Background: ATG4 is a cysteine peptidase crucial for macroautophagy.

Results: Gene deletion mutants show that the two ATG4s of Leishmania perform distinct roles, although there is some redundancy.

Conclusion: ATG4s are not individually essential but macroautophagy, a process important in the virulence of the parasite, requires one.

Significance: Highlights the distinct roles of ATG4 isoforms and their importance for autophagy and parasite infectivity.

Macroautophagy in Leishmania, which is important for the cellular remodeling required during differentiation, relies upon the hydrolytic activity of two ATG4 cysteine peptidases (ATG4.1 and ATG4.2). We have investigated the individual contributions of each ATG4 to Leishmania major by generating individual gene deletion mutants (Δatg4.1 and Δatg4.2); double mutants could not be generated, indicating that ATG4 activity is required for parasite viability. Both mutants were viable as promastigotes and infected macrophages in vitro and mice, but Δatg4.2 survived poorly irrespective of infection with promastigotes or amastigotes, whereas this was the case only when promastigotes of Δatg4.1 were used. Promastigotes of Δatg4.2 but not Δatg4.1 were more susceptible than wild type promastigotes to starvation and oxidative stresses, which correlated with increased reactive oxygen species levels and oxidatively damaged proteins in the cells as well as impaired mitochondrial function. The antioxidant N-acetylcysteine reversed this phenotype, reducing both basal and induced autophagy and restoring mitochondrial function, indicating a relationship between reactive oxygen species levels and autophagy. Deletion of ATG4.2 had a more dramatic effect upon autophagy than did deletion of ATG4.1. This phenotype is consistent with a reduced efficiency in the autophagic process in Δatg4.2, possibly due to ATG4.2 having a key role in removal of ATG8 from mature autophagosomes and thus facilitating delivery to the lysosomal network. These findings show that there is a level of functional redundancy between the two ATG4s, and that ATG4.2 appears to be the more important. Moreover, the low infectivity of Δatg4.2 demonstrates that autophagy is important for the virulence of the parasite.

Cysteine peptidases have been implicated in the development and pathogenesis of Eimeria. We have identified a single-copy cathepsin B-like cysteine peptidase gene in the genome database of Eimeria tenella (EtCatB). Molecular modeling of the predicted protein suggested that it differs significantly from host enzymes and could be a good drug target. EtCatB was expressed and secreted as a soluble, active, glycosylated mature enzyme from Pichia pastoris. Biochemical characterization of the recombinant enzyme confirmed that it is cathepsin B-like. Screening of a focused library against the enzyme identified three inhibitors (a nitrile, a thiosemicarbazone, and an oxazolone) that can be used as leads for novel drug discovery against Eimeria. The oxazolone scaffold is a novel cysteine peptidase inhibitor; it may thus find widespread use.

A crystallographic and biochemical study of L. major cysteine synthase, which is a pyridoxyl phosphate-dependent enzyme, is reported. The structure was determined to 1.8 Å resolution and revealed that the cofactor has been lost and that a fragment of γ-poly-d-glutamic acid, a crystallization ingredient, was bound in the active site. The enzyme was inhibited by peptides.

Cysteine biosynthesis is a potential target for drug development against parasitic Leishmania species; these protozoa are responsible for a range of serious diseases. To improve understanding of this aspect of Leishmania biology, a crystallographic and biochemical study of L. major cysteine synthase has been undertaken, seeking to understand its structure, enzyme activity and modes of inhibition. Active enzyme was purified, assayed and crystallized in an orthorhombic form with a dimer in the asymmetric unit. Diffraction data extending to 1.8 Å resolution were measured and the structure was solved by molecular replacement. A fragment of γ-poly-d-glutamic acid, a constituent of the crystallization mixture, was bound in the enzyme active site. Although a d-­glutamate tetrapeptide had insignificant inhibitory activity, the enzyme was competitively inhibited (K i = 4 µM) by DYVI, a peptide based on the C-­terminus of the partner serine acetyltransferase with which the enzyme forms a complex. The structure surprisingly revealed that the cofactor pyridoxal phosphate had been lost during crystallization.

Macroautophagy has been shown to be important for the cellular remodelling required for Leishmania differentiation. We now demonstrate that L. major contains a functional ATG12-ATG5 conjugation system, which is required for ATG8-dependent autophagosome formation. Nascent autophagosomes were found commonly associated with the mitochondrion. L. major mutants lacking ATG5 (Δatg5) were viable as promastigotes but were unable to form autophagosomes, had morphological abnormalities including a much reduced flagellum, were less able to differentiate and had greatly reduced virulence to macrophages and mice. Analyses of the lipid metabolome of Δatg5 revealed marked elevation of phosphatidylethanolamines (PE) in comparison to wild type parasites. The Δatg5 mutants also had increased mitochondrial mass but reduced mitochondrial membrane potential and higher levels of reactive oxygen species. These findings indicate that the lack of ATG5 and autophagy leads to perturbation of the phospholipid balance in the mitochondrion, possibly through ablation of membrane use and conjugation of mitochondrial PE to ATG8 for autophagosome biogenesis, resulting in a dysfunctional mitochondrion with impaired oxidative ability and energy generation. The overall result of this is reduced virulence.

Author Summary

Leishmaniasis is a disease of humans that is of major significance throughout many parts of the world. It is caused by the protozoan parasite Leishmania and mammals are infected through the bite of a sand fly in which the parasite develops. Parasite remodelling crucial for generation of the human-infective forms is aided by the catabolic process known as autophagy in which cell material is packaged within organelles called autophagosomes and subsequently broken down in the digestive lysosomal compartment. Here we show that autophagy in Leishmania requires the coordinated actions of two pathways, one of which involves a protein called ATG5. We have generated parasite mutants lacking this protein and shown that ATG5 is required for both autophagosome formation and also maintenance of a fully functional mitochondrion. The mutants lacking ATG5 have increased mitochondrial mass and phospholipid content, high levels of oxidants and reduced membrane potential, all being hallmarks of a dysfunctional mitochondrion with impaired ability for energy generation. Our results have thus revealed that a functional autophagic pathway is crucial for phospholipid homeostasis and mitochondrial function in the parasite and important for the parasite's differentiation, infectivity and virulence to its mammalian host.

The evolution of drug-resistance in pathogens is a major global health threat. Elucidating the molecular basis of pathogen drug-resistance has been the focus of many studies but rarely is it known whether a drug-resistance mechanism identified is universal for the studied pathogen; it has seldom been clarified whether drug-resistance mechanisms vary with the pathogen's genotype. Nevertheless this is of critical importance in gaining an understanding of the complexity of this global threat and in underpinning epidemiological surveillance of pathogen drug resistance in the field. This study aimed to assess the molecular and phenotypic heterogeneity that emerges in natural parasite populations under drug treatment pressure. We studied lines of the protozoan parasite Leishmania (L.) donovani with differential susceptibility to antimonial drugs; the lines being derived from clinical isolates belonging to two distinct genetic populations that circulate in the leishmaniasis endemic region of Nepal. Parasite pathways known to be affected by antimonial drugs were characterised on five experimental levels in the lines of the two populations. Characterisation of DNA sequence, gene expression, protein expression and thiol levels revealed a number of molecular features that mark antimonial-resistant parasites in only one of the two populations studied. A final series of in vitro stress phenotyping experiments confirmed this heterogeneity amongst drug-resistant parasites from the two populations. These data provide evidence that the molecular changes associated with antimonial-resistance in natural Leishmania populations depend on the genetic background of the Leishmania population, which has resulted in a divergent set of resistance markers in the Leishmania populations. This heterogeneity of parasite adaptations provides severe challenges for the control of drug resistance in the field and the design of molecular surveillance tools for widespread applicability.

Author Summary

Drug resistance is a serious problem that strikes at the core of infectious disease control. The mechanisms developed by pathogens to become resistant against existing drug treatments have been studied for many years but these studies have frequently scrutinized a few lines of the pathogen and rarely is it known whether the mechanisms identified occur in all pathogen populations present in endemic regions. In this study we assessed the diversity amongst drug-resistant parasites which emerged under treatment pressure in a natural parasite population. An extensive molecular and phenotypic characterisation of a collection of Leishmania donovani parasites isolated from leishmaniasis patients revealed that the parasites which are resistant to treatment have heterogeneous characters. The results provide evidence that how a parasite develops resistance under treatment pressure depends upon its genetic background. These findings provide key insights into the challenge that drug resistance poses for the control of infectious diseases like leishmaniasis.

Autophagy is the degradative process by which eukaryotic cells digest their own components using acid hydrolases within the lysosome. Originally thought to function almost exclusively in providing starving cells with nutrients taken from their own cellular constituents, autophagy is in fact involved in numerous cellular events including differentiation, turnover of macromolecules and organelles and defense against parasitic invaders. During the past 10–20 years, molecular components of the autophagic machinery have been discovered, revealing a complex interactome of proteins and lipids, which, in a concerted way, induce membrane formation to engulf cellular material and target it for lysosomal degradation. Here, our emphasis is autophagy in protists. We discuss experimental and genomic data indicating that the canonical autophagy machinery characterized in animals and fungi appeared prior to the radiation of major eukaryotic lineages. Moreover, we describe how comparative bioinformatics revealed that this canonical machinery has been subject to moderation, outright loss or elaboration on multiple occasions in protist lineages, most probably as a consequence of diverse lifestyle adaptations. We also review experimental studies illustrating how several pathogenic protists either utilize autophagy mechanisms or manipulate host-cell autophagy in order to establish or maintain infection within a host. The essentiality of autophagy for the pathogenicity of many parasites, and the unique features of some of the autophagy-related proteins involved, suggest possible new targets for drug discovery. Further studies of the molecular details of autophagy in protists will undoubtedly enhance our understanding of the diversity and complexity of this cellular phenomenon and the opportunities it offers as a drug target.

Cysteine proteases of the papain superfamily are present in nearly all eukaryotes and also play pivotal roles in the biology of parasites. Inhibition of cysteine proteases is emerging as an important strategy to combat parasitic diseases such as sleeping sickness, Chagas’ disease and leishmaniasis. Inspired by the in vivo antiparasitic activity of the vinyl sulfone based cysteine protease inhibitors (CPIs), a series of α-ketoheterocycles 1-15 has been developed as reversible inhibitors of a recombinant L. mexicana cysteine protease CPB2.8. The isoxazoles 1-3 and especially the oxadiazole 15 are potent reversible inhibitors of CPB2.8, however, in vitro whole-organism screening against a panel of protozoan parasites did not fully correlate with the observed inhibition of the cysteine protease.

The genome sequencing of several Leishmania species has provided immense amounts of data and allowed the prediction of the metabolic pathways potentially operating. Subsequent genetic and proteomic studies have identified stage-specific proteins and putative virulence factors but many aspects of the metabolic adaptations of Leishmania remain to be elucidated. In this study, we have used an untargeted metabolomics approach to analyze changes in the metabolite profile as promastigotes of L. donovani develop during in vitro cultures from logarithmic to stationary phase. The results show that the metabolomes of promastigotes on days 3–6 of culture differ significantly from each other, consistent with there being distinct developmental changes. Most notable were the structural changes in glycerophospholipids and increase in the abundance of sphingolipids and glycerolipids as cells progress from logarithmic to stationary phase.

Author Summary

Leishmania infections are considered neglected tropical diseases as the parasites affect millions of people worldwide but there are limited research efforts aimed at obtaining vaccines and new drugs. Leishmania has a digenetic life cycle alternating between promastigote forms, which develop in the sand-fly, the vector of the disease, and an amastigote form, which grows in mammals after being bitten by an infected sand-fly. In vitro studies with the promastigote forms are routinely used to gain insights about the parasite's cell biology. Little is known about how the different promastigotes forms are metabolically adapted to their particular micro-environment in the host or how they are pre-adapted metabolically for infecting a mammal, thus we have undertaken a study of the metabolite profile of L. donovani promastigotes in order to gain an understanding of the changes that occur during promastigote development. The analysis has revealed that the changes in promastigotes' metabolome between days 3 and 6 take place in a progressive manner; however major differences were observed when comparing the promastigotes on days 3 and 6. An increase in lipid abundance as promastigote development occurred was notable and is likely to reflect remodelling of the parasite's surface in readiness for infecting a mammal.

Leishmania major, an intracellular parasitic protozoon that infects, differentiates and replicates within macrophages, encodes two closely related MIF-like proteins, which have only ~20% amino acid identity with mammalian MIF. Recombinant L. major MIF1 and MIF2 have been expressed and the structures, resolved by X-ray crystallography, show a trimeric ring architecture similar to mammalian MIF but with some structurally distinct features. LmjMIF1, but not LmjMIF2, has tautomerase activity, indicating that the LmjMIFs have evolved potentially different biological roles. This is further demonstrated by the differential life cycle expression of the proteins. LmjMIF2 is found in all life cycle stages whereas LmjMIF1 is found exclusively in amastigotes, the intracellular stage responsible for mammalian disease. The findings are consistent with parasite MIFs modulating or circumventing the host macrophage response and thereby promoting parasite survival, however analysis of the L. braziliensis genome showed that this species lacks intact MIF genes - highlighting that MIF is not a virulence factor in all species of Leishmania.

The morphological events involved in the Leishmania major promastigote cell cycle have been investigated in order to provide a detailed description of the chronological processes by which the parasite replicates its set of single-copy organelles and generates a daughter cell. Immunofluorescence labeling of β-tubulin was used to follow the dynamics of the subcellular cytoskeleton and to monitor the division of the nucleus via visualization of the mitotic spindle, while RAB11 was found to be a useful marker to track flagellar pocket division and to follow mitochondrial DNA (kinetoplast) segregation. Classification and quantification of these morphological events were used to determine the durations of phases of the cell cycle. Our results demonstrate that in L. major promastigotes, the extrusion of the daughter flagellum precedes the onset of mitosis, which in turn ends after kinetoplast segregation, and that significant remodelling of cell shape accompanies mitosis and cytokinesis. These findings contribute to a more complete foundation for future studies of cell cycle control in Leishmania.

Background: Metacaspases are multifunctional cysteine peptidases.

Results: Trypanosoma brucei metacaspase 4 is a catalytically inactive metacaspase homologue required for parasite virulence, which interacts with an active parasite metacaspase during release from the cell.

Conclusion: Metacaspase 4 is a pseudopeptidase virulence factor.

Significance: Extracellular release and proteolytic processing provide novel insights into metacaspase function.

Metacaspases are caspase family cysteine peptidases found in plants, fungi, and protozoa but not mammals. Trypanosoma brucei is unusual in having five metacaspases (MCA1–MCA5), of which MCA1 and MCA4 have active site substitutions, making them possible non-enzymatic homologues. Here we demonstrate that recombinant MCA4 lacks detectable peptidase activity despite maintaining a functional peptidase structure. MCA4 is expressed primarily in the bloodstream form of the parasite and associates with the flagellar membrane via dual myristoylation/palmitoylation. Loss of function phenotyping revealed critical roles for MCA4; rapid depletion by RNAi caused lethal disruption to the parasite's cell cycle, yet the generation of MCA4 null mutant parasites (Δmca4) was possible. Δmca4 had normal growth in axenic culture but markedly reduced virulence in mice. Further analysis revealed that MCA4 is released from the parasite and is specifically processed by MCA3, the only metacaspase that is both palmitoylated and enzymatically active. Accordingly, we have identified that the multiple metacaspases in T. brucei form a membrane-associated proteolytic cascade to generate a pseudopeptidase virulence factor.

Oligopeptidase B is a clan SC, family S9 serine peptidase found in gram positive bacteria, plants and trypanosomatids. Evidence suggests it is a virulence factor and thus therapeutic target in both Trypanosoma cruzi and T. brucei, but little is known about its function in Leishmania. In this study L. major OPB-deficient mutants (Δopb) were created. These grew normally as promastigotes, had a small deficiency in their ability to undergo differentiation to metacyclic promastigotes, were significantly less able to infect and survive within macrophages in vitro, but were virulent to mice. These data suggest that L. major OPB itself is not an important virulence factor, indicating functional differences between trypanosomes and Leishmania in their interaction with the mammalian host. The possibility that an OPB-like enzyme (designated OPB2) in L. major might compensate for the loss of OPB in Δopb was investigated via by mapping its sequence onto the 1.6 Å structure of L. major OPB. This suggested that the residues involved in the S1 and S2 subsites of OPB2 are identical to OPB and hence the substrate specificity would be similar. Consequently there may be redundancy between the two enzymes.

Metacaspase (MCA) is an important enzyme in Trypanosoma brucei, absent from humans and differing significantly from the orthologous human caspases. Therefore MCA constitutes a new attractive drug target for antiparasitic chemotherapeutics, which needs further characterization to support the discovery of innovative drug candidates. A first series of inhibitors has been prepared on the basis of known substrate specificity and the predicted catalytic mechanism of the enzyme. In this Letter we present the first inhibitors of TbMCA2 with low micromolar enzymatic and antiparasitic activity in vitro combined with low cytotoxicity.

Leishmania mexicana cysteine peptidases (CPs) have been identified as important parasite virulence factors. More recently, a natural inhibitor of CPs (ICP) from L. mexicana has been characterized, and ICP mutants have been created. Infection of BALB/c mice with ICP null mutants or ICP reexpressing mutants resulted in nonhealing, progressively growing lesions albeit slightly attenuated compared with the growth of lesions produced by wild-type parasites. In contrast, BALB/c mice infected with mutants overexpressing ICP were able to significantly control lesion growth or heal. While BALB/c mice infected with wild-type parasites, ICP null mutants, or ICP reexpressing mutants produced significant antibody responses, including immunoglobulin E (IgE), no Th1 response, as indicated by antigen-induced splenocyte gamma interferon (IFN-γ) production, could be demonstrated. In contrast, BALB/c mice infected with mutants overexpressing ICP produced significantly less antibody, particularly IgE, as well as significantly reduced splenocyte interleukin-4 and enhanced IFN-γ production. BALB/c mice were able to resolve infection following infection with one ICP overexpressing clone, which was subsequently used for vaccination studies with BALB/c mice. However, no protection was afforded these mice when they were challenged with wild-type parasites. Nevertheless, two other mouse strains susceptible to L. mexicana, C3H and C57BL/6, vaccinated with overexpressing ICP mutants were able to control challenge infection associated with an enhanced Th1 response. This study confirms that L. mexicana CPs are virulence factors and that ICPs have therapeutic potential.

ICP is a chagasin-family natural tight binding inhibitor of Clan CA, family C1 cysteine peptidases (CPs). We investigated the role of ICP in Trypanosoma brucei by generating bloodstream form ICP-deficient mutants (Δicp). A threefold increase in CP activity was detected in lysates of Δicp, which was restored to the levels in wild type parasites by re-expression of the gene in the null mutant. Δicp displayed slower growth in culture and increased resistance to a trypanocidal synthetic CP inhibitor. More efficient exchange of the variant surface glycoprotein (VSG) to procyclin during differentiation from bloodstream to procyclic form was observed in Δicp, a phenotype that was reversed in the presence of synthetic CP inhibitors. Furthermore, we showed that degradation of anti-VSG IgG is abolished when parasites are pretreated with synthetic CP inhibitors, and that parasites lacking ICP degrade IgG more efficiently than wild type. In addition, Δicp reached higher parasitemia than wild type parasites in infected mice, suggesting that ICP modulates parasite infectivity. Taken together, these data suggest that CPs of T. brucei bloodstream form play a role in surface coat exchange during differentiation, in the degradation of internalized IgG and in parasite infectivity, and that their function is regulated by ICP.

Ecotin is a potent inhibitor of family S1A serine peptidases, enzymes lacking in the protozoan parasite Leishmania major. Nevertheless, L. major has three ecotin-like genes, termed inhibitor of serine peptidase (ISP). ISP1 is expressed in vector-borne procyclic and metacyclic promastigotes, whereas ISP2 is also expressed in the mammalian amastigote stage. Recombinant ISP2 inhibited neutrophil elastase, trypsin and chymotrypsin with Kis between 7.7 and 83 nM. L. major ISP2–ISP3 double null mutants (Δisp2/3) were created. These grew normally as promastigotes, but were internalized by macrophages more efficiently than wild-type parasites due to the upregulation of phagocytosis by a mechanism dependent on serine peptidase activity. Δisp2/3 promastigotes transformed to amastigotes, but failed to divide for 48 h. Intracellular multiplication of Δisp2/3 was similar to wild-type parasites when serine peptidase inhibitors were present, suggesting that defective intracellular growth results from the lack of serine peptidase inhibition during promastigote uptake. Δisp2/3 mutants were more infective than wild-type parasites to BALB/c mice at the early stages of infection, but became equivalent as the infection progressed. These data support the hypothesis that ISPs of L. major target host serine peptidases and influence the early stages of infection of the mammalian host.

Trichomonas vaginalis is an early divergent eukaryote with many unusual biochemical features. It is an anaerobic protozoan parasite of humans that is thought to rely heavily on cysteine as a major redox buffer, as it lacks glutathione. We report here that for synthesis of cysteine from sulphide, T. vaginalis relies upon cysteine synthase. The enzyme (TvCS1) can use as substrates either O-acetylserine or O-phosphoserine. The Kms of the enzyme for sulphide is very low (0.02 mM), suggesting that the enzyme may be a means of ensuring that sulphide in the parasite is maintained at a low level. T. vaginalis appears to lack serine acetyltransferase, the source of O-acetylserine in many cells, but has a functional 3-phosphoglycerate dehydrogenase and an O-phosphoserine aminotransferase that together result in the production of O-phosphoserine, suggesting that this is the physiological substrate. TvCS1 can also use thiosulphate as substrate. Overall, TvCS1 has substrate specificities similar to those reported for cysteine synthases of Aeropyrum pernix and Escherichia coli and this is reflected by sequence similarities around the active site. We suggest that these enzymes are classified together as type B cysteine synthases and we hypothesise that the use of O-phosphoserine is a common characteristic of these cysteine synthases. The level of cysteine synthase in T. vaginalis is regulated according to need, such that parasites growing in an environment rich in cysteine have low activity, whereas exposure to propargylglycine results in elevated cysteine synthase activity. Humans lack cysteine synthase, thus this parasite enzyme could be an exploitable drug target.

Leishmania major possesses, apparently uniquely, four families of ATG8-like genes, designated ATG8, ATG8A, ATG8B and ATG8C, and 25 genes in total. L. major ATG8 and examples from the ATG8A, ATG8B and ATG8C families are able to complement a Saccharomyces cerevisiae ATG8-deficient strain, indicating functional conservation. Whereas ATG8 has been shown to form putative autophagosomes during differentiation and starvation of L. major, ATG8A primarily form puncta in response to starvation - indicating a role for ATG8A in starvation-induced autophagy. Recombinant ATG8A was processed at the scissile glycine by recombinant ATG4.2 but not ATG4.1 cysteine peptidases of L. major and, consistent with this, ATG4.2-deficient L. major mutants were unable to process ATG8A and were less able to withstand starvation than wild type cells. GFP-ATG8-containing puncta were less abundant in ATG4.2 over-expression lines, in which unlipidated ATG8 predominated, which is consistent with ATG4.2 being an ATG8-deconjugating enzyme as well as an ATG8A-processing enzyme. In contrast, recombinant ATG8, ATG8B and ATG8C were all processed by ATG4.1, but not by ATG4.2. ATG8B and ATG8C both have a distinct subcellular location close to the flagellar pocket, but the occurrence of the GFP-labelled puncta suggest that they do not have a role in autophagy. L. major genes encoding possible ATG5, ATG10 and ATG12 homologues were found to complement their respective S. cerevisiae mutants, and ATG12 localised in part to ATG8-containing puncta, suggestive of a functional ATG5-ATG12 conjugation pathway in the parasite. L. major ATG12 is unusual as it requires C-terminal processing by an as yet unidentified peptidase.

We have evaluated the roles of key amino acids to the action of the natural inhibitor chagasin of papain-family cysteine peptidases. A W93A substitution decreased inhibitor affinity for human cathepsin L 100-fold, while substitutions of T31 resulted in 10-100-fold increases in the Ki for cruzipain of Trypanosoma cruzi. A T31A/T32A double mutant had increased affinity for cathepsin L but not for cruzipain, while the T31-T32 deletion drastically affected inhibition of both human and parasite peptidases. These differential effects reflect the occurrence of direct interactions between chagasin and helix 8 of cathepsin L, interactions that do not occur with cruzipain.

We have evaluated the roles of key amino acids to the action of the natural inhibitor chagasin of papain-family cysteine peptidases. A W93A substitution decreased inhibitor affinity for human cathepsin L 100-fold, while substitutions of T31 resulted in 10–100-fold increases in the Ki for cruzipain of Trypanosoma cruzi. A T31A/T32A double mutant had increased affinity for cathepsin L but not for cruzipain, while the T31-T32 deletion drastically affected inhibition of both human and parasite peptidases. These differential effects reflect the occurrence of direct interactions between chagasin and helix 8 of cathepsin L, interactions that do not occur with cruzipain.

Leishmania occurs in several developmental forms and thus undergoes complex cell differentiation events during its life cycle. Those are required to allow the parasite to adapt to the different environmental conditions. The sequencing of the genome of Leishmania has facilitated the identification of the parasite’s vast arsenal of proteolytic enzymes, a few of which have already been carefully studied and found to be important for the development and virulence of the parasite. This review focuses on these peptidases and their role in the cellular differentiation of Leishmania through their key involvement in a variety of degradative pathways in the lysosomal and autophagy networks.

Summary

The biological role of a natural inhibitor of cysteine peptidases (designated ICP) of Leishmania has been investigated by genetic manipulation of the parasite. Null mutants grew normally in vitro, were as infective to macrophages in vitro as wild-type parasites, but had reduced infectivity to mice. Mutants re-expressing ICP from a single gene gave partial restoration of virulence in vivo, whereas mutants over-expressing ICP secreted the inhibitor and showed markedly reduced virulence in mice. Promastigotes of the null mutants had similar cysteine peptidase activities as the wild-type parasites, suggesting that ICP is not required for the expression or processing of the enzymes. The only proteins found to bind to ICP in promastigote cell lysates were fully processed forms of CPA and CPB, showing that ICP does not bind in abundance either to zymogens of the cysteine peptidases or other leishmanial proteins. However, only a small proportion of ICP co-localised with CPA and CPB in the promastigote (in the endoplasmic reticulum and Golgi) and the majority of ICP resided in vesicles that are apparently distinct from endosomes and the multivesicular tubule (MVT)-lysosome. These data suggest that ICP has a role other than modulation of the activity of the parasite's own cysteine peptidases and their normal trafficking to the MVT-lysosome via the flagellar pocket. The finding that ICP partially co-localised with an endocytosed cysteine peptidase leads us to postulate that ICP has a role in protection of the parasite against the hydrolytic environment of the sandfly gut and/or the parasitophorous vacuole of host macrophages.

Leishmania occurs in several developmental forms and thus undergoes complex cell differentiation events during its life-cycle. Those are required to allow the parasite to adapt to the different environmental conditions. The sequencing of the genome of L. major has facilitated the identification of the parasite’s vast arsenal of proteolytic enzymes, a few of which have already been carefully studied and found to be important for the development and virulence of the parasite. This review focuses on these peptidases and their role in the cellular differentiation of Leishmania through their key involvement in a variety of degradative pathways in the lysosomal and autophagy networks.

Background

Visceral leishmaniasis caused by members of the Leishmania donovani complex is often fatal in the absence of treatment. Research has been hampered by the lack of good laboratory models and tools for genetic manipulation. In this study, we have characterised a L. infantum line (JPCM5) that was isolated from a naturally infected dog and then cloned. We found that JPCM5 has attributes that make it an excellent laboratory model; different stages of the parasite life cycle can be studied in vitro, it is accessible to genetic manipulation and it has retained its virulence. Furthermore, the L. infantum JPCM5 genome has now been fully sequenced.

Results

We have further focused our studies on LiCPA, the L. infantum homologue to L. mexicana cysteine peptidase CPA. LiCPA was found to share a high percentage of amino acid identity with CPA proteins of other Leishmania species. Two independent LiCPA-deficient promastigote clones (ΔLicpa) were generated and their phenotype characterised. In contrast to L. mexicana CPA-deficient mutants, both clones of ΔLicpa were found to have significantly reduced virulence in vitro and in vivo. Re-expression of just one LiCPA allele (giving ΔLicpa::CPA) was sufficient to complement the reduced infectivity of both ΔLicpa mutants for human macrophages, which confirms the importance of LiCPA for L. infantum virulence. In contrast, in vivo experiments did not show any virulence recovery of the re-expressor clone ΔLicpaC1::CPA compared with the CPA-deficient mutant ΔLicpaC1.

Conclusion

The data suggest that CPA is not essential for replication of L. infantum promastigotes, but is important for the host-parasite interaction. Further studies will be necessary to elucidate the precise roles that LiCPA plays and why the re-expression of LiCPA in the ΔLicpa mutants complemented the gene deletion phenotype only in in vitro and not in in vivo infection of hamsters.