesia). For the majority of species in the phylum the oocyst stage is of primary importance for dispersal, survival, and infectivity of the parasites. It is also the stage of major importance for detection, identification of the parasite and clarification of host specificity. Table II summarizes the biological characteristics, combined with the unique size and shape of the oocysts and their internal structures consisting of sporocysts and Table II Characteristic features of oocysts in the environment for genera of Cryptosporidium, Cyclospora, Isospora and Toxoplasma Time of sporulation (days)

Mean number of oocysts excreted during 24 h

Duration of infectivity under optimal condition (months)

Genera of parasite

Shape of oocyst*

Environmental form

Cryptosporidium

III

thick walled oocyst, measure 4–5 :m, immediately infectious upon excretion

0

oocyst measure 8–10 :m requires sporulation, not immediately infectious upon excretion

12–14

nd

II II

up to 1, humidity over 80%, temp. 22°C–32°C

oocyst measure 12×11 :m requires sporulation, not immediately infectious upon excretion

3–4

over 1 mil.

III III

from 4 to 24, humidity over 75%, temp. 5°C–12°C

Cyclospora

Isospora Toxoplasma Sarcocystis

* diagrammatic representation of relationship between oocysts, sporocysts and sporozoites nd – no data

over 10 mil. up to 4, humidity over 90%, temp. 1°C –15°C

Minireview

69

Contamination of the environment Effluents, slurry Effluents and liquids and sludge discharged on land discharged in water

Recreational use of land and water

Water treatment system

Farm lifestock

Indigenous wildlife

People

Pets

Fig. 2. Environmental reservoirs and potential patterns of transmission of Cryptosporidium, Cyclospora and Toxoplasma species

sporozoites, for each of the genera Cryptosporidium, Cyclospora, Isospora, Toxoplasma. The environmental reservoirs and potential patterns of transmission of these parasites are shown in Fig. 2. The reasons for emergence of infectious diseases, due to Cryptosporidium parvum and Toxoplasma gondii have been attributed mainly: (1) to zoonotic transmission, e.g. more animals and changes in agricultural practices have led to a higher probability of successful parasite transmission and spread from animals to humans; (2) sensitive or susceptible populations, e.g. there is an increasing number of older and immunocompromised individuals (AIDS and transplant patients), diabetics, infants, and pregnant women, all of whom may be more susceptible to infection with both parasites. Cryptosporidium spp. Cryptosporidiosis has rapidly emerged as a worldwide disease of man since the first cases were identified in 1976. Approximately 6000 cases of infection are reported in England and Wales each year, and the likely number of infections in the population is probably greatly underestimated. Cryptosporidum spp. are obligate intracellular parasites in the phylum Apicomplexa, order Eucoccidiida, family Cryptosporidae. C. parvum an intestinal parasite of mammals, which causes life-threatening diarrhoea in immunosuppressed humans and in young livestock animals is one of 13 valid species of Cryptosporidium: 7 in mammals, 3 in birds, 2 in reptiles and 1 species in fish, which are currently recognized and accepted according to the International Code of Zoological Nomenclature (Xiao et al., 2004). Table III shows the main characteristics of these species. Cryptosporidium is currently classified as an eimeriid coccidian, however there is mounting phenotypic and molecular phylogenetic evidence for a closer relationship with the gregarines and reclassification has been proposed but not yet adopted (Tenter et al., 2002). Also the complete genome sequence of C. parvum, type II isolate has been reported recently. Genomic analysis has identified extremely streamlined metabolic pathways and a reliance on the host for nutrients. In contrast to Plasmodium and Toxoplasma, the parasite lacks an apicoplast and its associated genome, and possesses a degenerate mitochondrion that has also lost its genomic components (Abrahamsen et al., 2004). However, it is quite clear that use of conventional taxonomic approaches can be informative with respect to understanding the biology of Cryptosporidium

70

Siñski E., Behnke J.M. Table III Characteristics of currently accepted species of Cryptosporidium* Species

Orginal host

Location**

Mean oocyst size (:m)

Status of infected host immuno immuno competent compromised

C. hominis

man

SI

4.9 × 5.2

+

+

C. parvum

mice

SI

5.0 × 4.5

+

+

C. felis

cat

SI

4.6 × 4.0

+

+

C. canis

dog

SI

5.0 × 4.7

+

+

C. wrairi

quinea-pig

SI

5.4 × 4.6

–

–

C. muris

mice

ST

8.4 × 6.3

–

+

C. andersoni

cattle

A

7.4 × 5.5

–

–

C. meleagridis

turkey

SI

5.2 × 4.6

+

+

C. baileyi

chicken

BF, CL

6.2 × 4.6

–

–

C. galli

birds

CL

6.0 × 4.5

–

–

C. serpentis

snakes

ST

6.2 × 5.3

–

–

C. saurophilum

lizard

SI

5.0 × 4.7

–

–

C. molnari

fish

SI, ST

4.3 × 3.3

–

–

* after Fayer et al., 2000; Chalmers et al., 2002 and Xiao et al., 2004, with some modifications ** A – abomasum, BF – bursa of Fabricius, CL – cloaca, ST – stomach, SI – small intestine

species, characterizing transmission dynamics, tracking infections and identifying sources of contamination and hence assessing the public health significance for humans, animals as well as for the environment, An essential stage in the life cycle of Cryptosporidium is the formation in the gastrointestinal tract of two types of oocysts, each containing four infectious sporozoites. Thin walled oocysts remain in the gut to prolong the infection. Thick walled oocysts are shed in apparently normal or diarrhoeic faeces to contaminate soil and water. C. parvum causes symptomatic illnesses mainly in young animals, although older animals may be carriers, and it is thought to be readily passed from animals to humans by the faecal-oral rout. The infective dose is relatively low, with an LD 50 of 132 oocysts reported for healthy adults (Du Pont et al., 1995). Cryptosporidium is resistant to disinfectants used in the water industry and has been implicated in over 20 waterborne outbreakes (Smith and Rose, 1998; Girdwood and Smith, 1999). The Milwaukee outbreak resulted in the death of 104 of 403 000 cases (Mac Kenzie et al., 1994). A number of biological features of Cryptosporidium affect its transmission and epidemiology: – the life cycle does not require dual or multiple hosts – the oocyst stage is shed in a fully sporulated state, so direct transmission can occur between hosts – autoinfection enables persistant disease in immunocompromised hosts – there is a large zoonotic (rodents, livestock) and human reservoir – the thick-walled oocysts are resistant to a wide range of pressures and can survive for long periods in the environment – the infectious dose is low and so small numbers of contaminating organisms are significant – hosts can shed large numbers of oocysts – there is a lack of specific drug therapy to clear infections efficiently. Cyclospora cayetanensis As with other coccodian parasites, C. cayetanesis (previously termed cyanobacterium like body) is an obligate intracellular parasite. It infects the cells of the upper portion of the small intestine causing recurring diarrhoeic disease – cyclosporosis in both immunocompetent and immunocompromised persons (Guerrant et al., 2001). Infections are endemic in many developing countries in South and Central America, Africa, India as well as in parts of Asia, and through trade have become a problem for many developed countries, e.g. USA, Canada. However, the true prevalence of the parasite in any population is unknown (Soave and Johnson 1995). Cyclosporosis appears to be a seasonal disease with periodicity linked to spring and early summer and seems to be both food and water borne. Moreover, C. cayetanesis is an important agent of

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traveler’s diarrhoea, most reported cases of cyclosporosis in Europe and Australia having been associated with international travel to endemic areas (Shields and Olson 2003). Toxoplasma gondii Toxoplasma gondii is an opportunistic intracellular parasite that, in the tachyzoite stage in the life cycle, is capable of infecting a wide range of nucleated cells of vertebrate hosts. The mode of T. gondii transmission remained unclear until 1970, when its life cycle was elucidated. This tissue-cyst forming coccidium (order Eucoccidiorida, family Eimeriidae) has a heterogeneous life cycle comprising an asexual phase in a variety of warm-blooded intermediate hosts and a sexual phase in the intestines of felines definitive hosts (Frenkel, 1973). In the intestinal cells of domestic cats the parasite undergoes both schizogony (asexual proliferation) and gametogony (sexual phase of the cycle), the latter resulting in the formation of immature oocysts. Cats can shed oocysts for 1–2 weeks in extremely large numbers, peaking at over a million a day, and these oocysts can remain viable for 1 year or even longer. Mature oocysts measured 12×11 :m, contain eight sporozoites and are infective to over 300 species of warm-blooded intermediate hosts, such as birds and mammals, including humans. When oocysts are ingested, the parasites undergo an asexual cycle with two stages: the tachyzoites (2 :m×6 :m) is the intracellular proliferative form which is present during the acute phase of infection, and the bradyzoite, the slowly dividing encysted tissue form (cyst 12 :m to 100 :m), is characteristic of the chronic phase of infection. Bradyzoites may persist throughout the life of intermediate hosts. Human infection with Toxoplasma is widespread, e.g. in the USA and UK estimates vary between 16 and 40% of the population being infected, while in Europe, Central and South America prevalence of infection ranges from 50 to 80% (Dubey and Beattie 1988). However, toxoplasmosis is generally asymptomatic in approximately 85% of immunocompetent persons, and for them there is no significant health risk. In immunodeficient individuals, including AIDS patients, organ transplant recipients and patients undergoing chemotherapy, central nervous system disease is common and chorioretinities or pneumonities may develop (Renold et al., 1992). T. gondii horizontal transmission via tissue cysts in humans is generally acquired by the oral route, after ingestion of raw or undercooked meat containing parasite cysts (lamb, pork and beef), and of vegetables or water contaminated with oocysts from infected cat faeces. Another route of transmission (vertical) is via transplacental passage of parasites from infected mothers to fetuses (congenital toxoplasmosis). Infection acquired during pregnancy, in the absence of prior immunity, may cause abortion, or congenital disease resulting in mental retardation or blindness in the infant (Remington et al., 2001). Contamination and survival in the environment of Cryptosporidium, Cyclospora and Toxoplasma oocysts The potential for environmental contamination depends upon a variety of factors including the geographic distribution of the parasite, number of infected human and non-human hosts, seasonal influence and duration of infection, the number of transmission stages excreted, agricultural practices, host behaviour and activity, socioeconomic and ethnic differences in human behaviour, and sanitation. Cryptosporidium oocysts have been detected in a variety of environmental matrices including farmyard manure, leachate, slurry, and soil. Also various water sources and foodstuffs (Rose and Slifco, 1999). The thick, two layered wall of Cryptosporidium oocysts ensures that oocysts are robust and resistant to a variety of environmental pressures particularly under cool, moist conditions. For example extremes of temperature and dehydration including freeze-drying, exposure to temperatures above 60°C and below –20°C for 30 minutes will all kill Cryptosporidium (Anderson, 1985), as will brief pasteurization (Harp et al., 1996). According to Blewett (1989) oocysts are killed by five minutes of exposure to moist heat of at least 60°C. Survival in manure heaps and slurry stores is adversely affected by the pH, temperature and ammonia characteristic of these environments (Jenkins et al., 1998). Although many common disinfectants used on farms, in hospitals or veterinary surgeries have little effect on Cryptosporidium, both hydrogen peroxide and ammonia inactivate oocysts (Casemore et al., 1989). Cyclospora oocysts have been detected in environmental samples, including water, wastewater and foods. Oocysts leave their host in an unsporulated non-infective form. In order to become infective they must

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sporulate for at least 12–14 days. Under appropriate conditions oocysts differentiate into two sporocysts, each containing two sporozoites and the rate of sporulation is probably influenced by environmental factors. However, it appears that sporulation dose not occur following exposure to –20°C for 24 hours or to 60°C for 1 hour thus rendering oocysts non-infective (Smith et al., 1997). Heating to 67°C kills toxoplasma tissue stages (tachyzoites and bradyzoites), but bradyzoites can survive refrigeration and some can survive freezing, maintaining infectivity after storage at –5°C. Oocysts of Toxoplasma are shed unsporulated in felid faeces, and after sporulation which requires some 1 to 5 days become infective. Oocysts of T. gondii have been detected in soil naturally contaminated with cat faeces (Ruiz et al., 1973) and in soil from gardens (Coutinho et al., 1982). According to studies conducted by Frenkel et al. (1975) oocysts may survive in soil for as long as 18 months. There is also evidence that oocysts can become distributed in the environment by mechanical spreading through the activities of invertebrate animals. However, there are only a few studies indicating that oocysts of Toxoplasma can survival in water; e.g. laboratory studies have shown they can survive for up to 4.5 years at 4°C (Dubey 1998). Survival for several months has been demonstrated in water, as has resistance to many disinfectants e.g. chlorine, freezing at –10°C, and drying, but they are killed by heating to 55– 60°C (Kuticic and Wikerhauser 1996). Morever, infections with T. gondii have been detected even in aquatic mammals-southern sea otters (Enhydra lutris nereis) and these imply that oocysts contaminating seawater can survive long enough for transmission to take place (Cole et al., 2000). The efficient transmission of cryptosporidiosis and cyclosporiosis is to a large extent dependent on the biological characteristics and especially the robust, and environmentally resistant oocysts of these parasites, which evolved specifically for this purpose. The potential risk of outbreaks of infection in human communities is dependent on precise local environmental and climatic conditions, and on farming practices (e.g. use of effluents to fertilise farm land) that facilitate access of oocysts to drinking water or result in contamination of food. Waterborne transmission is exacerbated particularly when climatic conditions affect the water resources of human communities (e.g. during periods of flooding) or when water treatment systems fail (e.g. employ inappropriate disinfectants or simply cannot cope with increases in human populations). This study was partly support by the Ministry of Scientific Research and Information Technology (KBN) grant 6P04C09721.

Literature A b r a h a m s e n M.S., T.J. T e m p l e t o n, S. E n o m o t o, J.E. A b r a h a n t e, G. Z h u, C.A. L a n c t o, M. D e n g, C. L i u, G. W i d m e r, S. T z i p o r i, G.A. B u c k, P. X u, A.T. B a n k i e r, P.H. D e a r, B.A. K o n f o r t o v, H.F. S p r i g g s, L. I y e r, V. A n a n t h a r a m a n, L. A r a v i n d and V. K a p u r. 2004. Complete genome sequence of the apicomplexan, Cryptopsoridium parvum. Science 304: 441–445. A n d e r s o n B.C. 1985. Moist heat inactivation of Cryptosporidium sp. Am. J. Public. Hlth. 75: 1433–1444. B l e w e t t D.A. 1989. Disinfection and oocysts, p. 107–115. In: Cryptosporidiosis: Proceedings of the First International Workshop. Agus K., D.A. Blewett (eds). Animal Disease Research Association, Edinburgh. C a s e m o r e D.P., D.A. B l e w e t t and S.E. W r i g h t. 1989. Cleaning and disinfection of equipment for gastro-intestinal flexible endoscopy. Gut 30: 1156. C h a l m e r s R.M., K. E l w i n, A. T h o m a s and D.H.M. J o y n s o n. 2002. Unusual types of cryptosporidia are not restricted to immunocompromised patients. J. Infect. Dis. 185: 270–271. Cole R.A., D.S. Lindsay, D.K. Howe et al. 2000. Biological and molecular characteristation of Toxoplasma gondii strains obtaned from southern sea otters (Enhydra lutris nereis). J. Parasitol. 86: 526–530. C o u t i n h o S.G., R. L o b o and G. D u t r a. 1982. Isolation of Toxoplasma from the soil during an outbreak of toxoplasmosis in a rural area of Brazil. J. Parasitol. 68: 866–868. D u b e y J.P. 1998. Toxoplasma gondii oocysts survival under defined temperatures. J. Parasitol. 84: 862–865. D u b e y J.P. and C.P. B e a t t i e. 1988. Toxoplasmosis of Animals and Man. CRC Press, Boca Raton. D u P o n t H., C.L. C h a p p e l l, C.R. S t e r l i n g, P.C. O k h u y s e n and W. J a k u b o w s k i. 1995. The infectivity of Cryptosporidium parvum in healthy volanteers. N. Engl. J. Med. 332: 855–859. F a y e r R., U. M o r g a n and S.J. U p t o n. 2000. Epidemiology of Cryptosporidium: transmission, detection and identification. Int. J. Parasitol. 30: 1305–1322. F r e n k e l J.K. 1973. Toxoplasmosis: parasite life cycle, pathology and immunology, p. 343–410. In: D. Hammond and P.L. Lond (eds). The coccdian. Eimeria, Isospora, Toxoplasma and related genera, University Park Press, Baltimore. F r e n k e l J.K., A. R u i z and M. C h i n c h i l l a. 1975. Soil survival of Toxoplasma oocysts in Kansas and Costa Rica. Am. J. Trop. Med. Hyg. 24: 439–443. G i r d w o o d R.W. and H.V. S m i t h. 1999. Cryptosporidium. In: R. Robinson, C. Batt (eds) Encyclopedia of Food Microbiology, p 946–954. Academic Press, London and New York.

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G u e r r a n t R.L., T. V a n G i l d e r, T.S. S t e i n e r, M.L. T h e l m a n, L. S l u t s k e r, T. H e n n e s s y, P.M. G r i f f i n, H. D u P o n t, R.B. S a c k, P. T a r r, M. N e i l l, I. N a c h a m k i n, I.B. R e l l e r, M.T. O s t e r h o l m, M.I. B e n n i s h and I.K. P i c k e r i n g. 2001. Practice guidelines for the management of infectious diarrhea. Clin. Infect. Dis. 32: 331–350. H a r p J.A., R. F a y e r, B.A. P e s c h et al. 1996. Effect of pasteurization on infectivity of Cryptosporidium parvum oocysts in water and milk. Appl. Environ. Microbiol. 62: 2866–2868. J e n k i n s M.B., D.D. B o w m a n and W.C. G h i o r s e. 1998. Inactivation of Cryptosporidium parvum oocysts by ammonia. Appl. Environ. Microbiol. 64: 784–788. K u t i c i c V. and T. W i k e r h a u s e r. 1996. Studies of the effect of various treatments on the viability of Toxoplasma gondii tissue cysts and oocysts, p. 261–265. In: Gross U. (ed.). Toxoplasma gondii, Springer-Verlag, Berlin. M a c K e n z i e W.R., N.J. H o x i e, M.E. P r o c t o r, et al. 1994. Massive waterborne outbreak of Cryptosporidium infecton associated with a filtered public water supply, Milwaukee, March and April, 1993. N. Eng. J. Med. 331: 161–167. M a r q u a r d t W.C., R.S. D e m a r e e and R.B. G r i e v e. 2000. Parasitology and vector biology. Harcourt Academic Press, San Diego, London, Boston, New York, Sydney, Tokyo, Toronto. R e m i n g t o n J.S., R. M c L e o d and G. D e s m o n t s. 2001 Toxoplasmosis, p. 205–346. In: J.S. Remington, J.O. Klein (eds), Infectious Diseases of the Fetus and Newborn, WB Saunders, Philadelphia. R e n o l d C., A. S u g a r, J.P. C h a v e et al. 1992. Toxoplasma encephalities in patients with the acquired immunodeficiency syndrome. Medicine 71: 224–239. R o s e J.B. and T.R. S l i f c o. 1999. Giardia, Cryptosporidium, and Cyclospora and their impact on foods: a review. J. Food Protect. 62: 1059–1070. R u i z A., J.K. F r e n k e l and L. C e r d a s. 1973. Isolation of Toxoplasma from soil. J. Parasitol. 59: 204–206. S h i e l d s J. M. and B.H. O l s o n. 2003. Cyclospora cayetanensis: a review of an emerging parasitic coccidian. Int. J. Parasitol. 33: 371–391. S m i t h H.V., C.A. P a t o n, M.M. M t a m b o et al. 1997. Sporulation of Cyclospora sp. oocysts. Appl. Environ. Microbiol. 63:1631–1632. S m i t h H.V. and J.B. R o s e. 1998. Waterborne cryptosporidiosis, current status. Parasitol. Today 14: 14–22. S o a v e R. and W.D. J o h n s o n. 1995. Cyclospora: conquest of an emerging pathogen. Lancet 345: 667:668. T e n t e r A.M., J.R. B a r t a, I. B e v e r i d g e, D.W. D u s z y n s k i, H. M e h l h o r n, D.A. M o r r i s o n, R.C.A. T h o m p s o n and P.A. C o n r a d. 2002. The conceptual basis for a new classification of the coccidian. Int. J. Parasitol. 32: 595–616. X i a o L., R. F a y e r, U. R y a n and S.J. U p t o n. 2004. Cryptosporidium taxonomy: recent advances and implications for public health. Clin. Microbiol. Rev. 17: 72–97.

Polish Journal of Microbiology 2004, Vol. 53, Suppl., 75–80

The Modulation of Transferrin Receptors Level on Mouse Macrophages and Fibroblasts by Toxoplasma gondii BO¯ENA DZIADEK, KATARZYNA DYTNERSKA-DZITKO and HENRYKA D£UGOÑSKA

Department of Immunoparasitology, Institute of Microbiology and Immunology, University of £ódŸ, Banacha 12/16 Str., 90-237 £ódŸ Abstract Macrophage-mediated early nonspecific immunological response is an important part of the immunity against intracellular parasite Toxoplasma gondii. The immunological functions of macrophages are closely connected with iron metabolism and acquiring of iron mainly from transferrin by the receptor-mediated endocytosis. The level of specific transferrin receptors can be modulated by different soluble exogenous and endogenous factors and also by microbial pathogens. The goal of our study was to determine the influence of T. gondii infection and toxoplasma lysate antigen (TLA) on the expression level of transferrin receptors (TfRs) on mouse macrophages and fibroblasts which can serve as host cells for the parasite replication. The level of TfRs was measured using CELISA assay. Strong down-regulation of the receptors level, started about 18 hours after infection of macrophages with a high number of freshly harvested tachyzoites T. gondii. Stimulation of the mouse cells with TLA antigen did not cause any changes in TfRs expression. In our studies we did not observe any differences in the TfRs level on mouse fibroblasts even after incubation with high concentrations of TLA antigen or inoculation with a high number of tachyzoites. K e y w o r d s: Toxoplasma gondii, transferrin receptors, macrophages functions.

Introduction An essential part of the immunity against intracellular parasite Toxoplasma gondii is early nonspecific T-cell independent immunological response mediated by macrophages, NK cells, dendritic cells and granulocytes. Macrophages are immune cells also responsible for the controlling of tachyzoite replication and the development of the later specific T-cell mediated response (Denkers and Gazzinelli, 1998; Denkers et al., 2004; Gazzinelli et al., 1996; Hauser and Tsai, 1986; Sher et al., 1993). The physiological and immunological functions of macrophages, like other mammalian cells, are strictly connected with iron metabolism. Changes in iron content affect macrophage microbicidal function and also macrophage-mediated cytotoxicity, for instance by involving iron in catalyzing the formation of highly toxic hydroxyl radicals via the Fenton reaction (Aisen et al., 2001; Weinberg, 2000). Most cells of vertebrate hosts acquire iron from the main serum iron-carrier, transferrin, by the receptormediated endocytosis. Transferrin is the physiological source of most of the iron required by different types of cells (Aisen et al., 1999; Qian and Tang, 1995). The uptake of iron on the transferrin-dependent pathway is initiated with the binding of iron-saturated protein (holo-transferrin) to the specific receptors on the cell membrane followed by the endocytosis of the receptor-holo-transferrin complex (internalization) and release of iron from the carrier-protein by a decrease in endosomal pH (Ponka and Lok, 1999; Qian and Tang, 1995). The transferrin receptors are membrane homodimeric glycoproteins with two subunits linked by two disulfide bonds and they are mainly responsible for the controlled cellular uptake of iron from transferrin (Ponka and Lok, 1999). With the exception of some cells, for example mature erythrocytes, resting T and B lymphocytes and circulating monocytes, transferrin receptors are expressed at various levels probably on all erythroid and non-erythroid cells including immune cells such as differentiated macrophages and activated Corresponding author: Bo¿ena Dziadek, Department of Immunoparasitology, Institute of Microbiology and Immunology, University of £ódŸ, Banacha 12/16 Str., 90-237 £ódŸ, phone: (042) 6354355, fax: (042) 6655818, e-mail: [email protected]

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lymphocytes (Galbraith and Galbraith, 1983; Ponka and Lok, 1999). The expression of the transferrin receptors on the macrophage surface could be modulated by various endogenous and exogenous stimuli. It was showed that J774 cells strongly suppressed the transferrin receptors expression after stimulation with IFN-( and LPS, and the observed down-regulation appeared to be NO-independent (Mulero and Brock, 1999). The decrease in the macrophage transferrin receptors synthesis was also found to be involved in the inhibition of multiplication of different microbial pathogens, like Legionella pneumophila and Mycobacterium avium (Byrd and Horwitz, 2000; Zhong et al., 2001). Additionally, it was noted that the intestinal epithelial cell line IEC-6 and rat primary enterocytes pretreated with IFN-( inhibited the intracellular replication of T. gondii and the observed inhibition was connected with the limitation of the availability of intracellular iron (Dimier and Bout, 1993; Dimier and Bout, 1998). The presented study documents that infection with parasite T. gondii which utilizes fibroblasts as well as macrophages as host cells for its replication could modulate the expression of transferrin receptors on the immune cells but not on fibroblasts. The observed down-regulation of the macrophage receptors expression might be an important mechanism of the macrophage-mediated host defense against protozoan parasite involved in the decreasing of intracellular iron and could result in the inhibition of T. gondii replication and even elimination of the pathogen. Experimental Materials and Methods Tissue culture media and reagents. Iscove’s medium (Sigma) supplemented with 5% FCS (Cytogen), 2 mM L-glutamine (Sigma), 50 :M 2-mercaptoethanol (Sigma), 100 U/ml penicillin and 100 :g/ml streptomycin (Polfa) for culturing of mouse fibroblast line L929; RPMI 1640 medium (The Institute of Immunology and Experimental Therapy PAN, Wroc³aw) with 10% FCS (Cytogen) or with 10% NCS (Sigma) with L-glutamine and antibiotics at concentration as above for culturing of mouse macrophages lines Ana-1 and J774, respectively; PBS without Ca 2+ and Mg2+ with 0.5% formaline for cell fixation. Cell lines. Murine fibroblasts line L929 obtained from the Institute of Medical Microbiology and Virology, University of Düsseldorf, Germany; two murine macrophages lines derived from two strains of inbred mice differing significantly in their natural resistance to toxoplasmosis: J774 (BALB/c origin) and Ana-1 (C57Bl/6 origin) received from the Institute of Medical Microbiology and Virology, University of Düsseldorf, Germany. Culture of Toxoplasma gondii BK (intraspecies subgroup I). Parasites were maintained by serial passage in confluent monolayers of mouse L929 fibroblasts grown in Iscove’s culture medium. Tachyzoites of T. gondii were harvested 3 or 4 days after infection, resuspended in culture medium appropriate for each cell line and after counting used for infection of murine macrophages or fibroblasts. Preparing of Toxoplasma Lysate Antigen (TLA). Washed twice and resuspended in PBS, tachyzoites of T. gondii were 10 times freeze-thawed in liquid nitrogen and at 37°C in warm water bath, respectively. Obtained extract was centrifuged at 10 000 × g (20 min, 4°C) to remove cellular debris and then the concentration of the protein was determined spectrophotometrically using Bradford standards. The average yield of the used procedure was 1 – 2 mg protein/10 9 parasites. The samples of TLA antigen were stored at –70°C and thawed directly prior to stimulation. CELISA assay. To detect expression of transferrin receptor (TfR) on mice fibroblasts (L-929) and macrophages (Ana-1 and J744) the cellular enzyme-linked immunosorbent assay (CELISA) was used. A total number of 2 × 10 6 cells was plated into tissue culture dishes ∅ 3 cm (Nunc) for 3 hours followed by incubation with four different concentrations (1, 3, 10 and 30 :g/ml) of TLA antigen or infection with freshly harvested tachyzoites of Toxoplasma gondii at the ratios 1:3, 1:10 and 1:30 parasites per mouse cell at 37°C in humidified atmosphere of 5% (RPMI 1640 medium) or 10% (Iscove’s medium) CO 2 in the air. Three, 6 or 18 hours postincubation the cells were collected using culture media, centrifuged at 65 × g for 10 min, washed and resuspended with PBS. After counting, the cells were fixed using 0,5% formaline (POCH) in PBS, aliquoted (1 × 10 4/well) into 96-well ELISA plate and dried at 37°C. Then the wells were blocked with 1% milk (Oxoid) at 4°C overnight and the rat IgG2a anti-mouse transferrin receptor monoclonal antibodies (obtained from postculture supernatants of hybridoma cell line R17.217.1.3., received from the Institute of Immunology, University of Mainz, Germany) were added. Incubation with primary antibodies was performed at 37°C for 2 h and followed by washing with PBS + 0.05% Tween-20 (Sigma). The reaction was developed using secondary peroxidaseconjugated goat anti-rat IgG + IgM polyclonal antibodies at dilution 1:2000 (Jackson ImmunoResearch Laboratories, Inc.) and ABTS [diammonium 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate), Sigma] as a chromogen. The absorbance values for all samples were determined at 8 = 405 nm using the automatic microtiter plate reader (Labsystems). The results are expressed as a mean of eight values at least from three independent experiments. Statisitcal analysis was performed by U Mann-Whitney test (p < 0.05).

Results Transferrin receptors expression on mouse fibroblasts and macrophages during T. gondii infection. In the study the CELISA assay was used to examine the changes in the expression of TfRs on murine fibroblasts and macrophages after T. gondii infection. To determine the ability of the parasite to modulate

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Transferrin receptors in toxoplasmosis 1.8 1.6

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Fig.1. Transferrin receptors level on mouse J774 (A) and Ana-1 (B) macrophages and L929 fibroblasts (C) after infection with Toxoplasma gondii BK.

K – control (no toxoplasms), 1:3, 1:10, 1:30 – the ratio of cells: Toxoplasma gondii; ▲ – 3 h; o – 6 h; ● – 18 h postinfection.

the level of expression, fibroblasts and Ana-1 macrophages were inoculated with viable T. gondii BK. Mouse J774 (Fig.1A) and Ana-1 (Fig. 1B) macrophages infected with a high number of the parasites at a ratio 1:30 displayed an essential decrease in TfRs level. A slight inhibition of TfRs expression on these cells occurred also after inoculation of cells with 10 tachyzoites per macrophage. A down-regulation of TfRs expression on mouse macrophages started about 18 hours postinfection; we did not find any changes in TfRs level 3 or 6 hours after inoculation of T. gondii. In the performed experiments we did not observe any alternation in the expression of TfRs on mouse fibroblasts (Fig. 1C) and there was no effect of low number of parasites (ratio 1:3) on the TfRs expression on mouse macrophages. Effect of TLA antigen on the expression of transferrin receptors. To determine the ability of T. gondii antigens to modulate the level of transferrin receptors, mouse macrophage lines J774 and Ana-1 were

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Fig.2. The expression of transferrin receptors on mouse J774 (■) and Ana-1 (●) macrophages after 18 h incubation with TLA antigen.

incubated with four different final concentrations (1, 3, 10 or 30 :g/ml) of the TLA for 18 h. The antigen stimulation of macrophages did not result in the changes of the expression of studied receptors compared to control unstimulated cells (Fig. 2). Even high concentrations of TLA antigen (10 and 30 :g/ml) used for experiments were unable to alter the transferrin receptors expression level on mouse macrophages. Discussion Toxoplasma gondii is an obligate intracellular pathogen that causes toxoplasmosis in many endothermic vertebrate hosts including humans. The essential part of the immunity against this parasitic infection is a nonspecific macrophage-mediated immunological response involved in the controlling of tachyzoite replication (Denkers and Gazzinelli, 1998). Iron is a crucial element that modulates the immunological functions of macrophages (Weinberg, 2000). On the other hand, iron is essential for multiplication of many bacterial and parasitic pathogens (Wilson and Britigan, 1998). Similarly to macrophages, pathogenic protozoa, such as Trypanosoma brucei, Trypanosoma cruzi, Leishmania spp. utilize mammalian iron-transporting protein, transferrin, as a source of iron during parasite replication in vertebrate hosts (Britigan et al., 1994; Lima and Villalta, 1990; Steverding et al., 1995). Recent research revealed that the possible way in which mammalian cells could inhibit the intracellular growth of T. gondii is limitation of the availability of intracellular iron, however, the mechanism of this limitation is not fully clear (Dimier and Bout, 1998). We examined the transferrin receptors (TfRs) level on mouse fibroblasts line L929 and macrophages line J774 and Ana-1 during infection with T. gondii BK or after stimulation with TLA antigen. We found that mouse macrophages but not fibroblasts inoculated with a high number of 30 tachyzoites per cell essentially decreased the expression of TfRs at 18 hours postinfection. The similar down-regulation of TfRs on mouse macrophages was noted during infection with M. avium. The treatment of the peritoneal cells with live mycobacteria resulted in a decreased TfRs level. Simultanously the expression of natural resistance-associated macrophage proteins Nramp1 mRNA and Nramp2 mRNA involved in the transporting of iron essential in the production of highly toxic hydroxyl radicals increased and correlated with the progress of infection (Zhong et al., 2001). These and our results could suggest that observed suppression in TfRs level on the immune cells during intracellular pathogens infections might be an important mechanism by which macrophages limit the availability of transferrin-associated iron. It would effect the growth of surviving parasite and result in the elimination of the pathogen. Also, experiments with human monocytes infected in vitro with L. pneumophila show that limiting of iron-saturated transferrin availability is crucial in the host antimicrobial immunological response (Byrd and Horwitz, 2000). Both macrophage lines used in our study responded very similarly to T. gondii infection what could suggest that the differences in natural susceptibility to toxoplasmosis between BALB/c (relatively resistant) and C57Bl/6 (relatively susceptible) mice are independent from the ability of macrophages to limit the TfRs expression. Contrary to the earlier study by Gail et al. (2001) we did not find any changes in the TfRs level on mouse fibroblasts. The dissimilarity in the obtained results could be a consequence of the different cell lines used

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for experiments and studying the TfRs expression by estimation of TfRs mRNA or very TfRs. Lack of any modification in fibroblasts TfRs expression might be related to the role of these cells in the organism. It is likely that opposite macrophages, the inhibition of transferrin-bound iron acquisition in fibroblasts did not result in the limitation of T. gondii replication. It could be connected with the presence of other proteins that serve as a source of intracellular iron as fibroblasts are very efficient permissive cells usually used in many laboratories to multiply toxoplasms in vitro. We did not find any influence of TLA antigen on the TfRs level on mouse macrophages. Inability of TLA to modify the TfRs expression might be related to the freeze-thawing procedure used for extracting the antigen. This technique is not sufficient for isolation of all, particularly membrane tachyzoite antigens families with a high yield. It is known that any surface T. gondii components (SAG and SRS-antigens, lectins, laminin) mediate adhesion of the parasite on the host cells (Boothroyd et al., 1998; Furtado et al., 1992; Grimwood and Smith, 1996; Jacquet et al., 2001; Manger et al., 1998; Ortega-Barria and Boothroyd, 1999) which could be correlated with the modification of the cellular mechanisms participating in the controlling of parasite replication. Moreover, stimulation with soluble toxoplasma antigens might be not adequate for TfRs modification that could require direct parasite-cell interaction. In conclusion, we postulate that down-regulation of TfRs by T. gondii infected-macrophages is one of the possible mechanisms responsible for inhibition of the parasite replication, and the observed decrease in TfRs level is time-dependent and correlated with progress of the infection. Literature A i s e n P., M. W e s s l i n g - R e s n i c k and E.A. L e i b o l d. 1999. Iron metabolism. Curr. Opin. Chem. 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