Polish Journal of Microbiology

POLSKIE TOWARZYSTWO MIKROBIOLOGÓW THE POLISH SOCIETY OF MICROBIOLOGISTS

Polish Journal of Microbiology 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)

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Polish Journal of Microbiology formerly Acta Microbiologica Polonica

2004, Vol. 53, No. 2

CONTENTS

MINIREVIEW

Classes and functions of Listeria monocytogenes surface proteins POPOWSKA M., MARKIEWICZ Z. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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ORIGINAL PAPERS

PCR-RFLP analysis of a point mutation in codons 315 and 463 of the katG gene of Mycobacterium tuberculosis isolated from patients in silesia, Poland WOJTYCZKA R.D, DWORNICZAK S., PACHA J., IDZIK D., KÊPA M., WYDMUCH Z., G£¥B S., BAJOREK M., OKLEK K.,

KOZIELSKI J. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Human papillomavirus (HPV) and Epstein-Barr Virus (EBV) cervical infections in women with normal and abnormal cytology SZKARADKIEWICZ A., WAL M., KUCH A., PIÊTA P. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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An attempt to protect winter wheat against Gaeumannomyces graminis var. tritici by using Rhizobacteria Pseudomonas fluorescens and Bacillus mycoides CZABAN J., KSIʯNIAK A., WRÓBLEWSKA B., PASZKOWSKI W.L.

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Induced resistance in tomato plants by IAA against Fusarium oxysporum lycopersici SHARAF E.F, FARRAG A.A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Optimization of carbon-nitrogen ratio for production of gibberellic acid by Pseudomonas sp. BAªIAÇIK KARAKOÇ S., AKSÖZ N. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Influence of DnaK and DnaJ chaperones on Escherichia coli membrane lipid composition SIEÑCZYK J., SK£ODOWSKA A., GRUDNIAK A. WOLSKA K.I. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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The carrier state of shiga-like toxin II (SLT II) and hemolysin-producing enteroaggregative Escherichia coli strain SOBIESZCZAÑSKA B.M., GRYKO R., DWORNICZEK E., KUZKO K. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Detection of CMV infected cells by flow cytometry – evaluation of MAbs CCH2 and AAC10 directed against early and late CMV antigens SIENNICKA J. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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BOOK REVIEW . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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INSTRUCTIONS TO AUTHORS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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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

Polish Journal of Microbiology 2004, Vol. 53, No 2, 75–88

Classes and Functions of Listeria monocytogenes Surface Proteins MAGDALENA POPOWSKA* and ZDZIS£AW MARKIEWICZ

Department of Bacterial Physiology, Institute of Microbiology, Warsaw University Miecznikowa 1, 02-096 Warsaw, Poland Received 15 March 2004 Abstract Listeria monocytogenes is an opportunistic pathogen that causes infections collectively termed listeriosis, which are related to the ingestion of food contaminated with these gram-positive rods. The pathogenicity of L. monocytogenes is determined by the following virulence factors: listeriolysin O, protein ActA, two phospholipases C, internalins (InlA and InlB), protein CwhA and a metalloprotease. The bacterium 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. Based on L. monocytogenes genome sequence analysis 133 surface proteins have been identified. In particular, the large number of proteins covalently bound to murein sets L. monocytogenes apart from other gram-positive bacteria. The ability of this pathogen to multiply in various environments as well as the possibility of its interaction with many kinds of eukaryotic cells is, in fact, made possible by the large number of surface proteins. K e y w o r d s: Listeria monocytogenes, virulence, murein, surface proteins

1. General characteristics of Listeria monocytogenes Listeria monocytogenes is a facultative anaerobe that grows best at 37°C and in neutral or slightly alkaline environment. 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). L. monocytogenes has also been detected in many food products (Schlech, 2000). The dissemination of the organism is related to the irrigation of rural areas with wastewaters, which results in its presence on vegetation and in products of animal origin. L. monocytogenes is pathogenic for people and animals and is the etiological factor of listeriosis, whose nature may be sporadic or epidemic. Infection is usually related to the ingestion of food products contaminated with the bacterium and for this reason listeriosis is considered a food-borne disease (Schlech, 2000). So far 13 serotypes of L. monocytogenes have been identified. On average, 90% of clinical infections are caused by serotypes Ia, Ib and IVb, the latter being the dominating type in Europe (Schlech, 2000). The most frequent forms of the disease are: meningoencephalitis, bacteremia and perinatal listeriosis. L. monocytogenes can also cause endocarditis, hepatitis, pleuritis, localized abscesses (e.g. in the brain) as well as muscular, skeletal and skin infections. Listeriosis is characterized by low incidence of the disease, but high mortality rate, 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 group of those at greatest risk embraces pregnant women, neonates, individuals over 60 years of age and people with impaired cellmediated immunity. The bacterial dose causing the disease in humans is not known, the infectious dose in mammals (monkey) is ≥109 cells (Farber, 1991). The incubation period for the disease in humans is from 11 to 70 days (Lorber, 1997). The pathogenicity of L. monocytogenes is largely determined by the following virulence factors (Portnoy et al., 1992): the internalins InlA and InlB, listeriolysin O (LLO), protein ActA, two phospholipases C * Corresponding author: e-mail: [email protected]

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Fig. 1. Physical and transcriptional organization of the central virulence gene cluster of L. monocytogenes. For explanations see text.

– a phospholipase specific for phosphatidylinositol (PI-PLC, PlcA) and phospholipase C specific for phosphosfatidylcholine (PlcB, lecithinase), and a metalloprotease. 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 (Fig. 1) (Chakraborty et al., 2000; VazquezBoland et al., 2001). The inlAB operon is located elsewhere (Lecuit et al., 2001). Moreover, such other determinants as protein CwhA (known earlier as p60 or Iap) (Park et al., 2000), catalase, superoxide dismutase, siderophores and protein LmaA are also required for full pathogenic activity of the bacterium. 1.1. Intracellular growth of Listeria monocytogenes L. monocytogenes is a model organism in studies on the pathogenesis of intracellular parasites. It is able to penetrate the cytoplasmic membrane of various eukaryotic cells, such as fibroblasts, epithelial cells, kidney cells and macrophages, grow in them and spread from one cell to another. Detailed studies of these events was possible as a result of the ability of L. monocytogenes to grow in macrophage cell lines. The individual stages of the life cycle of L. monocytogenes were first described in detail by Tilney and Portnoy (Portnoy et al., 2002). The first phase in the infection of J774 macrophages involves phagocytosis of the bacterial cell, combined with disruption of the cytoplasmic membrane (Cossart et al., 2003). This process is mediated by internalins A and B. Almost immediately after internalization of the bacterial cell the phagosome formed combines with the lysozome to digest the enclosed bacterial cell. To avoid the maturation of the phagosome to the phagolysosomal stage, L. monocytogenes brings about the disruption of the phagosomal membrane. This is brought about by the low pH inside this compartment, which results in the activation of listeriolysin O (Dramsi and Cossart, 2002) that together with other secreted enzymes (phospholipase B and C) and proteases causes lysis of the phagocytic vacuole and escape of the microorganism to the cytoplasm, where it replicates. In the case of nonphagocytic cells, a zipper-like mechanism is involved in the uptake of L. monocytogenes, in that the bacterium sinks into diplike structures on the host cell surface, until is finally engulfed (Cossart, 2002). While replicating in the cytosol, the cells become covered with actin filaments (Cossart and Lecuit, 1998), which rearrange to form relatively long (up to 40 mm) comet-like tails at one end of the bacterial cell. Some of the cells move towards the plasma membrane where they induce the formation of protrusions or pseudopods (listeriopods) that penetrate neighboring cells and are in turn engulfed by phagocytosis. These events result in the formation of a secondary phagosome surrounded by two membranes, with the inner one originating from the mother cell (Dabiri et al., 1990). The activity of listeriolysin O and phospholipase C (Smith et al., 1995) specific for phosophatidylocholine results in lysis of the newly formed vacuole and the listeriae escape to the cytoplasm where they can repeat their life cycle (Portnoy et al., 2002). 1.2. Therapy of listeriosis and prophylaxis Recommendations pertaining to choice of drug and duration of therapy in patients with listeriosis are based on results of studies with animal models, susceptibility of L. monocytogenes to antibiotics in vitro and the results of clinical trials embracing relatively small groups of affected individuals. L. monocytogenes is susceptible to most $-lactam antibiotics, except for the cephalosporins (Hof et al., 1997). However, new generation cephalosporins are commonly used to treat non-specific symptoms of bacteremia or in the therapy of meningoencephalitis, and for this reason treatment of listeriosis can sometimes be delayed until identification of the causative agent in blood sample or cerebrospinal fluid. The combination of ampicillin (or penicillin) and gentamicin is considered the therapy of choice in the treatment of listeriosis (Jones and MacGowan, 1995; Temple and Nahata, 2000). Ampicillin is considered bacteriostatic towards L. monocyto-

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genes cells but in concentrations achieved in the cerebrospinal fluid shows delayed bactericidal activity (within 48 hours) (MacGowan et al., 1998; Teuber, 1999). An alternative drug for patients intolerable to penicillin is co-trimoxazole (a combination of trimethoprim and sulfamethoxazole). Better outcomes in the case of patients with meningoencephalitis were observed when amoxicillin was used together with cotrimoxazole, compared to the combination of ampicillin and gentamicin (Merle-Melet et al., 1996). Erythromycin has also been found effective, especially in the case of pregnant women (MacGowan et al., 1998). 1.3. Features of the Listeria genome The complete nucleotide sequences of Listeria monocytogenes EGD genome (Acc. No AL591824) and a strain of the non-pathogenic L. innocua (Acc. No AL592022) were published in 2001 (Glaser et al., 2001). The G+C content of the chromosome is 39% for L. monocytogenes EGD. In the case of strain EGD 54 regions with lower, and 6 with higher G+ C content were also determined. It is suggested that the regions with different G+ C content than the rest of the chromosome were acquired via lateral transfer from other bacterial species (Glaser et al., 2001). Analysis of these regions shows that they code virulence and factors and surface proteins implicated in the adaptation and life cycle of the pathogen, which is able to live in various environmental conditions and different host cells (Buchrieser et al., 2003). All identified open reading frames (ORFs) take up 90.3% of the chromosome of both Listeria species. About 9.5% of the genes are regarded as specific for L. monocytogenes, and these genes code proteins related to virulence and adaptation to varied environmental conditions, Genes specific for L. innocua approximate 5%. For 35.3% genes of L. monocytogenes no potential function has yet been determined. L. monocytogenes has more surface proteins (with LPXTG sequence motif) than other gram-positive bacteria (Table I). 41 L. monocytogenes proteins with the LPXTG motif have been identified, 19 of which simultaneously have the LRR domain. Also, 7.3% of all L. monocytogenes genes, that is 209, are transcription regulators. The best-characterized regulator that activates many known virulence genes in L. monocytogenes, is protein PrfA, which is not present in L. innocua. In both Listeria genomes there is a large number of genes coding surface and secreted proteins, transporters and transcription regulators, which allow the bacterial cell to adapt to changing environmental conditions. The main difference between the two species lies in their surface protein composition (Buchrieser et al., 2003). Table I Surface proteins of several gram-positive bacterial species Genome size (Mb)

Proteins with LPXTG motifs

Proteins with GW modules

Listeria monocytogenes

2.94

41

9

Listeria innocua

1.01

31

9

Bacillus halodurans

4.20

3

0

Bacillus subtilis

4.21

1

0

Lactococcus lactis

2.37

9

0

Staphylococcus aureus

2.88

17

1

Streptococcus pneumoniae

2.04

11

0

Streptococcus pyogenes

1.85

13

0

Bacterial species

2. Overview of murein structure Bacterial cells, with very few exceptions, have a cell wall, whose main component is murein (syn. peptidoglycan). This polymer is composed of glycan chains in which alternating N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid are $-1,4 bound (1 →4). The structure of the glycan chains is relatively conserved, though certain modifications are known. The pentapeptide of the precursor, which may become shorted on incorporation into murein, is composed of amino acids in configuration both L and D. The D-lactyl groups of muramic acid in murein of the cell wall are usually amidated with L-alanyl-(-D-glutamylL-diaminoacyl-D-alanine stem tetrapeptides (or tripeptides). The peptide part of murein is far more varied and its composition is species-specific, but such factors as age of the cells and even growth conditions

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Fig. 2. Structure of the disaccharide-pentapeptide monomer of L. monocytogenes murein

may play an important role. In the murein, neighboring peptide chains may be peptide-bound either directly, between the diamino amino acid of one chain and usually D-alanine in position 4 of the other chain (4→3 mureins), or indirectly via a shorter or longer interpeptide bridge. Some bacteria, like Mycobacterium tuberculosis, have (3→ 3) mureins with crosslinks between adjacent meso-diaminopimelic acid (mDAP) residues, whereas others have mureins in which both (4 → 3) and (3 → 3) crosslinks occur. The crosslinkage of the murein of gram-negative is usually 20–30%, whereas in the case of gram-positive bacteria this value is much higher and can approach 100%. The primary structure of the murein of L. monocytogenes murein resembles that of Escherichia coli and, like E. coli murein, contains (m-DAP) acid as the diamino amino acid in the peptide side chain (Fig. 2). Also, like in the case of E. coli, both (4 → 3) and (3 → 3) crosslinks occur. Other modifications of L. monocytogenes murein include amidation of free (m-DAP) residues and partial de-N-acetylation of glucosoamine (this laboratory, unpublished). Also, typically for gram-positive bacteria, the cell wall of L. monocytogenes contains lipoteichoic acids, which in the case of the best studied serotype 1/2 are composed of polyribitol phosphate with N-acetylglucosamine and rhamnose substituents on ribitol and are bound to the muramic acid moiety of murein, lipoteichoic acids and numerous proteins, described in detail below, that are either anchored in the cytoplasmic membrane or cell wall or peptide-bound to the murein (Baba and Schneewind, 1998). 3. Proteins in the gram-positive cell wall Proteins are rather common in the cell wall of gram-positive bacteria where they can carry out many diverse functions. Their number, as well as functions, differ depending on given species and even strain. They can be involved in the adherence of the bacterial cells, protect them from phagocytosis by leukocytes, take part in motility and some of them show enzymatic activity. There are several known mechanisms of the exposure of these proteins on the cell surface and the nature of their interaction with the cell wall in grampositive bacteria. Each of these mechanisms depends on the nature of a given protein and the role it plays in the cell (Navarre and Schneewind, 1999). 3.1. Surface proteins of Listeria monocytogenes Analysis of the genome sequence of L. monocytogenes has allowed to determine the number and types of surface proteins of this bacterium (Cabanes et al., 2002), which are more abundant than in any other bacteria with known genome sequence. Among the L. monocytogenes surface proteins it is possible to distinguish

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Fig. 3. The major types of surface proteins found in L. monocytogenes: Proteins with LPXTG carboxy-terminal sorting signal; proteins anchored by a hydrophobic tail motif; proteins containing GW modules; lipoproteins. LTA – lipoteichoic acid, TA – teichoic acid. For explanations see text.

three major groups of proteins, that are characterized by specific structural features (Fig. 3). These are: 1. Proteins covalently linked to murein through their C-terminal domain (proteins with LPXTG sequence motif), 2. Proteins non-covalently bound by their C-terminal domain (GW proteins, hydrophobic tail proteins, CwhA-like proteins) and 3. Proteins linked to cell wall structures via their amino-terminal region (lipoproteins) (Dhar et al., 2000). 3.2. Proteins covalently linked to cell wall murein 3.2.1. Proteins containing the LPXTG motif It seems, at least so far, that the only mechanism allowing covalent binding of surface proteins to the cell murein of gram-positive bacteria requires a specific C-terminal signal (Fischetti et al., 1990), i.e. the LPXTG motif, which is strongly conserved and followed by a hydrophobic stretch of roughly 20 amino acids and a tail of positively charged amino acids. This sorting sequence keeps the protein in the cytoplasmic membrane until it is cleaved off between the TG residues of the LPXTG motif, followed by the formation of a peptide bond between threonine and diamino amino acid of murein. This reaction is catalyzed by a protein that has been termed a sortase (Navarre and Schneewind, 1999). The LPXTG motif has been found in over 100 bacterial surface proteins (Table I). The number of proteins of this type in L. monocytogenes (Buchrieser et al., 2003) is much higher than in many other grampositive species, e.g. 17 in Staphylococcus aureus (Kuroda et al., 2001), 13 in Streptococcus pyogenes, 11 in S. pneumoniae, 9 in Lactococcus lactis and 3 in Bacillus subtilis (Kunst et al., 1997). 3.2.2. Proteins with LRR domain The first identified protein of this group, which at the same time contains the LPXTG motif was internalin InlA (Jonquieres et al., 1998). The second protein (no LPXTG motif) was InlB (Jonquieres et al., 1999). inlA and inlB are the most thoroughly studied members of the so-called internalin multigene family. The genes are located in operon inlAB, which is transcribed from 4 promoters, only one of which is under the control of the regulator PrfA (Dramsi et al., 1997). Genetic inactivation of either inlA or inlB leads to a significant decrease in the virulence of L. monocytogenes.

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Fig. 4. Schematic domain organization of full-length InlA, InlB, InlC and InlH from L. monocytogenes. Homologous regions are the same sign coded. SS – signal sequence; cap – the cap domain; LRR – LRR domain; IR – inter-repeat-region; BR – B-repeats; Cws – cell wall spanning region; MA – membrane anchor; CR – C-repeats. Numerals indicate number of tandem repeats in the LRR domain. For explanations see text

Four other internalin-like proteins were identified more recently: InlE, InlF, InlG and InlH (Engelbrecht et al., 1998; Raffelsbauer et al., 1998). However, otherwise than internalin InlA, these four proteins are not involved in invasion but are important for colonization of host tissues in vivo (Portnoy et al., 2002). More recently, a new L. monocytogenes internalin-like protein, Lmo2026, has been identified, with a suggested role of the protein in invasion and multiplication in the brain (Autret et al., 2001). All these proteins belong to a large family which contains an amino-terminal LRR (leucine-rich repeat) domain, which is followed by a conserved IR (inter-repeat) region – a conserved region of repeats with similarity to immunoglobulin – several other repeats and the motif LPXTG (Fig. 4). It is not yet know whether the Ig-like domain plays any role in specific contacts with eukaryotic cell surface proteins or only has a role in stabilizing the LRR region. A comparison of the amino acid sequences of the known internalins indicates that the IR region is strongly conserved in these proteins (Schubert et al., 2002). The LRR and IR domains also occur in other bacterial proteins, which are mostly virulence factors. The fusion of these two domains in InlB and other internalins is an example of optimal adaptation of bacterial pathogens to their eukaryotic hosts in the course of evolution. Based on different lengths and consensus sequences in proteins from various kinds of cells at least 7 subfamilies of proteins with LRR repeats can be distinguished (Kajava and Kobe, 2002). Most of them contain a conserved N-terminal region with an LRR domain, preceded by a signal sequence - SS as well as an inter-repeat (IR) sequence that is like an immunoglobulin fold (Schubert et al., 2002). In some cases a second repeat region of up to three repeats of about 70 amino acids called B repeats may be present. The LRR domain of the listerial proteins consist of tandem repeats of 20–22 amino acids, being typical for the shorter bacterial sequences, with a large number of leucine or isoleucine residues. The C-terminal part of the internalin proteins contains a domain that anchors the protein on the cell surface or is absent, as in the case of secreted proteins. It has tandem ca 80 amino acid repeats that are highly basic and start with the dipeptide GW (Lecuit et al., 1997). The LRR domain is involved in many processes, such as adhesion, and signaling, for instance, but in general it can be said that it provides a versatile structural framework for the formation of protein-protein interactions. The consensus sequence of the 20 amino acids, being the repeat in the LRR domain has been determined: LXXLXLXXNXIXXIXXLXXL (Kajava and Kobe, 2002; Schubert et al., 2002). Genome sequence studies have shown that the internalin multigene family of L. monocytogenes consists of 24 genes. Besides five internalins that have been known for some time, 14 ORFs have been identified in the genome sequence that code for proteins with a signal sequence, an LRR domain and an LPXTG motif. Thirteen of these proteins have a second repeat domain and 6 the conserved IR domain. The large number of listerial proteins with the LRR domain and LPXTG motif distinguishes L. monocytogenes from among other bacteria. In addition, the L. monocytogenes genome codes for 5 proteins which contain the LRR domain but do not have the LPXTG motif. One of these is InlB, which contains the GW sequence and attaches to the bacterial cell surface by a different mechanism (Cabanes et al., 2002). Four other proteins are secreted proteins with the domain LRR, which include InlC (Dramsi et al., 1997; Engelbrecht et al., 1998). Internalin A Internalin A (InlA) is composed of 800 amino acids and is required for the entry of L. monocytogenes into only a few types of cells, including cells of the eukaryotic line Caco-2. The N-terminal part of the

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protein contains a typical for all internalins LRR region, followed by the IR region, but its C-terminus contains two and a half repeats of 75 amino acids, followed by a region that contains the LPXTG motif. Amino acids from 36 to 78 form a domain composed of three "-helixes – a cap domain. The LRR domain contains 15 and a half repeats of a 22 amino acid sequence, followed by a Ig-like domain between 415 and 495 aa. In terms of structure, this region is the most elastic part of the internalin domain, and when comparing InlA, InlB and InlH, only small differences in this region can be observed. The surface receptor for InlA is E-cadherin – or more specifically its extracellular part – which is expressed in epithelial cells (Jonquieres et al., 1998; Schubert et al., 2002). E-cadherin is a transmembrane glycoprotein, which contains 5 extracellular adherin domains, a transmembrane "-helix and intracellular domain. Intracellular E-cadherin is involved in the process of the formation of the actin cytoskeleton. The extracellular N-terminal domain of E-cadherin (EC1) is responsible for specific trans-interactions between identical cadherins of progeny cells, and is also the site recognized by InlA (Schubert et al., 2002). InlA forms a complex with this domain and specifically rearranges it. The reaction is Ca-dependent and involves Pro16, which is important for intramolecular rearrangements. The substitution of Pro16 in human EC1 by glutamine results in immunity to bacterial invasion (Lecuit et al., 1999). The interaction between InlA and E-cadherin leads to phagocytosis and not adherence. Why this is so is not entirely clear and so far, L. monocytogenes is the only pathogen that has been found to use E-cadherin to enter into cells. Other bacteria as a rule use other adhesion molecules, such as integrins. Using truncated forms of internalin AS in the noninvasive L. innocua, it was established that the LRR and IR regions are sufficient to mediate attachment and phagocytosis (Lecuit et al., 1997). The carboxy-terminal part of internalin A contains the motif LPXTG, which is responsible for the anchoring of the protein in the cell wall. Small amounts of internalin A can also be found in the culture supernatant, but most of the molecules are anchored via covalent linkages in the cell wall. Internalin B Besides InlA discussed above, L. monocytogenes also uses InlB to exploit another mammalian signaling pathway to enter the cell. The LRR region at the N-terminus of InlB embraces 213 amino acids, from 36 to 242. Amino acids 1–35 form a signal sequence that is cleaved off, so in the mature protein the LRR domain takes up the whole N-terminus. This region contains a hydrophilic cap composed of two $- and three "-helixes and eight LRRs. InlB, like many other internalin proteins, also contains a B repeat. Although the whole domain, including the B repeat, is necessary for efficient internalization, the N-terminal 213 residue region is sufficient to induce the entry of bacteria into cells and to activate signal transduction pathways (Shen et al., 2000). The cap at the N-terminus resembles a calcium-binding domain in calmodulin and related proteins but whether it binds calcium in InlB is still the subject of controversy (Bierne and Cossart, 2002; Marino et al., 2002). The amino acid sequence in this region is strongly conserved in all known internalins. InlB, in contrast to InlA, is loosely attached to the cell wall and is partially released to the environment (Jonquieres et al., 1999). InlB has been found to bind to three different kinds of receptors on mammalian cells. The receptor first identified was gC1qR, a glycoprotein with molecular weight 33 kDa that acts as a receptor for C1q, the first component of the complement cascade and occurs mainly in mitochondria and in the nucleus but can also be found on the surface of eukaryotic cells and in body fluids (Braun and Cossart, 2000). Binding is mediated by the Gly-Trp repeats of InlB (Marino et al., 2002). The second receptor is known as Met, which is a tyrosine kinase receptor. Met is a heterodimer composed of an extracellular alpha subunit and transmembrane beta subunit. Met is recruited and phosphorylated at the site of listerial entry. Notably, it has been found that Met and E-cadherin (see InlA above) co-localize when co-expressed in cells. Finally, the third type of ligand recognized by InlB are glycosaminoglycans, which “decorate” the proteoglycans that occur on the surfaces of all types of mammalian cells (Jonquieres et al., 1999). In this case too, binding is mediated by the Gly-Trp repeats. For activation of the receptors the N-terminal part of InlB, consisting of 213 amino acids out of the 595 amino acids of the whole protein (67 kDa) is required. This region, which contains the LRR motif is necessary and sufficient to activate phosphatidylinositol 3-kinase and bring about cytoskeletal rearrangements (Lecuit et al., 1999). To elucidate this effect the X-ray crystal structure of this domain was studied (Schubert et al., 2002; Kajava and Kobe, 2002). It was found that Ca is bound within it in a specific manner that may allow the metal can act as a bridge between InlB and the receptor on the surface of a mammalian cell (Bierne and Cossart, 2002). An analogous system occurs in InlH, where the LRR domain is longer by one 22 amino acid repeat, and an identical Ca-binding region is present. The C-terminal domain is between amino acids 263 and 343. Although

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the involvement of InlH in the survival of L. monocytogenes in the course of mouse infection has been established, the mechanism and function of InlH in the course of infection have so far not been elucidated. Calcium binding in the region of "-helix 1 and "-helix 2 does not cause conformational changes and plays no structural role. The binding of magnesium ions by "-helix 1 in the absence of calcium ions has also been shown. The Ca-binding region has been postulated as the site of protein-protein interactions. The metal has been proven to be required in the process of adhesion to host cells, which is a fairly common mechanism in pathogens that induce phagocytosis. The C-terminal region of InlB contains three highly basic tandem repeats of about 80 amino acids, beginning with the dipeptide GW (Bierne and Cossart, 2002). The GW repeats are involved in loose association of the protein with the bacterial surface, mainly though non-covalent interactions with lipoteichoic acid present on the surface of most gram-positive bacteria. Interestingly, they also confer on InlB the unusual property of adhering to bacteria when added to the bacterial growth medium. Internalin C InlC is a secreted protein with molecular weight of 30 kDa with five consecutive leucine-rich repeats, which is required for the full virulence of L. monocytogenes in the mouse cell infection model. Similar small, secreted internalins were identified in the culture supernatant of L. ivanovii. The expression of gene inlC occurs via two different modes – independent of the transcription activator PrfA and dependent on it. The main promoter from which inlC is transcribed is strictly dependent on PrfA and contains a PrfA-binding site at position –40 from the transcription start site. The gene inlC is not located adjacent to other virulence genes and is flanked by two so-called housekeeping genes, rplS and infC. In L. ivanovii gene i-inlC with 92% similarity to inlC has been identified. InlC, like InlA and InlB, contains the typical LRR motif (Engelbrecht et al., 1998). No InlC-binding receptor has yet been found on the surface of eukaryotic cells, but it is equally possible that such a receptor may occur inside the host cells, especially since the synthesis of this particular internalin is preferentially induced in the cytosol of mammalian cells. 3.3. Proteins with the RGD motif The L. monocytogenes protein Lmo1666, besides the LPXTG motif and 10 PKD repeats contains a RGD (Arg-Gly-Asp) motif (Cabanes et al., 2002). This motif has been found in proteins participating in adhesion to eukaryotic cells and has been shown to be the core recognition sequence for many integrins. They are present in a variety of integrin ligands, including pathogen surface proteins from Leishmania and Bordetella pertussis (Finlay and Cossart, 1997). These data may indicate the role of protein Lmo1666 in the invasion of the host cells. Besides protein Lmo1666, the RGD motif has also been found in two other L. monocytogenes surface proteins: ActA and in a lipoprotein with unknown function, Lmo0460. The role of the RGD sequence in the former is unknown but probably does not mediate the attachment of L. monocytogenes to the host cell (Alvarez-Dominguez et al., 1997). 3.4. Proteins with PKD repeats Eleven L. monocytogenes proteins with the motif LPXTG contain PKD repeats at their C-terminal end (Cabanes et al., 2002). The domain PKD was first identified in the human protein polycystin-1 (PKD1) encoded by gene PKD1, whose function is unknown (Huan and van Adelsberg, 1999). The protein contains a signal peptide, the LRR domain, lipoprotein A module (LDL-A), a calcium-dependent domain and 16 PKD repeats consisting of about 80 amino acids each. PKD domains have also been found in extracellular regions of proteins from higher organisms, bacteria and archeons. It has been shown that domain PKD contains an Ig-like fold and this type of domain has been shown to form ligand binding sites in cell surface proteins (like in the case of IR sequences of internals – see above), and it is therefore suggested that PKD domains carry out a similar function. Two internalin proteins of L. monocytogenes Lmo0331 and Lmo0333 contain both LRR and PKD domains, which may indicate their role in adhesion or signaling. 3.5. Sortases Surface proteins with the LPXTG motif are anchored in the cell wall by specific cysteine proteases that have been termed sortases (Cossart and Jonquieres, 2000). The first studied sortase of Staphylococcus aureus is a 206 amino acid protein, with a potential N-terminal signal peptide that could also act as a membrane

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anchor (Ilangovan et al., 2001). It also has a cysteine at position 184, within a catalytic TLXTC signature, which has been shown to be critical for the functioning of the enzyme. Sortase recognizes the sequence LPXTG of proteins translocated through the cell wall, catalyzes its proteolysis and subsequent covalent attachment of the protein to murein. The purified enzyme catalyzes the formation of a hydroxylaminesensitive acyl enzyme intermediate, which in the presence of murein precursors allows a transpeptidation reaction to proceed (Ton-That et al., 1999). Sortase thus has both protease and transpeptidase activities. The enzyme cleaves the sequence LPXTG between threonine and glycine and the carboxyl group of threonine is then amide-linked to the amino group of cross-bridges within murein precursors. In S. aureus the pentaglycine interpeptide bridge is covalently bound to the lysine of the side peptide of the cell wall murein. This general scheme applies to all proteins covalently linked to murein but since the structure of bacterial murein is species-specific, the LPXT part of the protein may bind differently in the case of different bacteria. For example, Streptococcus pyogenes murein contains a dialanine bridge to which the protein binds, whereas in L. monocytogenes the binding has been found to be between the threonine of LPXT and m-(DAP) acid (Cossart and Jonquieres, 2000). The sortase genes of various bacteria are frequently located next to the gene that codes for their substrate. Analysis of the genome sequence of L. monocytogenes has identified two sortase genes, one being similar to srtA of Staphylococcus aureus, and consequently also termed srtA and the other srtB, at a distance of 1300 kb from srtA in the Listeria genome (Garandeau et al., 2002). The sequence around cysteine 184 (based on S. aureus numbering) in the L. monocytogenes SrtA was strikingly conserved compared to the S. aureus sortase with the TLXTC motif being evident. The enzyme is 222 amino acids long and in the L. monocytogenes genome its structural gene is flanked by genes coding for proteins similar to Bacillus subtilis ORFs, Yhfl of unknown function and YxiJ, a 3-methyladenine DNA glycosylase. The construction of a L. monocytogenes mutant lacking functional SrtA sortase showed that the enzyme is indispensable for the anchoring of the invasion protein internalin A to murein. The mutation also prevented the proper sorting of several other murein-anchored proteins with the LPXTG motif. The srtA mutant is defective in entering epithelial cells and its virulence in the mouse model is strongly attenuated. The second L. monocytogenes sortase gene, srtB, is adjacent to two genes coding LPXTG proteins (Bierne et al., 2004). The enzyme is 246 amino acids long and is 23% identical to SrtA, which is lower than could be expected. It too, contains the TLXTC sequence and putative signal sequence region but in addition contains two stretches of 13 and 31 amino acids that are not present in SrtA. The second S. aureus sortase, SrtB, is needed to anchor proteins with the NPQTN motif. Gene srtB is part of an iron-regulated region, which carries genes for surface proteins with the LPXTG motif and the NPQTN domain and an iron transporter gene (Jonsson et al., 2003). In L. monocytogenes gene srtB is also part of an area that codes for LPXTG proteins and an iron transporter but does not contain any gene for proteins with the NPQTN motif. Moreover, it is not known whether this region is subject to similar regulation. These and other observations suggest the existence of two, if not more, distinct families of sortases in gram-positive bacteria (Paterson and Mitchell, 2004). 3.6. Proteins non-covalently linked to the cell wall 3.6.1. Proteins interacting with LTA A prime example of this type of protein is L. monocytogenes internalin B, which has been described in detail above. It interacts with lipoteichoic acids (LTAs) via the GW modules (dipeptide Gly-Trp) at its C-terminal end (Bierne and Cossart, 2002). The conserved GW modules also interact with glycosaminoglycans on mammalian cells. InlB is partly anchored in the wall, using lipoteichoic acid as a ligand (Jonquieres et al., 1999). This attachment is relatively weak and consequently internal B can be released from the cells on incubation in InlB Tris/HCl with high concentration, even though the enzyme cannot be released from the cell wall after its digestion with muramidase. Eight GW modules are also present in the well-characterized cell surface amidase of L. monocytogenes. The larger number of modules presumably allow for tighter binding to the cell surface (Navarre and Schneewind, 1999). Genome sequence analysis has identified a further seven other proteins in L. monocytogenes containing the GW motif. Interestingly, six of them, similarly to Ami (Lmo2558) (Jacquet et al., 2002) contain the amidase domain (Lmo1215, Lmo1216, Lmo2203, Lmo1521, Lmo2591, lmo1076). Internalin InlB is the only protein of this group that has both GW modules and an LRR domain. In L. innocua there are 9 GW proteins and GW modules similar to the listerial ones have been found in the autolysin Atl from Staphylococcus aureus (Oshida et al., 1995) and in three other surface murein hydrolases: AtlC from Staphylococcus caprae, AtlE from S. epidermidis and Aas from S. saprophyticus (Heilmann et al., 2003).

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These four autolysins share the same organization, two amidase domains linked GW modules (Cabanes et al., 2002). These observations may suggest that the mechanism of autolysin anchoring on the cell surface using specific GW modules is common to staphylococci and listeriae (Baba and Schneewind, 1998). 3.6.2. Cwha and Cwha-like proteins L. monocytogenes protein CwhA (previously Iap or p60) participates in the invasion of host cells but also has murein hydrolase activity and has been shown to be participate in cell division (Park et al., 2000). The authors of an early paper maintained that a mutation in the structural gene for CwhaA (iap) was lethal for L. monocytogenes (Wuenscher et al., 1993). However, subsequent reports (Wisniewski and Bielecki, 1999; Pilgrim et al., 2003) showed that the deletion of iap was not lethal but led, amongst others, to impaired cell division and actin-based motility. Protein CwhA contains two LysM domains, a SH3 domain (bacterial Src homology 3) and the C-terminal domain NLPC/P60. Domain LysM is present in many enzymes degrading cell wall murein. The main function of this domain is binding to murein. The bacterial SH3 domain (SH3b) is homologous to eukaryotic SH3 domains and is also found in CwhA-like proteins in other Listeria species (Whisstock and Lesk, 1999) as well as in other bacteria, such as Bacillus subtilis, Escherichia coli, Chlamydia trachomatis, Haemophilus influenzae, Helicobacter pylori, Staphylococcus aureus and Streptococcus pyogenes. The function of this domain has not yet been determined but the murein hydrolase lysostaphin from Staphylococcus simulans contains a C-terminal SH3 domain which is responsible for the attachment of the protein to the cell wall (Baba and Schneewind, 1998). This observation suggests such a role for other bacterial proteins containing the SH3b domain. The NLPC/P60 domain (100–110 amino acids), was first determined in Cwha and in the E. coli lipoprotein precursor NlpC. The function of this domain is not clear but it has since been found in several other lipoproteins and bacterial surface proteins. A search for the NLPC/P60 domain in the L. monocytogenes genome revealed a similar sequence in three other proteins, one of which, termed P45, has been characterized as a protein with murein hydrolyzing activity. The enzymes is both present on the cell surface of L. monocytogenes and secreted to the growth medium of the bacterium. For this reason the gene encoding it has been named spl (secreted protein with lytic property) (Schubert et al., 2000). Proteins cross-reacting with a monoclonal antibody that reacts with P45 were present in strains of all seven Listeria species investigated, except L. grayi. Protein Lmo0394, besides the NLPC/P60 domain also contains a SH3 domain. The third listerial protein containing the NLPC/P60 domain is Lmo1104. CwhA-like proteins have also been found in L. innocua, as well as in other gram-positive bacteria, such as B. subtilis, L. lactis, S. aureus, M. leprae and M. tuberculosis where they presumably function as murein hydrolases (Cabanes et al., 2002). 3.6.3. Proteins with hydrophobic tail Eleven proteins of L. monocytogenes have a carboxyl terminus consisting of a hydrophobic domain, followed by positively charged amino acids and this „tail” serves to attach the proteins to the bacterial cell surface (Domann et al., 1992). The best studied of these proteins is ActA, which plays a crucial role in actin-based motility of listeriae since it is involved in the polymerization of actin fibers in the host cell. Expression of the ActA polypeptide is controlled by the PrfA regulator protein and its structural gene is located between the metalloprotease (mpl) and phosphatidylcholine-specific phospholipase C (plcB) genes. (Pistor et al., 1995). The C-terminal region of the protein contains a hydrophobic stretch of 22 amino acids followed by a tail with positive charge. Protein L. monocytogenes SvpA (Lmo2185) – described in 2001, is also attached to the cell surface by means of a hydrophobic tail, similarly to ActA and is required for the escape of L. monocytogenes from the phagosome formed inside a macrophage which is indispensable for the intracellular survival of the bacterial and implicates the protein in virulence (Borezee et al., 2001). The L. monocytogenes genome encodes 9 other proteins (Lmo0058, Lmo0082, Lmo0528, Lmo0552, Lmo0586, Lmo0701, Lmo0821, Lmo2061 and Lmo2186) with C-terminal hydrophobic region. Protein ActA and Lmo0082 are the only ones of this group that are not present in L. innocua (Buchrieser et al., 2003). 3.7. Lipoproteins Bacterial lipoproteins are characterized by a specific signal peptide, which as a rule is shorter than the “classical” signal sequences, contains more hydrophobic amino acids in its central region and is followed by a cysteine. These proteins are synthesized in the form of a prolipoprotein, which is then cleaved by a lipoprotein-specific peptidase (proliprotein peptidase or peptidase II), to produce the mature form of the

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protein. Lipoproteins can be anchored by their fatty acids in the cytoplasmic membrane, though free periplasmic forms are known, e.g. in Escherichia coli. Moreover, sometimes, like in the case of the E. coli lipoproteins, known as Braun’s lipoprotein, the peptide part of the lipoprotein can be covalently attached to the diaminopimelic acid of the murein. Bacterial lipoproteins are involved in initiation of the immune response in mammalian cells (Aliprantis et al., 1999). They are capable of activating many types of cells, including monocytes, macrophages, neutrofils and B cells, thus playing a significant role in pathogenesis. In L. monocytogenes 68 genes (that is 2.5% of all L. monocytogenes genes) coding lipoproteins have been identified. The number of these genes in L. monocytogenes is the highest among all pathogenic gram-positive bacteria, e.g. M. tuberculosis codes for 65 lipoproteins (1.7% of all genes). In the case of the Mycoplasma pneumoniae and M. genitalium genomes 6.8% and 4.5% of all open reading frames, respectively, code for lipoproteins (Himmelreich et al., 1996), though of course the size of the genomes of these two species chromosomes is very much smaller than that of L. monocytogenes. Only a few of L. monocytogenes lipoproteins have been studied and their putative roles identified. Lipoprotein TcsA has been found to activate T cells (Sanderson et al., 1995), whereas lipoprotein OppA (Borezee et al., 2000), similarly to its counterparts in many other bacterial species, e.g. Escherichia coli, participates in the transport of oligopeptides and is required for growth of the cells at low temperature. Other L. monocytogenes lipoproteins that can participate in host cell-pathogen interactions, like Lmo1847 and Lmo1800, have been identified but their specific roles remain unclear. The latter is likely a tyrosine phosphatase. 3.8. Diverse proteins similar to surface proteins of other gram-positive species L. monocytogenes protein, Lmo1799 that contains the LPXTG motif also contains 226 Ala-Asp (AD) tandem repeats. Proteins with Ser-Asp (SD) repeats have been found in bacteria belonging to the genus Staphylococcus. In S. aureus, CifA that binds to fibrinogen contains a SD repeat region which is predicted to span the cell wall and expose the ligand on the outer surface of the bacterium. The AD repeats in Lmo1799 could play a similar role, though the role of the putative protein in L. monocytogenes remains to be elucidated. Another L. monocytogenes protein containing the LPXTG motif, Lmo0842, is present also in L. innocua and shows similarity to other proteins, R28 from Streptococcus pyogenes, Rib from S. agalactiae and Esp from Enterococcus faecalis (Shankar et al., 1999). All four proteins contain the motif LPXTG and 6 to 12 repeats of about 80 amino acids that show high sequence identity (up to 42% identity). R28 is an adhesin that may play a role in virulence. Esp is a surface protein with as yet unknown function, which participates in the formation of biofilms by E. faecalis. The putative protein Lmo0842 could thus play a role in virulence and/or the formation of biofilms. 4. Conclusions The L. monocytogenes genome is unusual, compared to most other bacteria, in that 4.7% of all its genes code for a total of 133 surface proteins. The genes encoding these proteins are predominantly located in the first 25% of the L. monocytogenes genome, which region would seem to have higher plasticity and greater capacity for accommodating lateral gene transfer. However, the answer to the question why this particular region stands out is, at this stage, purely speculative. The large number of surface proteins and varied systems of their anchoring in the cell wall is very likely related to the ability of L. monocytogenes to survive in a broad range of environmental conditions that are frequently detrimental, and to interact with diverse types of eukaryotic host cells. Surface proteins play an important role in interactions between a microorganism and its environment as well as in the consecutive stages of the infection process. Among the main virulence factors of L. monocytogenes are the surface proteins internalin B, which is required for the induction of phagocytosis and penetration of the bacteria into macrophage cells, and protein ActA, which stimulates the accumulation and polymerization of actin into fibers in the host cell and subsequently enables the movement of L. monocytogenes in the cytoplasm and the migration of the bacteria from one eukaryotic cell to another. Most of the known virulence genes of L. monocytogenes are regulated by protein PrfA – a transcription regulator that binds to the palindromic sequence TTAACAnnTGTTAA (PrfA-box), located in the promoter region (Bockmann et al., 2000). Genome sequence analysis revealed the presence of 19 genes coding surface proteins preceded by the

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PrfA-box sequence: 10 of them code proteins with the LPXTG motif, 2 proteins with the GW motif, 1 a hydrophobic protein and the remaining 6 lipoproteins (Buchrieser et al., 2003; Glaser et al., 2001). 25% genes coding proteins with the motif LPXTG are preceded by PrfA-box sequence, whereas this sequence has been found in the case of only 10% of all L. monocytogenes genes. These data suggest that proteins with the LPXTG motif are involved in virulence and being surface proteins, responsible for contact with an eukaryotic cell, initiate a cascade of events ultimately resulting in infection. Consequently, they would be an ideal target for modern-day chemotherapeutics. Similarly, knowledge of the mechanism of the attachment of surface proteins to murein, mediated by sortase, as well as the increasing knowledge of the function of surface proteins in pathogenesis, is currently the base for murein studies aimed at creating a “magic bullet” acting on proteins involved in the first stage of infection – the adhesion of the bacterial cell to the eukaryotic host cell. One of such targets would thus be the internalins as proteins inducing and initiating this process. Another, of arguably greater importance, would be the family of sortase enzymes, which play a crucial role in the sorting of proteins to cell wall structures of gram-positive bacteria and therefore are of prime importance for pathogenesis. Moreover, these proteins are ubiquitous in all gram-positive pathogens studied thus far. Literature A l v a r e z - D o m i n g u e z C., J.A. V a z q u e z - B o l a n d, E. C a r r a s c o - M a r i n, P. L o p e z - M a t o and F. 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Interactions of Listeria monocytogenes with mammalian cells during entry and actin-based movement: bacterial factors, cellular ligands and signaling. EMBO J. 17: 3797–3806. C o s s a r t P., J. P i z a r r o - C e r d a and M. L e c u i t. 2003. Invasion of mammalian cells by Listeria monocytogenes: Functional mimicry to subvert cellular functions. Trends in Cell Biol. 1: 23–31. C o s s a r t P. 2002. Molecular and cellular basis of the infection by Listeria monocytogenes, an overview. Int. J. Med. Microbiol. 291: 401–409. D a b i r i G.A., J.M. S a n g e r, D.A. P o r t n o y and F.S. S o u t h w i c k. 1990. Listeria monocytogenes moves rapidly through the host-cell cytoplasm by inducing directional actin assembly. Proc. Natl. Acad Sci. USA. 87: 6068–6072. D h a r G., K.F. F a u l l and O. S c h n e e w i n d. 2000. Anchor structure of cell wall surface proteins in Listeria monocytogenes. Biochemistry 39: 3725–3733. D r a m s i S., P. D e h o u x, M. L e b r u n, L. 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G l a s e r P. and The Listeria Consortium. 2001. Comparative Genomics of Listeria species. Science 294: 849–852. H e i l m a n n Ch., G. T h u m m, G.S. C h h a t w a l, J. H a r t l e i b, A. U e k ö t t e r and G. P e t e r s. 2003. Identification and characterization of a novel autolysin (Aae) with adhesive properties from Staphylococcus epidermidis. Microbiol. 149: 2769–2778. H i m m e l r e i c h R., H. H i l b e r t, H. P l a g e n s, E. P i r k l, B.C. L i and R. H e r r m a n n. 1996. Complete sequence analysis of the genome of the bacterium Mycoplasma pneumoniae. Nucleic Acids Res. 24: 4420–4449. 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 u a n Y. and J. v a n A d e l s b e r g. 1999. Polycystin-1, the PKD1 gene product, is in a complex containing E-cadherin and the catenins. J. Clin. Invest. 104: 1459–1468. I l a n g o v a n U., H. T o n - T h a t, J. I w a h a r a, O. 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Polish Journal of Microbiology 2004, Vol. 53, No 2, 89–93

PCR-RFLP Analysis of a Point Mutation in Codons 315 and 463 of the katG Gene of Mycobacterium tuberculosis Isolated from Patients in Silesia, Poland ROBERT D. WOJTYCZKA1, SZYMON DWORNICZAK2*, JERZY PACHA1, DANUTA IDZIK1, MA£GORZATA KÊPA1, ZENOBIA WYDMUCH1, SWIET£ANA G£¥B1, MAGDALENA BAJOREK3, KAZIMIERZ OKLEK2 and JERZY KOZIELSKI2 1 Department

of Microbiology, Faculty of Pharmacy, The Medical University of Silesia, Sosnowiec, [email protected], ul. Jagielloñska 4, 41-200 Sosnowiec, Poland 2 Department of Lung Diseases and Tuberculosis, Faculty of Medicine, The Medical University of Silesia, ul. Kozio³ka 1, 41-803 Zabrze, Poland, tel./fax +48 32 2745664 3 Mycobacteriology Laboratory of Specialists’ Hospital, ul. Padarewskiego 2, 41-500 Chorzów, Poland Received in revised form 13 March 2004 Abstract Resistance to antituberculous agents is an important cause of ineffectiveness of antimicrobial therapy. The resistance of M. tuberculosis to antituberculous agents is a result of mutations in genes participating in those agent’s action. The antituberculous drug – isoniazid can be activated by Mycobacterium tuberculosis either through a hydroperoxidase I/II or a superoxide-dependent oxyferrous pathway. The present study analyzed the frequency of the mutations occurring in codons 315 and 463 in katG gene of Mycobacterium tuberculosis strains, isolated from patients with pulmonary tuberculosis from Silesia, Poland. In this study 23 isoniazid-resistant Mycobacterium tuberculosis strains were analyzed. For RFLP analysis, a 620 bp amplified fragment of katG gene was digested with restriction endonuclease MspI. Among 24 isoniazid-resistant strains, isolated from patients between 2000– 2001, point mutations were found in 30% of analyzed isoniazid-resistant strains in codons 315 or 463 (7 strains). In contrast, no mutations in codons 315 and/or 463 katG gene were found in 16 strains (70%). Obtained results suggests that point mutations S315T (AGC→ACC) and R463L in katG gene are infrequent in the analyzed population. K e y w o r d s: M. tuberculosis resistance, isoniazyd activation, katG mutations

Introduction Tuberculosis is one of the main health problems in the world today. Mycobacterium tuberculosis is still the most important human pathogen with about 10 million new cases of tuberculosis and 30 million deaths during the last decade (Szczuka, 2000). These deaths occur primarily in the developing world, where access to effective antituberculous therapy is limited. The increase in the number of drug-resistant strains has also complicated the management of tuberculosis. Recently there has been observed a rise in the rate of resistant strains to isoniazid (INH) (Zwolska et al., 2000), one of the front-line drugs of choice for tuberculosis treatment (Bass, Jr. et al., 1994). The resistance of M. tuberculosis to at least one drug in Poland between 2000 and 2001 expressed as a rate per 100 000 population reached the level of approximately 0.8. The resistance to INH in the same period reached almost 12% of all isolated resistant strains (Augustynowicz-Kopeæ et al., 2002). Intracellular pathogenic bacteria, including M. tuberculosis, frequently have multitiered defense mechanisms, ensuring their survival in host phagocyte cells. One such defense determinant in M. tuberculosis is the katG gene, which encodes an enzyme with catalase, peroxidase and peroxinitritase activities (Master et al., 2001). * Author for correspondence: Dr n.med. Szymon Dworniczak, Katedra i Klinika Chorób P³uc i GruŸlicy Œl¹skiej Akademii Medycznej, ul. Kozio³ka 1, 41-803 Zabrze, tel./fax +48 32 2745664, e-mail: [email protected]

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An important issue related to association between the katG gene product and isoniazid sensitivity is the nature of hypersensitivity of the M. tuberculosis complex (MTC) to isoniazid. Strains in MTC are often sensitive to 0.02 µg ml–1 of INH but other species of mycobacteria are much less sensitive to isoniazid and species from other genera are relatively insensitive to the drug (Bass, Jr. et al., 1994). Interestingly, the only M. tuberculosis katG gene product is capable of activating the prodrug-isoniazid (Master et al., 2001). The M. tuberculosis resistance to INH could be established by the specific mutations of several genes (Dobner et al., 1997; Mdluli et al., 1998; Petrini and Hoffner, 1999; Rinder et al., 1998). The high level isoniazid resistance (MIC > 0.2 :g/ml) is linked to mutations at codons 315 and 463 of catalase-peroxidase gene (katG), which encodes an enzyme with catalase, peroxidase and peroxinitritase activities (Morris et al., 1995). The frequency of these mutations range from 64 to 71% depending on the world region (Victor et al., 1996). In some cases, detection of specific mutation could serve as a predictor of geographical origin (Haas et al., 1997). Several methods have been promulgated to analyze these mutations, including DNA sequencing and restriction fragment length polymorphisms (RFLP) of polymerase chain reaction (PCR) products (Cockerill et al., 1995; Rinder et al., 1999; Rodriguez et al., 2000). The PCR-RFLP methods seem to be relatively feasible and convenient to detect such point mutations. As mentioned above, the resistance to isoniazid can be a result of mutations in the katG gene. So an objective of our research was to determine the frequency of the mutations in codons 315 and 463 of the katG gene, by PCR-RFLP technique, among INH resistant strains of M. tuberculosis isolated from patients of Silesia, Poland. Furthermore we were tried to evaluate this method for the fast detection of INH resistant strains in hospital population. Experimental Material and Methods Bacterial strains. All of the M. tuberculosis strains used in this study were taken from patients living in the Silesia region of Poland. The microbiological laboratory of the Department of Lung Diseases and Tuberculosis, Faculty of Medicine, the Medical University of Silesia collected mycobacterial strains from all second degree microbiological laboratories in the region from 2000 to 2001. All isolates were grown on Löwenstein-Jensen (L-J) medium and examined for growth rate, gross and microscopic colony morphology, and pigmentation. The strains were identified as Mycobacterium tuberculosis using conventional biochemical tests (Pichula, 1977) and PCR techniques (Almeda et al., 2000; Weil et al., 1996). The primers corresponding to portions of Mycobacterium tuberculosis IS6110 and mtp40 were purchased from DNA-Gdañsk II s.c. (Gdañsk, Poland). The amplification conditions were optimized for the highest signal to background ratio giving the 209 bp fragment of IS6110 and 317 bp fragment of mtp40. The amplification products were analyzed by electrophoresis through 8% polyacrylamide gels with Tris-borate-EDTA buffer and visualized using standard sliver stain method. Isoniazid resistance testing was performed by the standard proportion method in L-J medium at drug concentration 0.2 µg of isoniazid per 1 cm3 as previously described (Scheller et al., 1998). Totally 290 strains of M. tuberculosis were examined. From this population 23 isoniazid resistant, one standard strain H37Rv from ATCC No 25618 and one INH susceptible clinical isolate (for control purposes) were introduced to the study. DNA isolation. One loop of mycobacterial colony was scraped from the L-J medium and resuspended in 1 cm 3 of 0.9% NaCl solution and 200 µl of the suspension was used for isolation. DNA was extracted using QIAamp Tissue Kit (QIAGEN GmbH, Germany). Primer construction and PCR. Sequence information of the M. tuberculosis katG gene deposited at GenBank database under accession number X68081 was used. The primers kG904 (5’-AGC TCG TAT GGC ACC GGA AC-3’) and kG1523 (5’-TTG ACC TCC CAC CCG ACT TG-3’) (GENSET SA, Paris, France) were used to amplify 620 bp katG gene fragment. The primers asymmetrically encompass codons 315 and 463. The PCR reaction was done by using Hot Start Taq Master Mix Kit (QIAGEN GmbH, Germany). The most sensitive signal to background ratio was observed after 35 cycles of denaturing at 94°C for 1 min, annealing 65°C for 1 min and extension at 72°C for 1 min. Reaction products were visualiFig. 1. The results of representative RFLP patterns zed in 6% polyacrylamide gel after silver staining method. of PCR products obtained by MspI digestion of the RFPL analysis. For RFLP analysis, a 620 bp amplicon fragment katG 620-bp katG PCR fragment. gene was digested with restriction endonuclease MspI (MBI Fermentas, Lanes: 1, 3, 6, 8 no mutations; 2 mutation in position 315; Germany). The characteristic sequence for their endonuclease activity is 4, 5, 7 mutation in position 463; 9 – molecular size marker CCGG. Mutation S315T (AGC→ACC) in codon 315 leads to the appear(all values as base pairs)

2

91

katG gene of M. tuberculosis from patients in Poland

ing of this hot place in the katG gene (AGC GGC→ACC GGC). PAG electrophoresis of digested product revealed four fragments: 228, 137, 132, 65 bp. In case of codon 463 the mutation R463L leads to loss of characteristic place for MspI endonuclease activity (ATC CGG→CTN (N = A, G, C, T) or TTA/G). PAG electrophoresis revealed three fragments of digestion: 228, 202, 153bp. When two mutations occur simultaneously 315/463 the pattern of digestion is as follow: 228, 202, 132 bp. In case of luck of mutations occurence in the katG gene, mentioned above, PAG electrophoresis of digested PCR product revealed four fragments: 228, 153, 137, 65 bp. The digestion products were separated in a 8% polyacrylamide gel and stained with the silver method according to procedures proposed by others (Cockerill et al., 1995). The results of the representative sample of this analysis are presented in Figure 1.

Results and Discussion The emergence of resistance to antituberculous drugs is a relevant matter worldwide, but the retrieval of antibiograms for Mycobacterium tuberculosis is severely delayed when phenotypic methods are used. Genotypic methods allow earlier detection of resistance, although conventional approaches are cumbersome or lack sensitivity or specificity. In 56% (n = 13) of analyzed cases the isoniazid-resistance was observed in never treated or previous tuberculosis patients – the primary resistance. 6 of 23 selected strains (25%) were multi-drug resistant. It represents 2% of the whole subjected population. In the examined population the mtp40 gene was present in 22 of 24 resistant strains (91%) contrary to data noticed by A. Weil et al. (Weil et al., 1996) suggesting the phenomena of higher incidence of resistant strains lacking the mtp40 gene. The results are shown in Table I. Table I Characteristics of the Mycobacterium tuberculosis strains included in to the study Strain Strain Previous Niacin IS6110 mtp40 ID treatment test number fragment gene

INH

RFP

SM

EMB

Mutation in codon 315

Mutation in codon 463

Double mutations 315/463

1

9

No

+

+

+

R

S

S

S

–

–

–

2*

23

Yes

+

+

+

R

R

R

S

+

–

–

3

30

No

+

+

+

R

S

R

S

–

–

–

4

45

No

+

+

–

R

S

S

S

–

+

–

5

63

No

+

+

–

R

S

R

S

–

+

–

6

66

No

+

+

+

R

S

S

S

–

–

–

7

69

Yes

+

+

+

R

S

R

S

–

+

–

8

72

No

+

+

–

R

S

S

S

–

–

–

9

86

Yes

+

+

+

R

S

R

S

–

–

–

10

91

Yes

+

+

+

R

R

R

S

–

–

–

11

99

Yes

+

+

+

R

R

R

S

–

–

–

12

130

No

+

+

+

R

S

R

S

–

–

–

13

145

Yes

+

+

+

R

S

R

S

–

+

–

14

154

No

+

+

+

R

S

R

S

–

–

–

15

164

Yes

+

+

+

R

R

R

S

–

–

–

16

165

No

+

+

+

R

S

R

S

–

–

–

17

167

Yes

+

+

+

R

R

R

S

–

–

–

18

168

No

+

+

+

R

S

S

S

–

–

–

19

169

No

+

+

+

R

S

S

S

–

–

–

20

175

No

+

+

+

R

S

R

S

–

+

–

21

190

No

+

+

+

R

S

S

S

–

–

–

22

201

Yes

+

+

+

R

S

S

S

+

–

–

23

205

Yes

+

+

+

R

R

R

S

–

–

–

24

137

Yes

+

+

+

S

S

R

S

–

–

–

H37Rv

301

NA

+

+

+

S

S

S

S

–

–

–

Legend: S – strain susceptible; R – strain resistant; (+) positive reaction; (–) – negative reaction; INH – isoniazid; RFP – rifampicin; SM – streptomycin; EMB – etambuthol * Bold faced rows – multi-drug resistant strains

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Previous investigators have hypothesized that isoniazid is a pro-drug that requires in vivo activation by katG gene product and that resistance to isoniazid strongly correlates with deletions or point mutations in this gene (Dobner et al., 1997). One such mutation, katG (S315T), is found in approximately 50% of clinical isolates exhibiting isoniazid resistance (Wengenack et al., 1998). In the present study we analyzed the frequency of the occurrence of the mutations in codons S315T and R463L in katG gene in isoniazid-resistant strains Mycobacterium tuberculosis isolated from patients in Silesia. Among 23 isoniazid-resistant strains point mutation was found in 30% of analyzed isoniazidresistant strains in codons 315 or 463 (7 strains). In contrast, no mutations in codons 315 and 463 katG gene were found in 16 strains (70%) phenotypicaly resistant to INH. Also no mutations in katG gene were found in H37Rv and the wild INH susceptible strain. Mutations in the katG gene of M. tuberculosis have been associated with resistance to INH in clinical isolates. Previous studies of Rouse et al. have identified specific missence mutations in katG gene at 13 different codons in M. tuberculosis katG gene resulting in INH resistance (Rouse et al., 1996). It has been reported by DNA sequencing studies that 64– 75% of INH resistant M. tuberculosis strains have the mutation in codons 315, 463 or both (Dobner et al., 1997; Musser et al., 1996). Study of Pretorius (Pretorius et al., 1995) suggests approximately equal proportions of INH-resistant isolates from South Africa (16%) and from other geographical regions (21%) with the mutations in the katG gene. But later this point of view was revised (Victor et al., 1996), indicating geographical differentiation of the appearance of analyzed mutations (Haas et al., 1997). In our study mutations in codon R463L were observed in 5 isoniazid-resistant strains (21.7%). Another common mutation observed in isoniazid-resistant isolates is the substitution of a threonine for a serine at codon 315. In our study such mutations in position 315 were observed in 2 resistant strains (8.7%). In subjected population we did not detect the simultaneous mutation in both positions, which was observed by others (Cockerill et al., 1995; Haas et al., 1997). In Cockerill study (Cockerill et al., 1995) mutations in codon 463 were identified in 20–40% of drug resistant isolates. However R463L type of mutation in this codon is the most frequent there is another type of mutations possible – R463G (CGG → CCG) (Musser et al., 1996;Rouse et al., 1996), but in this case the MspI endonuclease hot spot will be preserved. Point mutation S315T (AGC → ACC) is relatively the most frequent, but this type of substitution could also be realized by another type of mutation AGC → AAC, which is un-detectable with the aide of proposed method. The most convenient solution of these problems are to sequence the obtained amplimers (Dobner et al., 1997). These studies have shown that about 30% of INH-resistant strains isolated from patients in the Silesia region have the mutations S315T or R463L at the katG gene. The proposed method for rapid and relatively easy detection of mutations predictive of isoniazid resistance could help to introduce very early the isolation procedure for patients infected with resistant strains. However, molecular methods are not yet capable of complete replacing more traditional methods of susceptibility testing for M. tuberculosis but can serve as a valuable supplementation. Acknowledgments. We would like to thank Mrs Jolanta Kieda-Szurkowska, M.D., Head of the First Department of Tuberculosis and Lung Diseases of Specialists’ Hospital and Mrs Danuta Korniak, M.D., the Director of Specialists’ Hospital in Chorzów for giving the opportunity for collaboration. This work was supported by a grant from the Medical University of Silesia number NN-1-140/01.

Literature A l m e d a J., A. G a r c i a, J. G o n z a l e z, L. Q u i n t o, P.J. V e n t u r a, R. V i d a l, G. R u f i, J.A. M a r t i n e z, M.T. J i m e n e z - d e - A n t a, A. T r i l l a and P.L. A l o n s o. 2000. Clinical evaluation of an in-house IS6110 polymerase chain reaction for diagnosis of tuberculosis. Eur. J. Clin. Microbiol. Infect. Dis. 19: 859–867 A u g u s t y n o w i c z - K o p e æ E., Z. Z w o l s k a, A. J a w o r s k i, E. K o s t r z e w a, M. K l a t t, A. Œ w i d e r s k a and I. D r o z d. 2002. Frequency of drug resistant tuberculosis in Poland in 2000 as compared to 1997 (In Polish). Pneumonol. Alergol. Pol. 70: 193–202 B a s s J.B., Jr., L.S. F a r e r, P.C. H o p e w e l l, R. O’ B r i e n, R.F. J a c o b s, F. R u b e n, D.E. S n i d e r, Jr. and G. T h o r n t o n. 1994. Treatment of tuberculosis and tuberculosis infection in adults and children. American Thoracic Society and The Centers for Disease Control and Prevention. Am. J. Respir. Crit. Care Med. 149: 1359–1374 C o c k e r i l l F.R., J.R. U h l, Z. T e m e s g e n, Y. Z h a n g, L. S t o c k m a n, G.D. R o b e r t s, D.L. W i l l i a m s and B.C. K l i n e. 1995. Rapid identification of a point mutation of the Mycobacterium tuberculosis catalase-peroxidase (katG) gene associated with isoniazid resistance. J. Infect. Dis. 171: 240–245

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D o b n e r P., S. R u s c h - G e r d e s, G. B r e t z e l, K. F e l d m a n n, M. R i f a i, T. L o s c h e r and H. R i n d e r. 1997. Usefulness of Mycobacterium tuberculosis genomic mutations in the genes katG and inhA for the prediction of isoniazid resistance. Int. J. Tuberc. Lung Dis. 1: 365–369 H a a s W.H., K. S c h i l k e, J. B r a n d, B. A m t h o r, K. W e y e r, P.B. F o u r i e, G. B r e t z e l, V. S t i c h t - G r o h and H.J. B r e m e r. 1997. Molecular analysis of katG gene mutations in strains of Mycobacterium tuberculosis complex from Africa. Antimicrob. Agents Chemother. 41: 1601–1603 M a s t e r S., T.C. Z a h r t, J. S o n g and V. D e r e t i c. 2001. Mapping of Mycobacterium tuberculosis katG promoters and their differential expression in infected macrophages. J. Bacteriol. 183: 4033–4039 M d l u l i K., R.A. S l a y d e n, Y. Z h u, S. R a m a s w a m y, X. P a n, D. M e a d, D.D. C r a n e, J.M. M u s s e r and C.E. B a r r y. 1998. Inhibition of a Mycobacterium tuberculosis beta-ketoacyl ACP synthase by isoniazid. Science 280: 1607–1610 M o r r i s S., G.H. B a i, P. S u f f y s, L. P o r t i l l o - G o m e z, M. F a i r c h o k and D. R o u s e. 1995. Molecular mechanisms of multiple drug resistance in clinical isolates of Mycobacterium tuberculosis. J. Infect. Dis. 171: 954–960 M u s s e r J.M., V. K a p u r, D.L. W i l l i a m s, B.N. K r e i s w i r t h, D. v a n S o o l i n g e n and J.D. v a n E m b d e n. 1996. Characterization of the catalase-peroxidase gene (katG) and inhA locus in isoniazid-resistant and -susceptible strains of Mycobacterium tuberculosis by automated DNA sequencing: restricted array of mutations associated with drug resistance. J. Infect. Dis. 173: 196–202 P e t r i n i B. and S. H o f f n e r. 1999. Drug-resistant and multidrug-resistant tubercle bacilli. Int. J. Antimicrob. Agents 13: 93–97 P i c h u l a K. 1977. Acid fast bacilli detection by culture methods. p. 117–147. In: M. Janowiec (ed.) Microbiology of tuberculosis (in Polish). PZWL, Warszawa P r e t o r i u s G.S., P.D. v a n H e l d e n, F. S i r g e l, K.D. E i s e n a c h and T.C. V i c t o r. 1995. Mutations in katG gene sequences in isoniazid-resistant clinical isolates of Mycobacterium tuberculosis are rare. Antimicrob. Agents Chemother. 39: 2276–2281 R i n d e r H., A. T h o m s c h k e, S. R u s c h - G e r d e s, G. B r e t z e l, K. F e l d m a n n, M. R i f a i and T. L o s c h e r. 1998. Significance of ahpC promoter mutations for the prediction of isoniazid resistance in Mycobacterium tuberculosis. Eur. J. Clin. Microbiol. Infect. Dis. 17: 508–511 R i n d e r H., K. F e l d m a n n, E. T o r t o l i, J. G r o s s e t, M. C a s a l, E. R i c h t e r, M. R i f a i, V. J a r l i e r, M. Va q u e r o, S. R u s c h - G e r d e s, E. C a m b a u, J. G u t i e r r e z and T. L o s c h e r. 1999. Culture-independent prediction of isoniazid resistance in Mycobacterium tuberculosis by katG gene analysis directly from sputum samples. Mol. Diagn. 4: 145–152 R o d r i g u e z J.C., G. R o y o and F. R o d r i g u e z - V a l e r a. 2000. Application of four molecular techniques for typing outbreak-associated Mycobacterium tuberculosis strains. APMIS 108: 231–236 R o u s e D.A., J.A. D e V i t o, Z. L i, H. B y e r and S.L. M o r r i s. 1996. Site-directed mutagenesis of the katG gene of Mycobacterium tuberculosis: effects on catalase-peroxidase activities and isoniazid resistance. Mol. Microbiol. 22: 583–592 S c h e l l e r S., H. K a w a l s k i, K. O k l e k, S. D w o r n i c z a k, T. M a t s u n o, K.W. K l i m m e k, M. R a j c a and J. S h a n i. 1998. Correlation between virulence of various strains of Mycobacteria and their susceptibility to ethanolic extract of propolis. Z. Naturforsch. 53c: 1040–1044 S z c z u k a I. 2000. Tuberculosis in Poland and the World at the beginning of third millennium (in Polish). Przegl. Epidemiol. 54: 9–24 V i c t o r T.C., G.S. P r e t o r i u s, J.V. F e l i x, A.M. J o r d a a n, P.D. v a n H e l d e n and K.D. E i s e n a c h. 1996. katG mutations in isoniazid-resistant strains of Mycobacterium tuberculosis are not infrequent. Antimicrob. Agents Chemother. 40: 1572 W e i l A., B.B. P l i k a y t i s, W.R. B u t l e r, C.L. W o o d l e y and T.M. S h i n n i c k. 1996. The mtp40 gene is not present in all strains of Mycobacterium tuberculosis. J. Clin. Microbiol. 34: 2309–2311 W e n g e n a c k N.L., S. T o d o r o v i c, L. Y u and F. R u s n a k. 1998. Evidence for differential binding of isoniazid by Mycobacterium tuberculosis KatG and the isoniazid-resistant mutant KatG(S315T). Biochemistry 37: 15825–15834 Z w o l s k a Z., E. A u g u s t y n o w i c z - K o p e æ and M. K l a t t. 2000. Primary and acquired drug resistance in Polish tuberculosis patients: results of a study of the national drug resistance surveillance programme. Int. J. Tuberc. Lung Dis. 4: 832–838

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Polish Journal of Microbiology 2004, Vol. 53, No 2, 95– 99

Human Papillomavirus (HPV) and Epstein-Barr Virus (EBV) Cervical Infections in Women with Normal and Abnormal Cytology ANDRZEJ SZKARADKIEWICZ, MARZENA WAL, ALICJA KUCH and PRZEMYS£AW PIÊTA

Poznañ University of Medical Sciences, Wieniawskiego 3, 61-712 Poznañ, Poland Received 1 March 2004 Abstract In 48 adult women, subdivided into group 1 with no cervical intraepithelial neoplasia (CIN-negative) and group 2 (CIN-positive), endocervical scrapes were tested for the presence EBV DNA and HPV DNA using PCR-ELISA. In addition, attempts were made to detect HPV 16 and HPV 18 using other PCR amplification techniques. In parallel, in biopsies of uterine cervix obtained from group 2 patients, presence of EBER was documented by RNA in situ hybridization (ISH). Sera of all patients were tested for anti-EBV antibodies. In group 1, presence of EBV DNA was noted in the material obtained from 8 women (30.8%), while HPV DNA was detected in 2 women (7.7%). In group 2, EBV DNA was present in the material obtained from 11 patients (50%), including 7 (31.8%) with HPV DNA also identified. In 5 women (22.7%) of group 2 only HPV DNA was detected. The identifical HPV DNA in all cases belonged to HPV 16 type. Both in group 1 and in group 2, all patients were found to carry serum IgG-anti-VCA and IgG-anti-EBNA antibodies. The results allow to conclude that, co-infection with EBV and HPV 16 may be of cervical significance in etiopathogenesis of uterine cervical cancer. K e y w o r d s: human papillomavirus, Epstein-Barr virus, cervical neoplasia, carcinogenesis

Introduction At present, both human papillomavirus (HPV) and Epstein-Barr virus (EBV) infections are known to be associated with potential development of epithelioid malignancies (zur Hausen, 1991; Anagnostopoulos et al., 1996; Szkaradkiewicz, 2003). Currently, more than 100 types of HPV have been recognised, among which HPV 16 and HPV 18 represent the major causative agent of uterine cervical cancer (Bosch et al., 1995; Bosch et al., 2002). In the process of carcinogenesis, two oncoproteins, representing products of E6 and E7 HPV genes seem to play a significant role. E6 is a protein capable of inactivating p53 gene while activity of E7 protein leads to inactivation of pRb gene, the key controller of cell cycle at the G1 phase (Munger et al., 1992). As demonstrated by in vivo experiments, E6 and E7 may act synergistically, inducing tumour development (Munger et al., 1992; Furumoto et al., 2002). Another virus, the infectious mononucleosis-inducing EBV, may also play a significant role in carcinogenesis. Such a role for EBV has been documented in the etiopathogenesis of endemically manifested nasopharyngeal carcinoma (NPC) (Le Roux et al., 1998), and recent studies point to its association with some cases of oropharyngeal cancers (Szkaradkiewicz et al., 2002), and with pathogenesis of cervical tumours (Landers et al., 1993). In the process of neoplastic transformation of epithelial cells, an important part seems to be fulfilled by the latent membrane protein-1 (LMP-1), produced by EBV both in the course of a productive and a latent infection. A direct effect of the interaction involves activation of cellular gene transcription, mediated by NF6B, and expression of the anti-apoptotic bcl-2 gene (Wensing et al., 2000). Considering the above data, in the present study we investigated development of uterine cervix infections with HPV and/or EBV in women with normal and abnormal cytology. Corresponding author: prof. Andrzej Szkaradkiewicz, Department of Medical Microbiology, University of Medical Sciences, Wieniawskiego 3, 61-712 Poznañ, Poland, tel./fax: 048-61-8536128, e-mail address: [email protected]

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Experimental Materials and Methods Patients. Results of gynaecological, cytological and histological studies permitted to distinguish 2 groups of women: group 1 including 26 women at the age of 21– 43 years (mean 29,9 ± 5,9), which were free of cervical intraepithelial neoplasia (CIN-negative, Papanicolau II°), and group 2 including 22 patients at the age of 24–44 years (mean 35 ± 6,3) with histologically demonstrated cervical intraepithelial neoplasia (CIN-positive). Considering the histology, in the group 2 the subgroup 2a of 12 patients with mild dysplasia (CIN I) or a moderate dysplasia (CIN II) and the subgroup 2b of 10 patients with severe dysplasia or with carcinoma in situ (CIN III) were distinguished. Endocervical scrapes for cytological examination were collected from a disc of the vaginal portion and from the external orifice of the canal using Cytobrush Cell Collector (Medscand Medical). The material was applied to the microscope slide and immediately fixed with the Cytofix preparation (Biochetest). The preparations were classified as described by Papanicolau (Papanicolau, 1954). In all the patients in whom cytological III° result was obtained, samples of uterine cervix disc were also obtained and histological examination was performed. Material for virological studies was sampled from a disc of uterine portion and from external orifice of cervical canal using brush collectors with a transportation medium (Cervical Sampler, Digene). In all studied women of groups 1 and 2 samples of 2 ml peripheral blood were obtained to perform serological tests for presence of anti-EBV antibodies. Extraction of DNA. DNA was isolated from the uterine cervix material using the Perfect gDNA Blood Mini Isolation Kit (Eppendorf). Presence of genomic DNA was verified employing a primer pair for $-globin, coding a region of 326 bp (TIB MolBiol, Poznañ). Determination of EBV DNA. EBV-DNA was detected using PCR-ELISA technique (Sharp Signal System; Digene). PCR reaction was conducted according to Digene procedure, using EBV1 primer and biotinylated EBV2 primer. The obtained biotinylated PCR product was estimated using a microplate colorimetric technique with single-stranded RNA probe. The immobilised hybrids were detected using anti-RNA-DNA antibodies, conjugated with alkaline phosphatase. The absorbance was recorded using Behring Microstrip Reader, _ _ at 8 = 405 nm. The cut-off value was calculated as recommended in the manufacturer’s instruction: _ 2×XNPCR + 0.100, where X NPCR represented mean absorbance for PCR product in the negative control. In our studies, XNPCR = 0.098, which corresponded to the cut-off value of 0.296. Determination of HPV DNA. HPV-DNA was detected using PCR-ELISA technique (Sharp Signal System; Digene). PCR reaction was conducted according to Digene instruction with the defined primers (MY09 and the biotinylated primer MY11) for HPV types: 6/11/42/43/44 and 16/18/31/33/35/39/45/51/52/56/58. The obtained biotinylated PCR product was analysed by a colorimetric microplate technique using a complementary single-stranded probe. The immobilised hybrids were detected using alkaline phosphatase-conjugated anti-RNA-DNA antibodies. The absorbance was quantitated using the Behring _ _ Microstrip Reader, at 8 = 405 nm. The cut-off value was calculated as recommended by the instruction: 2×XNPCR + 0.100, where XNPCR represents mean absorbance _ for PCR product in the negative control. In our studies, XNPCR = 0.086, which corresponded to positive cut-off value of 0.272. Determination of HPV 16 and HPV 18. DNA sequences specific for HPV 16 (160 bp) and 18 (240 bp) genomes were detected using PCR amplification employing primers homologous to E6 region (TIB Molbiol). The detection was performed in 2% agarose gel, using ethidium bromide (Sigma). Detection of EBER (EBER 1 and EBER 2) in tissue material. EBV DNA product in the form of untranslated RNA (EBER 1 and EBER 2) particles was detected in tissue material using in situ hybridization (ISH) (Howe et al., 1986). The tissue material was fixed in formalin and embedded in paraffin. It originated from uterine cervix samples obtained from women with Papanicolaustained cytological smears graded IIIo. Five :m thick sections were deparaffinised and digested with proteinase K for 30 min at 37°C, washed in DEPC. This was followed by inactivation of proteinase K in 0.4% PFD solution for 20 min at 4°C. The hybridisation was performed using fluorescein-labelled RNA probe of 15 nucleotides in length (PNA Probe/FITC; DakoCytomation) for 15 h at 37°C. After a thorough washing in SWS solution (DakoCytomation) the product was detected using anti-FITC/AP antibodies. BCIP/NBT (PNA ISH Detection Kit; DakoCytomation) was used as a substrate. Determination of serum anti-EBV antibodies. Anti-EBV antibodies were quantitated by ELISA. Sera of studied women were tested employing kits for antibodies directed to the early antigen, IgG-anti-EA (ETI-EA-G; DiaSorin), antibodies directed to viral capsid antigen, IgM-anti-VCA (ETI-VCA-M; DiaSorin), IgG-anti-VCA (ETI-VCA-G; DiaSorin) and antibodies reactive with EBV nuclear antigen, IgG-anti-EBNA (ETI-EBNA-G; DiaSorin). Absorbance was read at 8 = 450/630 nm, using the Behring Microstrip Reader. Results were expressed in AU/ml, where AU corresponded to an arbitrary unit established by comparison with antibody standard. Values ≥ 20 AU/ml in the above described tests were considered positive. Statistical analysis. Differences in frequencies of positive results were assessed by Fisher’s exact test and were considered significant at P < 0.05.

Results Results of viral DNA quantitation by PCR technique in the uterine cervix material in the distinguished groups of women are shown in Table I. In group 1, presence of EBV DNA was disclosed in material obtained from 8 women (30.8%), while HPV DNA was present in 2 women (7.7%). In none of women in the group could presence of both viral genomes, EBV and HPV was disclosed. In group 2, EBV DNA was detected in material obtained from 11 patients (50%), including 7 (31.8%) in whom HPV DNA was also identified (in 3 patients in the subgroup 2a and in 4 patients in the subgroup 2b). In 5 women (22.7%) of group 2 (2 patients of subgroup 2a and 3 patients of subgroup 2b) only HPV DNA was detected. The

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HPV and EBV cervical infections Table I EBV DNA and HPV DNA in group 1 (CIN-negative) women and group 2 (CIN-positive ) patients Studied group/subgroup

Number of cases DNA DNA DNA DNA EBV(+) / HPV(+) EBV(+) / HPV(–) EBV(–) / HPV(+) EBV(–) / HPV(–)

GRUP 1 (n = 26)

0

8

2

16

GRUP 2 (n = 22) subgroup 2a (CIN I or CIN II) (n = 12)

3

1

2

6

subgroup 2b (CIN III ) (n = 10)

4

3

3

0

identified HPV DNA in all cases (2 women of group 1 and 12 women of group 2) belonged to HPV 16 type. In 16 women (61.5%) of group 1 and in 6 women (27.3%) of group 2 (subgroup 2a) genomes of EBV and/or HPV could not be detected. HPV DNA or HPV DNA and EBV DNA were significantly more frequent in group 2 (p < 0.05). On the other hand, no significant differences between the two groups were found in frequencies of EBV DNA (p> 0.05).

Fig. 1. Mild dysplasia (CIN I) in cervical epithelium. Note positive reaction for EBER in most of cell nuclei

Fig. 2. Cervical carcinoma with individual cell nuclei with EBER

98 300

Antibody titres (AU/ml)

2

Szkaradkiewicz A. et al.

280

IgG anti-EA

260

IgM anti-VCA

240

IgG anti-VCA

220

IgG anti-EBNA

200 180 160 140 120 100 80 60 40

Cut-off value

20 0

Group 1 (CIN-negative)

Group 2 (CIN-positive)

Fig. 3. Anti-EBV serum antibodies (AU/ml) in studied group of women

In turn, in uterine cervix samples obtained from 22 patients of group 2, ISH technique permitted to demonstrate EBER presence in 11 women (4 patients of subgroup 2a and 7 patients of subgroup 2b), in the form of uniform deposits in cell nuclei of the stratified multilayered flat epithelium, manifesting various grades of dysplasia and carcinoma (CIN I, CIN II or CIN III) (Figures 1 and 2). In parallel, EBV DNA was identified by PCR in uterine cervical samples obtained in the women, consistent with identification of the DNA by ISH approach in the patients. Results of the search for IgG-anti-EA, IgM-anti-VCA, IgG-anti-VCA and IgG-anti-EBNA antibodies in sera of patients of the two groups are presented in Figure 3. In all studied patients of both group 1 and group 2 presence of serum antibodies of IgG-anti-VCA and IgG-anti-EBNA types was disclosed. On the other hand, in none of the cases could presence of serum IgG-anti-EA or IgM-anti-VCA antibodies be documented. Discussion In present study, manifestation of HPV and/or EBV viral infections has been analysed in the two distinguished groups of CIN-negative and CIN-positive women. The studies, performed in all the patients by PCR technique on uterine cervix-sampled material, have demonstrated a significant link between infection with HPV 16 or HPV 16 plus EBV on one hand and cervical intraepithelial neoplasia on the other. On the other hand, incidence of EBV infection has not significantly differed between the two groups of women. Thus, confirming significance of HPV 16 infection in etiopathogenesis of uterine cervix carcinoma, the results have pointed to possible co-operation of EBV in the pathogenesis. The suggestion seem to be confirmed by studies of Sasagawa et al. (2000), who demonstrated HPV infection and EBV co-infection in 39% patients with invasive cancer of uterine cervix and documented strong in situ expression of latent EBV genes, expressed by production of EBER-1 and of EBNA-2 and LMP-1 proteins. EBV latency has been well documented to involve function of its selected genes (Anagnostopoulos et al., 1996). Cells of epithelial tumours linked to EBV infection manifested expression of genes coding for EBER, BART transcripts and EBNA-1 protein only (type I latency), or also of LMP-1 and LMP-2 (type II latency), as well as various variants thereof (I/II type latency). However, expression of all EBV latent genes, including the gene coding for EBNA-2 protein (type III latency) takes place in B cells (Rowe et al., 1992; Anagnostopoulos et al., 1996). In view of the data it seems probable that the quoted above authors obtained falsely positive result for EBNA-2 protein. On the other hand, mixed infection with HPV could have resulted in activation of various latent EBV genes.

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Studies presented in this paper indicate that the infection with EBV alone does not constitute a causal factor for uterine cervix pathology. The conclusion has been corroborated by results of in situ hybridisation which documented presence of EBER particles both in cell nuclei of cells manifesting mild dysplasia or moderate dysplasia (CIN I, CIN II), and in cells of severe dysplasia or cancer. The data may confirm in part results of the other authors (Ammatuna et al., 2000; de Oliveira et al., 1999), who demonstrated no relation between detection of EBV DNA and intraepithelial pathology of uterine cervix. Analysis of serological tests in the two groups has demonstrated presence of IgG-anti-VCA and IgG-anti-EBNA antibodies. The detected profile of humoral reaction has indicated an experienced in the past EBV infection, and presence of anti-viral antibodies in all the patients is consistent with the ubiquitous presence of EBV in human population (Anagnostopoulos et al., 1996; Trzciñska et al., 2001). Therefore, it is possible that acute EBV infection is followed by secondary persistent infection in epithelial cells of uterine cervix, which promotes carcinogenesis with additional effects of other factors (co-infection with HPV, genetic factors, environmental effects) (Sixbey et al., 1986; Herrmann et al., 2003). All the data obtained allows to suggest that, in most cases, acute EBV infection results in persistent infection of epithelial cells in uterine cervix. While cervical intraepithelial neoplasia is linked mainly to infection with HPV 16, EBV may co-operate with HPV in induction of uterine cervix pathology. Literature A m m a t u n a P., L. G i o v a n n e l l i, D. G i a m b e l l u c a, S. M a n c u s o, E. R u b i n o, P. C o l l e t t i, G. M a z z o l a, P. B e l f i o r e and R. L i m a. 2000. Presence of human papillomavirus and Epstein-Barr virus in the cervix of woman infected with the human immunodeficiency virus. J. Med. Virol. 62: 410–415. A n a g n o s t o p o u l o s I. and M. H u m m e l. 1996. Epstein-Barr virus in tumours. Histopathology 29: 297–315. B o s c h F.X., A. L o r i n c z, N. M u n o z, C.J. M e i j e r and K.V. S h a h. 2002. The causal relation between human papillomavirus and cervical cancer. J. Clin. Pathol. 55: 244–265. B o s c h F.X., M.M. M a n o s, N. M u n o z, M. S h e r m a n, A.M. J a n s e m, J. P e t o, M.H. S c h i f f m a n, V. M o r e n o, R. K u r m a n and K.V. S h a h. 1995. Prevalence of human papillomavirus in cervical cancer: a worldwide perspective. International Biological Study on Cervical cancer (IBSCC) Study Group. J. Natl. Cancer Inst. 87: 796–802. d e O l i v e i r a D.E., T.A.F. M o n t e i r o, W.A. d e M e l o, M.A.R. M o r e i r a, M. A l v a r e n g a and C.E. B a c c h i. 1999. Lack of Epstein-Barr virus infection in cervical carcinomas. Arch. Pathol. Lab. Med. 123: 1098–1100. F u r u m o t o H. and M. I r a h a r a. 2002. Human papilloma virus (HPV) and cervical cancer. J. Med. Invest. 49: 124–133. H e r r m a n n K. and G. N i e d o b i t e k. 2003. Epstein-Barr virus-associated carcinomas: facts and fiction. J. Pathol. 199: 140–145. H o w e J.G. and J.A. S t e i t z. 1986. Localization of Epstein-Barr virus-encoded small RNAs by in situ hybrydization. Proc. Natl. Acad. Sci USA 83: 9006–9010. L a n d e r s R.J., J.J. O’ L e a r y, M. C r o w l e y, I. H e a l y, P. A n n i s, L. B u r k e, D. O’ B r i e n, J. H o g a n, W.F. K e a l y and F.A. L e w i s. 1993. Epstein-Barr virus in normal, pre-malignant, and malignant lesions of the uterine cervix. J. Clin. Pathol. 46: 931–935. L e R o u x F. and I. J o a b. 1998. 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Polish Journal of Microbiology 2004, Vol. 53, No 2, 101–110

An Attempt to Protect Winter Wheat Against Gaeumannomyces graminis var. tritici by the use of Rhizobacteria Pseudomonas fluorescens and Bacillus mycoides JANUSZ CZABAN*, ANDRZEJ KSIʯNIAK, BARBARA WRÓBLEWSKA and WOJCIECH L. PASZKOWSKI

Department of Agricultural Microbiology, Institute of Soil Science and Plant Cultivation, Pu³awy, Poland Received in revised form 2 February 2004 Abstract Pseudomonas fluorescens strains III107 and II21 and Bacillus mycoides strains JC192 and K184, stimulating growth of winter wheat, were chosen for the studies. The bacterial strains inhibited on agar nutrient medium the growth of Gaeumannomyces graminis var. tritici (Ggt) – the pathogenic fungus causing take-all on wheat. Both strains of pseudomonads synthesized relatively high amounts of Fe3+ chelators. The strains of bacilli were characterized by the very fast spreading on agar media. Furthermore, strain II21 was highly cyanogenic, and strain JC192 highly chitinolytic. Bacterization of winter wheat seeds (especially with strains III107 and JC192) significantly reduced the percentage of the plants infested with the pathogen in the 28 day glasshouse pot experiment. In the plot experiment, the winter wheat seeds were inoculated with a mixture of strains III107, II21 and JC192. Due to the bacterization the yield of wheat grain and straw was higher in comparison to the series with Ggt alone by 122% and 75%, respectively, but it amounted only to 45% and 43% of the control series not contaminated with Ggt. The decrease of percentage of wheat ears with weight less than 500 mg from 61% in Ggt-series to 25% in Ggt-bacterized-series, and especially the decrease of percentage of wheat ears with weight less than 200 mg from 43% to 14% additionally indicate the partial protection of the winter wheat against Ggt by the rhizobacteria. In the experimental series not contaminated with Ggt the percentage of these wheat ears fractions did not exceed 3% and 0.5%, respectively. K e y w o r d s: take-all biocontrol, winter wheat, Pseudomonas fluorescens, Bacillus mycoides.

Introduction Take-all, caused by Gaeumannomyces graminis (Sacc.) Arx & Olivier var. tritici J. Walker (Ggt) is the most significant root disease of wheat (Triticum aestivum L.) worldwide (Weller et al., 1997). The increasing occurrence of this disease is a consequence of increasing area of winter wheat cultivation (Kuœ and Mróz, 1996). There are no effective sources of cultivar resistance or chemical control (Sarniguet et al., 1992). Biological control by naturally existing antagonistic microorganisms is an alternative strategy, which in certain circumstances might be integrated with other strategies (Hornby, 1998). There have been many reports that bacterial isolates from rhizosphere soil or plant roots (especially fluorescent pseudomonads, because of their excellent root colonization ability, and bacilli, because of their ability to survive in unfavourable conditions, and because of ability of both groups of bacteria to produce a range of compounds inhibiting Ggt growth) are able to control the plant disease or directly stimulate crop growth (Hornby, 1998; Kim et al., 1997; Mariano et al., 1997; Mróz et al., 1994; Ryder et al., 1999; Tsuchiya, 1997; Weller, 1988; Weller et al., 1997; Wenhua and Hetong, 1997; Wong, 1994). Fluorescent pseudomonads may also have a role in natural form of biocontrol – take-all decline (Weller, 1988). The purpose of our work was a preliminary evaluation of the potential of some strains of Pseudomonas fluorescens (Trevisan 1889) Migula 1895 and Bacillus mycoides Flügge, 1886, isolated from rhizosphere of winter wheat, to control Gaeumannomyces graminis var. tritici (Ggt) on wheat. * Corresponding author: Janusz Czaban, Department of Agricultural Microbiology, Institute of Soil Science and Plant Cultivation, 8 Czartoryskich St., 24-100 Pu³awy, Poland; e-mail: [email protected]

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Experimental Materials and Methods Inoculum of the pathogen. The pathogenic fungus, Gaeumannomyces graminis var. tritici (Ggt) was isolated from wheat infested with the pathogen. The inoculum of Ggt was prepared by growing the fungus on autoclaved oat grains. In a pot experiment the inoculum was mixed with the whole mass of soil at the rate of 1% (w/w). In a plot experiment the inoculum was mixed with the upper (20 cm) layer of the soil (75 g/m2). The source of the rhizobacterial strains. The rhizobacterial strains (P. fluorescens – III107 and II21, and B. mycoides – JC192 and K184) were isolated from the winter wheat roots according to the method describing by Kobus et al. (1993). Metabolic activities of the rhizobacteria. Antagonistic activity of the isolated strains against Ggt was determined on Petri plates (9 cm diameter) with Difco potato-dextrose agar (PDA). The plugs of agar with Ggt (4 mm diameter) were placed in the middle of the plates, and the tested bacteria were inoculated at the distance of 3.5 cm from the middle of the plates. The zones of Ggt inhibition were measured after 7 days of the incubation at 28°C. Chitinolytic activity was determined in tubes with agar medium (Strzelczyk et al., 1990) containing 0.5% (w/v) of colloidal chitin. The bacteria were inoculated at the top of the agar medium. The depth of clear zone indicating the rate of chitin hydrolysis was measured after 28 days of incubation at 28°C. Ability to synthesize Fe+3 complexing compounds was studied in mineral nutrient medium containing 2.5% (v/v) of glycerol (Ksiê¿niak and Kobus; 1993). After 21 day incubation at 28°C, to 1 cm 3 of the culture filtrates 0.3 cm3 of 6% (w/v) FeCl3 . 6H2O in 0.1N HCl was added. After 1 hour the optical density of the mixture was analyzed at the wavelength of 520 nm, and the concentration of Fe +3 complexing compounds was estimated from the calibration curve, prepared for a synthetic iron chelator – deferoxamine mesylate USP (Desferal – CIBA-GEIGY) after its reaction with FeCl3 (Jaroszuk-Œcise³ and Kurek, 2001). The capability of the bacterial strains for HCN production was studied according to the method described by Paszkowski et al. (1996). Preparation of the rhizobacterial inocula. In the pot experiment the inocula of the strains were prepared by rinsing off the bacterial cells after 48 hours growth on solid King’s medium B, with 1% (w/v) solution of CM-cellulose. Before sowing, the seeds were soaked in the bacterial suspensions (109 CFU/ml) for 30 min. The seeds from the series non-inoculated with the bacteria were soaked for 30 min in 1% solution of CM-cellulose after rinsing off the plates containing the sterile nutrient medium. In the plot experiment the inocula of the strains (III107, II21 and JC192) instead of the CM-cellulose solution were rinsed off the Petri plates with sterile water to obtain suspensions of the individual strains containing 10 9 CFU/ml. The winter wheat seeds were sprayed with the mixture (1:1:1 v/v) of the bacterial suspensions. Characteristics of the soils. In the pot experiment a field black diluvial soil developed from sandy slight loam (pHKCl 6.8; organic C content 0.96%; total N 0.09%; CEC 16.01 meq 100g –1; clay 11%; silt 7% and sand 82%) was used. The soil, sieved through a 2 mm screen, was placed in the pots (1 kg per pot). A brown soil developed from sandy loam (pHKCl 6.0; organic C content 1.13%; total N 0.11%; CEC, 16.35 meq 100g–1; clay 17%; silt 33% and sand 50%) was used in the plot (0.8 m 2 area) experiment. Plant tests. The pot experiment was conducted in November in a glasshouse with additional electric lighting. In this experiment 20 seeds of winter wheat cv. Kobra per pot were sown. The number of emerged plants was determined everyday between the 7th and 14th day after sowing. The number of plants was reduced to 8 per pot on the 14th day after sowing. The plants were harvested after 28 day of growth. Then the dry weights of the plant shoots and roots, and the percentage of infested wheat plants and the percentage of roots infested with the pathogen were determined. These measurements were done with 4 replicates. After 21, 25 and 28 days of incubation the percentage of wheat plants with morbid symptoms (yellowing, browning and withering) on aboveground parts was determined from all 4 pots of each experimental series excluding replications. In the microplot experiment the soil was enriched with multiple mineral fertilizer Azofoska (100 g per pot) and with potassium chloride (8 g per pot). Seeds of winter wheat cv. NAD 899 were sown in the amount of 200 per one microplot on the 15 th of September. After the harvest, yield of the grain and the straw, number of ears and the weight of each individual ear were determined. All determinations were done with 4 replicates. The distribution of the ear weights of winter wheat was done for the ears of all plots of the individual experimental series. Statistical evaluations. All the data (in 4 replicates) were subjected to analysis of variance and separated with Student’s t-test (P = 95%). The values of percentage of healthy plants were transformed for statistical evaluation according to the equation y = arc sin √x

Results and Discussion From among many various strains of fluorescent pseudomonads and bacilli isolated in our laboratory from winter wheat roots, the representatives of Pseudomonas fluorescens and Bacillus mycoides were chosen for the studies. The bacteria belonging to the former species were the most numerous group of isolates from the surface and the inside of the winter wheat roots and they were metabolically very active (Czaban, 2001). The endospore-forming bacteria belonging to B. mycoides are known as very fast growers, which spread rapidly on the solid nutrient media. These rhizobacteria were the fastest colonizers of the agar media from the pieces of soil free winter wheat roots (soil was rinsed off) put on the surface (Czaban, 2001). Based on this, we expected that strains belonging to these groups of bacteria would be good colonizers of wheat roots. The capability of bacteria for colonizing the plant roots is essential for biocontrol of root pathogens (Weller, 1988). The four bacterial strains (P. fluorescens – III107 and II21, and B. mycoides – JC192 and K184) were chosen because of their ability to reduce the growth of Ggt on potato-dextrose agar medium (Table I) and to

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Use of rhizobacteria to protect wheat against take-all Table I Selected properties of the rhizobacterial strains chosen to the vegetation experiments with Gaeumannomyces graminis. var. tritici (Ggt) Bacterial species

Pseudomonas fluorescens Bacillus mycoides

The strain symbol

Inhibition Fe3+ chelating Chitinase synthesis HCN synthesis of Ggt. growth compounds (clearing zone in mm) (Scale 0–5) (inhibition zone in mm) (:M Desferal l–1)

III107

10

125

0

0

II21

17

190

0

5

JC192

0*

12

25

0

K184

0*

18

0

0

* – the lack of the inhibition zone, but the growth of Ggt was very strongly inhibited by intensively expanding bacteria.

stimulate the growth of winter wheat (up to 44% in a case of B. mycoides K184) in sterile sand enriched with Hoagland’s nutrient medium and in nonsterile soils, not contaminated with plant pathogens (Czaban, 2001). The zones of Ggt growth inhibition on PDA (Table I) suggest the synthesis of some antibiotics by the examined pseudomonad strains. Both strains of P. fluorescens also produced relatively high amounts of Fe3+ complexing compounds, and P. fluorescens II21 produced high amount of HCN (Table I). Weller (1988) and Weller et al. (1997) claimed that the antibiotic production was one of the most important features of bacteria with regard to take-all control on wheat. The important role of siderophores in suppression of take-all was described by Hornby (1998), Tazawa-Isogami et al. (1997) and Wong and Baker (1984). Also, in the opinion of some authors, the bacterial synthesis of HCN may help to suppress the disease of cereals caused by Gaeumannomyces graminis (Hornby, 1998; Ross and Ryder, 1994; Paszkowski, 1998). Keel et al. (1990) suggested that the suppression of soil-borne pathogens by an effective biocontrol agent – P. fluorescent strain CHA0 was a multifactorial mechanism which was mainly due to the production of antibiotics, siderophores and HCN. Strain JC192 of B. mycoides was capable for intensive degradation of chitin (Table I). Chitinolytic ability of this bacteria may play a role in control of Ggt, because antagonistic B. mycoides caused lysis of hyphae of Ggt (Bednáøová-Civínová et al., 1981; Campbell and Faul, 1979 – cited by Kim et al., 1997). In the pot experiment on soil not contaminated with the pathogenic fungus, bacterization of the wheat seeds with all tested bacterial strains had favorable influence on the seedlings emergence (Fig. 1) and on the plant biomass, especially the dry root weight (Fig. 2 and 3). These results confirm our earlier findings (Czaban, 2001) that these strains are PGPRs (plant growth promoting rhizobacteria). Ggt significantly decreased winter wheat seedlings emergence (Fig. 1), reduced dry weight of plant shoots and roots (Fig. 2 and 3) and infested roots of almost all plants (Fig. 4). In the experimental series with Ggt the negative influence of the pathogen on winter wheat seedlings emergence was intensified by bacterization of wheat seeds until 9– 10 day of the incubation (Fig. 1). It was probably caused by the synergistic effect of phytohormone-like substances produced by the fungus and the bacterial strains. At the end of the 28-day pot experiment the bacterial strains slightly increased the dry plant weight (except for B. mycoides K184) in comparison to series with Ggt alone, but this stimulation was statistically insignificant (Fig. 2 and 3). The abilities of the bacteria to protect wheat plants against Ggt were more distinctly visible in the shape of decreased percentage of plants with infested roots and in the shape of decreased percentage of infested roots of all plants (Fig. 4), as well as the percentage of plants with morbid symptoms on shoots (Fig. 5). The bacterial strains P. fluorescens III107 and B. mycoides JC192 were the best plant protectors against Ggt. In the opinion of many authors, the use of bacterial strains in their combination will improve the effectiveness of biological control treatments against many plant pathogens, including Ggt (de Boer et al., 1997; Duffy et al., 1996; Lemanceau et al., 1992; Pierson and Weller, 1994). Increasing the genetic diversity of the biological control system through the use of microbial mixtures may result in the treatments that persist longer in the rhizosphere and utilize a wider array of biocontrol mechanisms under a broader range of environmental conditions (Pierson and Weller, 1994). A synergism between the antibiotic producing bacteria and the chitinase producing bacteria to inhibit R. solani was reported by Sung and Chung (1997). Moreover, Bednáøová-Civínová et al. (1981) showed that while the population of the biocontrol agent – P. putida (introduced on wheat grain) declined with the age of the wheat plants, the population of the native chitinolytic B. mycoides increased in the rhizosphere (only in the bacterized series). This suggests that the B. mycoides having a mycolytical effect on Ggt could

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Czaban J. et al. 14 Control

Number of emerged plants per pot

12

Ggt III 107

10

Ggt + III 107 8

II 21 Ggt + II 21

6

JC 192 4

Ggt + JC 192 K 184

2

Ggt + K 184

0 7

8

9

10 11 12 Days after sowing

13

14

Fig. 1. Winter wheat seedlings emergence as an effect of soil contamination with Ggt and wheat seed inoculation with rhizobacteria: Pseudomonas fluorescens (strains III107 and II21) and Bacillus mycoides (strains JC192 and K184) in the 28 day glasshouse pot assay

130 120 110

ab ab

% of the control series % o t e co t o se es

100 90

a

aa

a

b b

130% 112%

80

100%

70 60 50

cd

40

111%

c

88% cd

cd d

30 20 10 0 Control III 107

II 21 JC192

K184

Ggt

Ggt + Ggt + Ggt + Ggt + III 107 II21 JC192 K184

Fig. 2. Dry weight of winter wheat shoots as an effect of soil contamination with Ggt and wheat seed inoculation with rhizobacteria: Pseudomonas fluorescens (strains III107 and II21) and Bacillus mycoides (strains JC192 and K184) in the 28 day pot experiment. The mean weights of the shoots expressed in columns as percentage of the control value [350 mg per pot] with different letters are significantly different at P < 0.05.

protect the wheat against take-all in the later stages of the plant growth following the initial protection by P. putida. Also Wong (1994) suggested that there might be a role for combining species of Pseudomonas and Bacillus to extend the period of biocontrol of take-all.

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105

Use of rhizobacteria to protect wheat against take-all 150 140 130

% of the control series

120

ab abc c

110 100 90

a a b b

bb

131%

cc

80 70

119% 114%

100%

60 50 40

108%

d d

d

d

d

30 20 10 0 Control III107

II21

JC192

K184

Ggt

Ggt + III107

Ggt + Ggt + II21 JC192

Ggt + K184

Fig. 3. Dry weight of winter wheat roots as an effect of soil contamination with Ggt and wheat seed inoculation with rhizobacteria: Pseudomonas fluorescens (strains III107 and II21) and Bacillus mycoides (strains JC192 and K184) in the 28 day pot experiment. The mean weights of the roots expressed in columns as percentage of the control value [86 mg per pot] with different letters are significantly different at P