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Current Genomics, 2003, 4, 109-121

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Leishmania Cysteine Proteinases: From Gene to Subunit Vaccine Rafati, S.1,*, Fasel, N.2, Masina, S.2 1

Department of Immunology, Pasteur Institute of Iran, Tehran, Iran and Lausanne, Epalinges, Switzerland

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Institute of Biochemistry, University of

Abstract: Whole genome sequences of microbial pathogens present new opportunities for clinical application. Presently, genome sequencing of the human protozoan parasite Leishmania major is in progress. The driving forces behind the genome project are to identify genes with key cellular functions and new drug targets, to increase knowledge on mechanisms of drug resistance and to favor technology transfer to scientists from endemic countries. Sequencing of the genome is also aimed at the identification of genes that are expressed in the infectious stages of the parasite and in particular in the intracellular form of the parasite. Several protective antigens of Leishmania have been identified. In addition to these antigens, lysosomal cysteine proteinases (CPs) have been characterized in different strains of Leishmania and Trypanosoma, as new target molecules. Recently, we have isolated and characterized Type I (CPB) and Type II (CPA) cysteine proteinase encoding genes from L. major. The exact function of cysteine proteinases of Leishmania is not completely understood, although there are a few reports describing their role as virulence factors. One specific feature of CPB in Leishmania and other trypanosomatids, is the presence of a C-terminal extension (CTE) which is possibly indicative of conserved structure and function. Recently, we demonstrated that DNA immunization of genetically susceptible BALB/c mice, using a cocktail of CPB and CPA genes, induced long lasting protection against L. major infection. This review intends to give an overview of the current knowledge on genetic vaccination used against leishmaniasis and the importance of CP genes for such an approach.

LEISHMANIA AS A PUBLIC HEALTH PROBLEM AND THE LEISHMANIA GENOME NETWORK INITIATIVE

sequencing [6*]. The expected completion date for the sequence of the entire L. major genome is in 2003.

Leishmania parasites belong to the order Kinetoplastidae. They are the causative agents of a large spectrum of diseases, varying from localized cutaneous infection to visceral dissemination, the latter is often fatal if not treated [1,2]. Leishmaniasis currently affects some 12 million people, mostly children and young adults, in 88 countries on all continents. 350 million people are exposed to the risk of infection and the annual incidence of new cases is about 2 million [1]. Recently, there has been an increase in the overlap of visceral leishmaniasis (VL) and HIV infection due to the spread of the AIDS pandemic [3,4]. In Sudan, 100,000 and in Eastern India, more than 200,000 people have died over the past years due to Leishmania/HIV co-infection [4]. In Southern Europe, 25 to 70% of adult VL cases are related to HIV infection and 1.5 to 9.5% of AIDS cases suffer from reactivated VL [5]. Thus, leishmaniasis constitutes a major threat to human health. In 1994, the World Health Organization, in its Tropical Disease Research program, decided to prioritize parasite genome analysis. International genome networks for Filariae, Schistosoma, Leishmania, Trypanosome brucei (T. brucei) and Trypanosma cruzi (T. cruzi) were established. The Leishmania reference strain, L. major MHOM/IL/81/Friedlin, was selected by the Leishmania Genome Network (LGN; http://www.ebi.ac.uk./ parasites/leish.html) in 1995 for use in genome mapping and

THE LEISHMANIA GENOME AND ITS APPLICATION TO VACCINE DEVELOPMENT

*Address correspondence to this author at the Department of Immunology, Pasteur Institute of Iran, P.O. Box 11365-6699, Tehran, Iran; Tel: 0098 21 6468761-4 Ext. 2111; Fax: 0098 21 8742314-5; E-mail: [email protected]

1389-2029/03 $41.00+.00

Leishmania parasites exist in two basic developmental forms, promastigotes (flagellated) and amastigotes (nonflagellated). Promastigotes multiply and differentiate in the gut of the sandfly vector, whilst the amastigote replicates within macrophage phagolysosome vacuoles in the mammalian host. Old World Leishmania species such as L. major and L. infantum have 36 chromosomes [7]. Several chromosomes have been completely sequenced from the L. major Friedlin reference strain. Partial sequence is available for several other chromosomes. Gene expression in trypanosomatids appears to be primarily regulated at the post-transcriptional level. Trypanosomatid genes are arranged in polycistronic transcription units which are processed to mono-cistronic mRNAs by trans-splicing a “mini-exon” (providing the 5’ cap structure) to the primary transcript [8-10]. It is likely that this mechanism is coupled with polyadenylation of the mRNA at its 3’ end. This RNA processing generates multiple mRNAs which can be differentially regulated in the free-living flagellated promastigote form, present in the gut of the insect, or in the non-motile amastigote form specialized for growth and survival in the vertebrate host macrophage. Intron sequences, to date, have not been identified within any of the Leishmania protein-coding genes, although one intron has been found in T. brucei and T. cruzi [11]. Comparison of the Leishmania genomic sequence

©2003 Bentham Science Publishers

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and cDNAs will provide additional information which will determine the relevance of cis-splicing in Kinetoplastidae. A remarkable feature deduced from the completed chromosome sequences for Leishmania is that they have large clusters of adjacent genes with dissimilar functions that are found on the same DNA strand. Convergent and divergent junctions have been seen on several completed and uncompleted chromosomes [12]. This coding strand polarity is also present in the genomes of Trypanosoma [13]. Leishmania chromosomes range in size from 300 kb to over 2.5 Mb, yielding a genome size of about 35 Mb [7] with a GC content of 63%. Preliminary sequencing data and coding potential predictions suggest a density of one gene every 3.2 kb. Therefore, it was estimated that the Leishmania genome contains up to 9’800 genes [12**]. Based on their putative biological function, 13 categories of genes have been designated from greater than 650 genes identified through cosmid sequencing (for additional information see the Leishmania Genome network homepage: http://www.ebi. ac.uk./parasites/leish.html). The largest (69%) category of genes are those that are unclassified, with 40% potentially specific to Leishmania [12**]. Therefore, upon completion of the Leishmania genome sequence, greater than 5000 genes with parasite-specific functions may be discovered [12**]. One of the expected benefits of genome analysis of any pathogenic organism with regard to human health is the development of new vaccine and antimicrobial agents. For leishmaniasis, chemotherapy by pentavalent antimony containing drugs or amphotericin B are at present the primary means for controlling the disease [14]. The effectiveness of these compounds, however is eroded by the emergence of drug resistance [15]. Therefore, other strategies, such as vaccination, need to be developed for the control of this disease. For a detailed review on Leishmania vaccine development [16]. Several protective antigens of Leishmania have already been identified [17]. These antigens can have various functions in the parasite which extend from cell surface molecules (e.g. HASPB1) [18] to nuclear proteins (e.g. histone H1) [19]. Many of the vaccines which have been developed in the past require in vitro culture of the pathogen followed by the identification of suitable vaccine candidates using biochemical, immunological, microbiological and genetic analysis. In successful cases, this conventional approach often takes years or even decades to identify, produce and characterize a protective antigen. The availability of sequences from the genome of Leishmania and other pathogens, will provide an alternative approach to conventional vaccine development. “Reverse vaccinology” refers to a genome-based approach to vaccine design whereby computer analysis is used to predict antigens which are most likely to be vaccine candidates [20]. Serogroup B meningococcus (MenB) was the first pathogen example where reverse vaccinology has been used successfully to identify highly conserved proteins which will be the basis for clinical development of a vaccine against MenB [21]. Therefore, access to the complete genome sequence of Leishmania and the deciphering of gene functions will give rise to new vaccine candidates and identification suitable drug targets. In the vaccine field, the access to numerous DNA sequences will favour the development of genetic

Rafati et al.

vaccination when considering its simple use, low cost of production and flexibility of combining multiple genes in one construct. RECENT DEVELOPMENTS IN GENETIC VACCINATION FOR LEISHMANIASIS Genetic vaccination has in recent years provided promising new approaches to vaccination. Protective responses with DNA vaccines against several pathogens have been demonstrated [22*, 23]. In comparison to the recombinant protein and attenuated organism production, a DNA vaccine is relatively simple and inexpensive to produce, this is an advantage when considering vaccination plans for the developing countries. In addition, genetic vaccines effectively engage both major histocompatibility class (MHC) I and MHC II pathways, thereby allowing the induction of both CD8+ and CD4+ T cells [23]. This feature is particularly attractive for a disease like leishmaniasis which relies on a cell-mediated response for protection. Other unique features that make DNA vaccination particularly attractive are the long-lived production of the antigen, which is similar to the situation in natural Leishmania infection, together with improved the immunological memory. Moreover, the parasite antigen may be produced in its native conformation which may be important for the induction of host protection [23]. The first Leishmania vaccine delivered as a plasmid encoded the cDNA from the Leishmania surface glycoprotein gp63 [24]. In this study, peripheral blood lymphocytes from the immunized mice proliferated to in vitro stimulation with freeze/thaw parasites and produced IFN-γ but not IL-4. Following challenge with infectious L. major parasites, significantly smaller lesions developed in mice immunized with gp63 plasmid DNA as compared to the control groups. Subsequent to this work, the protective efficacy of LACK DNA immunization in comparison with immunization of LACK protein and IL-12 was investigated [25]. It was shown that the LACK gene construct induced a strong protective response comparable to that achieved when LACK protein plus recombinant IL-12 were used as immunogens, and better protection than that seen by injection of LACK protein alone. Moreover, it was demonstrated that the depletion of CD8+ cells at the time of vaccination or infection abolished the protective response induced by LACK DNA vaccination, suggesting a role for CD8+ T cells in DNA vaccine induced protection toward L. major. Several studies have tried to understand the reasons for the enhanced efficacy of DNA vaccination when compared to protein and adjuvant vaccination. The low level presentation of antigen and/or induction of IL-12 through CpG motifs found in plasmid DNA, are the two interesting findings. In fact, the first in vivo study demonstrating that CpG triggered a Th-1 response was performed using a Leishmania infection as the classic model for a Th-2 type disease [26**]. In this study, when BALB/c mice were simultaneously injected with infectious Leishmania parasites and CpG DNA, lesion sizes were controlled and a Th-1 type response was mounted. Other studies have revealed the

Leishmania Cysteine Proteinases

influence of toll like receptors such as TLR9 as being essential for the recognition of bacterial DNA-containing CpG motifs and in the induction of Th-1 responses in vivo [27,28]. In recent years, several more studies have supported earlier reports concerning the protective capacity of DNA vaccination in a Leishmania model of disease. A novel strategy to identify Leishmania vaccine candidates through sequential immunization with a cDNA library of L. donovani was recently described [29]. In this work, BALB/c mice were immunized with plasmid DNA isolated and pooled from 15 cDNA sublibrairies. From these sublibrairies several groups of cDNAs giving protection against L. donovani challenge were identified. This protection was seen to correlate with the production of a specific Th-1 immune response. In another study [30], genetically resistant C57BL/6 mice were immunized with a mixture of plasmid DNA encoding the Leishmania antigens LACK, LmSTI11 and TSA. The mice were subsequently challenged under so called “natural challenge” by inoculation of 100 metacyclic promastigotes in the ear dermis. The results from this study suggest that under natural challenge conditions, DNA vaccination has the capacity to confer complete protection against cutaneous leishmaniasis and to prevent the establishment of infection reservoirs. The cysteine proteinases are another example of immunodominant Leishmania antigens which have been extensively studied as both DNA and protein immunogens in susceptible mice. This work will be elaborated in the following sections. CYSTEINE PROTEINASES OF LEISHMANIA Cysteine Proteinases (CPs) are enzymes that belong to the papain superfamily. They are one of the four major classes of proteolytic enzymes which are produced by a variety of organisms from prokaryotes to mammals [31,32]. They function in a number of normal, physiological processes and can also be involved in the pathogenesis of several diseases [33-37], and more recently in cell death [38]. The MEROPS database (http://www.merops.co.uk/) provides a useful source of information and additional links to all the known proteases. Interesting findings have been reported concerning the CPs of protozoa such as Plasmodium [39], Trichomonads [40], Entamoeba [41], Cryptosporidium [42], Eimeria [43], Toxoplasma [44], and Naegleria [45]. The majority of the proteinases detected in T. cruzi, T. brucei and various Leishmania species are also cysteine proteinases. Through these extensive studies, it is evident that cysteine proteinases are attractive drug and/or vaccine targets as they are involved in parasite survival, replication and onset of disease [46]. In 1982, Coombs was the first to report that amastigotes of L. mexicana contain unusually high cysteine proteinase activity and multiple soluble enzymes with an apparent size range of 16-36 kDa [47]. These enzymes account for over 90 percent of the total proteinase activity of the parasite, and

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were shown to locate within unusual lysosome-like organelles called megasomes [48]. Amastigotes contain as many as 34 of these organelles which can comprise 15 percent of the total cell volume. Similar structures showing multiple high activities of cysteine proteinases are also seen in amastigotes of L. pifanoi and L. amazonensis [49] and in soluble extracts of amastigotes from L. major [50]. The high level of sequence conservation in the active sites of eukaryotic cysteine proteinases, enabled the design of degenerate oligonucleotides for the isolation of CP genes from Leishmania. The characteristics of each type of cysteine proteinase is further discussed below. TYPE I CYSTEINE PROTEINASES OF LEISHMANIA Most of the proteinase activity detected in different Leishmania species belong to the Type I (encoded by the CPB gene) cysteine proteinases which are homologues of Cathepsin L-like enzymes. Analysis of cDNA and the deduced protein sequence isolated from L. mexicana, showed that Type I cysteine proteinases could be divided into four domains, the pre-, pro-, central domains and a Cterminal extension (CTE) domain which is found only in Kinetoplastidae [51]. The Type I cysteine proteinases have been identified in different Leishmania species, L. mexicana [47], L. pifanoi [52], L. amazonesis [53] and L. major [54], and are highly stage regulated, with the highest level of activity occurring in the amastigote form which lives in a parasitophorous vacuole of a host macrophage. In other trypanosomatids such as T. cruzi, the Type I cysteine proteinases are generally known as cruzipain and are present in all life-cycle stages, with the most abundant found in the replicating forms and the insect epimastigote stage [55]. In contrast to the 130 copies of Type I genes present in all trypanosomatid species, the L. mexicana Type I cysteine proteinases appear as a single tandem array of 19 copies [56]. The genes encode a pre-pro-enzyme that are subsequently processed to give the mature enzyme. The distinguishable feature of Type I enzymes from other CPs of the papain superfamily is the presence of an unusual CTE [57]. In T. cruzi, this CTE is 130 amino acids long, in T. brucei 110, and in different Leishmania species it is 100 amino acids in length. The CTE is joined to the catalytic domain by a specific motif consisting of threonine, proline and serine residues which form a flexible region accessible to proteolytic cleavage [57]. Comparison of the full sequence of Type I CPs between different Leishmania species shows that the highest variability is found in the CTE as shown in Fig. (1). In some instances the CTEs are glycosylated and may partly be removed by proteolytic cleavage during processing of the enzyme to its mature form. It has been demonstrated that the CTE is not essential for enzyme activity, as proteinases that lack the full extension are active [58]. This information has been demonstrated for recombinant T. brucei, T. cruzi and L. mexicana Type I CPs. A recent study with Leishmania showed that the CTE is not essential for intracellular trafficking of the enzyme to the lysosome [57]. This was supported by the fact that truncated forms of CPA and CPB, lacking the CTE, were still found to localize in the

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L.major L.mexicana L.pifanoi

1 1 1

L.major L.mexicana L.pifanoi

31 31 31

K G C K T T V I P T K E C L P N G A G G S F Q M E C G D H Q 60 R G C R K T L I K A N E C H K N G G G G A C M I K C S P Q K 60 Q G C R K T L I K A N E C H K N G G G G A S M I K C S P Q K 60

L.major L.mexicana L.pifanoi

61 61 61

V L K L T Y T S M N C T G E A K Y T V T R E G K C G I S W S 90 V T M C T Y S N E F C V G G G L C F E T H D G K C S P Y F F 90 V T M C T Y S N E F C V G G G L C F E T P D G K C A P Y F L 90

L.major L.mexicana L.pifanoi

91 91 91

G S S K S I C Q Y V 100 G S I M N T C H Y T 100 G S I M N T C H Y T 100

P T P G P N T T T T T H A P K R V T V K Q I T C T D Y F C R A A P G T S T S S E T P A P R P V V V E Q V I C F D K N C R A A P G T S T S S E T P A P R P V M V E Q V I C F D K N C T

30 30 30

Fig. (1). Alignment of CPB C-terminal extension sequences from Old (L. major) and New World (L. mexicana, L. pifanoi) Leishmania species. Shaded areas represent identities.

lysosome [59*]. Instead of the CTE, pro-domains in both Leishmania and T. cruzi are sufficient and necessary for targeting CPs on lysosomes [57]. The exact function of the CTE is not known, however, there is some evidence to suggest that the CTE has a role in immune evasion. It is postulated as being highly immunogenic and, therefore, may play a role in the diversion of the host immune response [60]. TYPE II CYSTEINE PROTEINASES OF LEISHMANIA Type II cysteine proteinases (encoded by the CPA gene) of Leishmania are also Cathepsin L-like [61]. Type II cysteine proteinases were first reported for L. mexicana [61], and have subsequently been described for other Leishmania species such as L. major [62] and L. donovani [63]. In all the reported Type II cysteine proteinase gene products, there is a unique three amino acid insertion (GVM) near the Nterminus of the central domain [61]. The close proximity of the three amino acid insertion to the N-terminus of the mature protein might have an influence on the activity of the enzyme. Type II CPs are coded by a single copy gene. The gene is transcribed in a stage-regulated manner and the maximal level is seen in the amastigote form of the parasite, with lower expression in the stationary phase promastigote. Unlike Type I cysteine proteinases, Type II have a very short CTE of 10 amino acids, which is highly conserved between different species of Leishmania as shown in Fig. (2). Although the Type II cysteine proteinase protein has not been purified or shown to be a functional enzyme, it seems likely that the enzyme has narrow substrate specificity [61]. TYPE III CYSTEINE PROTEINASES OF LEISHMANIA The third type of cysteine proteinase identified in Leishmania (encoded by the CPC gene) shares greater

similarity to the mammalian Cathepsin B family of enzymes than to the other mammalian cysteine proteinases, and have been detected in Leishmania parasites and Trypanosoma [http://www.ebi.ac.uk/parasites/leish.html]. L. major and L. mexicana Type III cysteine proteinases share 82% identity at the amino acid level. CPC is a single copy gene and present in all life cycle stages of Leishmania, with elevated expression in the multireplicative promastigote form. It contains three possible N- glycosylation sites, one in the pro-region and two in the mature domain. Type III cysteine proteinases do not have a CTE, and are inactive towards gelatin, as are the Type II cysteine proteinases [64]. The Type III cysteine proteinases show marked differences in substrate specificity from the extensively studied Type I CP enzymes [65]. The role of the Type III enzymes currently remains unknown.

L.major L.donovani L.mexicana

1 1 1

N T S H V P T T A A 10 N T S H V P T T A A 10 H T P H V P T T T A 10

Fig. (2). Alignment of CPA C-terminal extension sequences from Old (L. major, L. donovani) and New World (L. mexicana) Leishmania species. Shaded areas represent identities.

CYSTEINE PROTEINASES AS VACCINE CANDIDATES In recent years there has been a surge of interest in investigating the immunogenic properties of CPs. There is evidence to suggest that CPs may act as modulators of the host immune response in favor of parasite survival and proliferation. In addition, cysteine proteinases have been described as potential immunogens with the ability to divert

Leishmania Cysteine Proteinases

the immune response to a Th-1 type. Taken together, these findings suggest that cysteine proteinases could be a suitable Leishmania vaccine candidate. The immunogenicity of the native form of the L. amazonensis cysteine proteinase, termed p30 was evaluated in BALB/c mice [66]. In this study, it was demonstrated that lymphoproliferation due to the p30 antigen was stage specific. Moreover, no difference was observed in the proliferation response of p30 between L. amazonensis susceptible or resistant strains of mice, and the predominant T-cells in the lymphocyte cultures were found to be CD4+. BALB/c susceptible mice were immunized with p30 and challenged with L. amazonensis amastigotes. A low level of infection was developed, thus indicating a potential protective role for p30. A fraction containing the amastigote cysteine proteinase (ACP) of L. major with an apparent molecular weight of 24kDa was used to immunize BALB/c mice [50]. Following challenge with L. major metacyclic infectious parasites, the animals developed significantly smaller lesions than in control animals. Spleen cells from the immunized mice showed a significant proliferative response to ACP and produced high levels of IFN-γ. Recently, we were able to demonstrate that the cysteine proteinase rich fraction of ACP from L. major was able to induce a strong Th-1 response in recovered cases of cutaneous leishmaniasis (CL). In contrast, in patients with chronic CL, IFN-γ was infrequently observed in response to this cysteine proteinase rich fraction. These findings suggest that this ACP fraction is a strong inducer of a primed human immune response to L. major and therefore may have a potential protective role against leishmaniasis through the activation of a Th-1 type immune response [67]. Wolfram et al. [68] were the first to describe the use of cysteine proteinases as defined recombinant antigens. In this study the L. mexicana CPB gene (LmCPB), was over expressed as a recombinant protein in E.coli and used as an antigen to induce and establish a T helper cell line. This T cell line recognized epitopes shared by native cysteine proteinases isolated from parasites and the recombinant CPB. Following this investigation it could be concluded that the L. mexicana cysteine proteinase was a T-cell immunogen and could be used to develop a potentially protective Th-1 cell line [68]. Recently, we were able to demonstrate that recombinant cysteine proteinases of L. major are recognized by the immune system of human patients infected by Leishmania [62]. In this study, humoral immune responses to cysteine proteinases were detected when analyzing sera from the individuals with active or recovered cases of CL [69]. In addition (Rafati et al., unpublished results), both recombinant CPA and recombinant CPB were able to stimulate the production of a Th-1 type CD4+T cell/IFN-γ response without the induction of IL-4 in peripheral blood monocyte cells from the recovered individuals. Thus, recombinant cysteine proteinases were able to stimulate the production of a Th-1 type immune response. The protective properties of recombinant cysteine proteinases against L. major infectious challenge was demonstrated using suceptible BALB/c mice (Rafati et al.,

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submitted). In this study, recombinant CPB and recombinant CPA proteins were used in combination with the poloxamer 407 adjuvant for immunizations. Subcutaneous immunization with recombinant CPB but not recombinant CPA induced protective immunity. Interestingly, depletion of CD8+ T cells at the time of infection abolished the protective response induced by the recombinant CPB vaccination. In addition, the frequency of IFN-γ producing CD8 + cells in the protected group was equivalent to that observed in resistant C57BL/6 mice. The protective effect and immune response of DNA encoding L. major Type 1 and Type II cysteine proteinases were recently investigated in our laboratory [62]. In this study, DNA plasmids encoding CPA and CPB were either co-injected or injected separately into BALB/c mice. Mice receiving CPA DNA alone were not protected from challenge with L. major promastigotes, whereas mice which received an immunization with CPB DNA alone showed a delay for up to 11 weeks in footpad swelling. Co-injection of CPA and CPB genes resulted in long lasting protection. At the same time, we investigated the protective effect when using a combinational DNA and protein immunization approach. Mice were firstly immunized with a cocktail of CPA/CPB plasmid encoding DNA and then boosted with a recombinant CPA/CPB mixture in incomplete Freund’s adjuvant (IFA). The mice in this group showed a similar protective response to those immunized several times with CPA/CPB plasmid DNA alone. Therefore, this strategy of DNA immunization followed by recombinant protein boost presents an alternative means of immunization and vaccine development. CONCLUSIONS AND FUTURE DIRECTIONS The global contribution into the study of Leishmania genomics will help to understand the mechanisms underlying the aetiology of this parasite which will lead to the generation of vaccine targets. Cysteine proteinases of Leishmania are one of the target molecules that have been studied extensively in recent years. L. major cysteine proteinases are strong stimulators of cells from human leishmaniasis patients and demonstrate protective capacity in mouse models of the disease. CPs could thus serve as the basis of a sub-unit vaccine for CL. Further work into the study of CPs for other Leishmania diseases is required. For example, with the increasing overlap of visceral leishmaniasis and HIV infection, immediate attention is required to identify the protective role of the CP enzymes for L. infantum, the causative agent of visceral leishmaniasis. To this end, knowledge of the L. infantum genome sequence would help to identify potential visceral leishmaniasis antigens for use in a genetic vaccination approach to control this life threatening disease. ACKNOWLEDGEMENTS This investigation received financial support from UNDP/World Bank/WHO Special Program for research and Training in Tropical Disease (TDR) # 970556 and A10115 to SR and FNRS grant (No 31-59450.99) to NF.

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Table I: Cysteine proteinase genes of Leishmania

Type

Organism

Cathepsin L-like (Type I)

Cathepsin L-like (Type II)

Cathepsin B (Type III)

Gene

L. mexicana

Accession Number

LmxCPB1

Z49963.2

LmxCPB2.8

Z49962.2

LmxCPB19

Z49965.1

L. pifanoi

LpCYS2

L. major

T.

=

Trypanosoma

Th

=

T helper cell

VL

=

Visceral leishmaniasis

REFERENCES [1]

Desjeux, P., Herwaldt, B. Leishmaniasis. Public health aspects and control. Clin. Dermatol. 1996, 14: 417-423 .

L00718.1

[2]

Herwaldt, B. Leishmaniasis. Lancet, 1999, 354: 1191-1199.

LmjCPB

U43706

[3]

L. chagasi

LdcCYS1

AF004592.2

Solbach, W., Laskay, T. The host response to Leishmania infection. Adv. Immunol. 2000,74:275-317.

[4] L. mexicana

LmxCPA

X62163.1

Desjeux, P. UN AIDS: Leishmania and HIV in Gridlock., Geneva WHO and the UN AIDs, WHO/CTD/LEISH/98 1998, Add. 1 and UNAIDs/98, 23.

L. major

LmjCPA

AJ130942

[5]

L. pifanoi

LpCYS1

L00717.1

Albrecht, H. Redefining AIDS: towards a modification of the current AIDS case definition. Clin. Infect. Dis. 1997, 24: 64-74.

L. chagasi

LdcCYS2

AF004593.2

[6*]

L. mexicana

LmxCPC

Z48599.1

Myler, P., Stuart, K. Recent developments from the Leishmania genome project. Curr. Opin. Microbiol. 2000, 3: 412-416.

L. major

LmjCPC

U437051

This paper repots that more than 600 completely sequenced new genes have been identified, representing 8% of the total gene complement of Leishmania. A large proportion of the gene remain unclassified with 40% of these being potentially Leishmania specific. It describes that the genes are organized into large polycistronic clusters of adjacent genes on the same DNA strand. Chr1 contains two such clusters organized in a divergent manner, where as Chr3 contains two convergent clusters with a singel divergent genes at one telomere with the two large clusters separated by a tRNA gene.

ABBREVIATIONS ACP

=

Amastigote cysteine proteinase

CL

=

Cutaneous leishmaniasis

CP

=

Cysteine protease

CPA

=

Cysteine protease A

CPB

=

Cysteine protease B

CTE

=

C-terminal extension

LGN

=

Leishmania genome network

IFN-γ

=

Interferon gamma

IL-4

=

Interleukin 4

IL-12

=

Interleukin 12

IFA

=

Incomplete Freund’s adjuvant

Kb

=

Kilobase

KDa

=

Kilodalton

L.

=

Leishmania

MHC

=

Major histocompatibility class

Mb

=

Megabase

[7]

Wincker, P., Ravel, C., Blaineau, C., Pages, M., Jauffret, Y., Dedet, J.P., Bastien, P. The Leishmania genome comprises 36 chromosomes conserved across widely divergent human pathogenic species. Nucleic Acids Research, 1996, 24:1688-1694.

[8]

Graham, S.V., Barry, J.D. Transcriptional regulation of metacyclic variant surface glycoprotein gene expression during the life cycle of Trypanosoma brucei. Mol. Cell Biol. 1995,15: 5945-5956.

[9]

Pays, E., Vanhamme, L. in ed. Developmental regulation of gene expression in African trypanosomes. (Smith, D. F. a. P., M., Oxford, UK) 1996, 88-114.

[10]

Ullu, E., Tschudi, C., Günzl, A. in ed. Trans-splicing in trypanosomatid protozoa. (Smith, D. F. a. P., M., Oxford, UK) 1996, 115-133.

[11]

Mair, G., Shi, H., Li, H., Djikeng, A., Aviles, H., Bishop, J., Falcone, F. Gavrilescu, C., Montgomery, J., Santori, M., Stern, L., Wang, Z., Ullu, E., Tschudi, C. A new twist in trypanosome RNA metabolism: cis-splicing of pre-mRNA. RNA, 2000, 6: 163-169.

[12**] Myler, P., Audleman, L., deVos, T., Hixson, G., Kiser, P., Lemley, C., Magness, C., Rickel, E., Sisk, E., Sunkin, S., Swartzell, S., Westlake, T., Bastien, P., Fu, G., Ivens, A., Stuart, K. Leishmania major Friedlin chromosome 1 has an

Leishmania Cysteine Proteinases

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unusual distribution of protein-coding genes. Proc. Natl. Acad. Sci. U.S.A. 1999, 96: 2902-2906.

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