Flagellar Pocket Research Articles

Membrane protein SMP-1 is required for normal flagellum function inLeishmania

Eukaryotic flagella and cilia are surrounded by a membrane that is continuous with, but distinct from, the rest of the plasma membrane. In Leishmania parasites, the inner leaflet of the flagellar membrane is coated with the acylated membrane protein, SMP-1. Here, we provide evidence that SMP-1 stabilizes the flagellar membrane and is required for flagella elongation and function. The expression and flagella targeting of SMP-1 is tightly associated with flagella elongation during amastigote to promastigote differentiation. Deletion of the genes encoding SMP-1 and the flagellar pocket protein SMP-2, led to the production of short flagella and defects in motility. Alterations in the physical properties of the smp-1/smp-2(-/-) flagellar membrane were suggested by: (1) the accumulation of membrane vesicles in the flagellar matrix, and (2) further retraction of flagella following partial inhibition of sterol and sphingolipid biosynthesis. The flagella phenotype of the smp-1/smp-2(-/-) null mutant was reversed by re-expression of SMP-1, but not SMP-2. SMP-1 contains a jelly-roll beta-sheet structure that is probably conserved in all SMP proteins, and forms stable homo-oligomers in vivo. We propose that the SMP-1 coat generates and/or stabilizes sterol- and sphingolipid-rich domains in the flagellar membrane.

Evaluation of antileishmanial activity of eupomatenoid-5, a compound isolated from leaves of Piper regnellii var. pallescens

Infection with Leishmania spp. causes a disease with multifaceted clinical manifestations in humans. The treatment for leishmaniasis is dependent on a limited range of drugs. Here we investigated the antileishmanial activity of eupomatenoid-5, a neolignan isolated from leaves of Piper regnellii var. pallescens. We showed that eupomatenoid-5 had a dose-dependent activity during 72 h of treatment, exhibiting IC 50 of 9.0 µg/mL and 13.0 µg/mL for promastigote and axenic amastigote forms, respectively, and IC 50 of 5.0 µg/mL for intracellular amastigote forms of Leishmania amazonensis. When L. amazonensis was treated with eupomatenoid-5, it underwent considerable ultrastructural alterations, as shown by transmission electron microscopy. Among the alterations was the appearance of intense exocytic activity in the region of the flagellar pocket, myelin-like figures, and vacuoles in the cytoplasm of parasites treated with 9.0 µg/mL. Cells treated with 25.0 µg/mL showed a very large structure, apparently an extension of the endoplasmic reticulum. Also, mitochondrial swelling was detected at this concentration, indicating damage and significant change in this organelle. A cytotoxicity assay showed that the action of the isolated compound is more specific for protozoa and it is not toxic to macrophages. Our studies indicated that eupomatenoid-5 might be a potential new drug for the treatment of leishmaniasis, because this compound displays interesting antileishmanial activity in vitro against promastigote, axenic amastigote, and intracellular amastigote forms of L. amazonensis.

Proteomic examination of Leishmania chagasi plasma membrane proteins: Contrast between avirulent and virulent (metacyclic) parasite forms

About two million new cases of leishmaniasis with 50 000 associated deaths occur worldwide each year. Promastigotes of the causative Leishmania spp. develop from the procyclic stage to the highly virulent metacyclic stage within the sand fly vector. We hypothesized that proteins important for promastigote virulence might be uniquely represented in the plasma membrane of metacyclic, but not procyclic, promastigotes. Procyclic (logarithmic) promastigotes and purified metacyclic promastigotes from stationary phase cultures of Leishmania chagasi were used to prepare membrane preparations either by surface biotinylation-streptavidin affinity separation or by octyl glucoside detergent extraction. These membrane fractions were enriched over 130- and 250-fold, respectively, as estimated by Western blotting for the plasma membrane's major surface protease. Hundreds or dozens of proteins were identified by LC-MS/MS in the surface biotinylation or detergent extraction, respectively. Confocal microscopy suggested the difference between the lists was due to the fact that proteins localized both on the surface membrane and within the flagellar pocket were accessible to surface biotinylation, whereas only proteins on the membrane were obtained by detergent extraction. Using detergent extraction, we found different proteins were present in membranes of the procyclic stage compared to metacyclic stage promastigotes. Several dozen were stage specific. These data provide a foundation for identifying virulence factors in the plasma membranes of Leishmania spp. promastigotes during metacyclogenesis.

Cysteine peptidases fromPhytomonas serpens: biochemical and immunological approaches

Phytomonas serpens, a phytoflagellate trypanosomatid, shares common antigens with Trypanosoma cruzi. In the present work, we compared the hydrolytic capability of cysteine peptidases in both trypanosomatids. Trypanosoma cruzi epimastigotes presented a 10-fold higher efficiency in hydrolyzing the cysteine peptidase substrate Z-Phe-Arg-AMC than P. serpens promastigotes. Moreover, two weak cysteine-type gelatinolytic activities were detected in P. serpens, while a strong 50-kDa cysteine peptidase was observed in T. cruzi. Cysteine peptidase activities were detected at twofold higher levels in the cytoplasmic fraction when compared with the membrane-rich or the content released from P. serpens. The cysteine peptidase secreted by P. serpens cleaved several proteinaceous substrates. Corroborating these findings, the cellular distribution of the cruzipain-like molecules in P. serpens was attested through immunocytochemistry analysis. Gold particles were observed in all cellular compartments, including the cytoplasm, plasma membrane, flagellum, flagellar membrane and flagellar pocket. Interestingly, some gold particles were visualized free in the flagellar pocket, suggesting the release of the cruzipain-like molecule. The antigenic properties of the cruzipain-like molecules of P. serpens were also analyzed. Interestingly, sera from chagasic patients recognized both cellular and extracellular antigens of P. serpens, including the cruzipain-like molecule. These results point to the use of P. serpens antigens, especially the cruzipain-like cysteine-peptidases, as an alternative vaccination approach to T. cruzi infection.

Depletion of 14-3-3 proteins in bloodstream-form Trypanosoma brucei inhibits variant surface glycoprotein recycling

Bloodstream-form Trypanosoma brucei have two 14-3-3 proteins, which are required for parasite multiplication. We here describe the effects of 14-3-3 depletion on vesicular transport of variant surface glycoprotein (VSG). 14-3-3 depletion had no detectable effect on de novo synthesis and trafficking of VSG to the cell surface, or on VSG endocytosis. Despite strong inhibition of cell division, the flagellar pocket was not enlarged and the ultrastructure of internal organelles appeared normal. The Rab11-positive recycling endosome compartment was, however, fivefold smaller than normal, and the rate of return of recycling VSG to the surface was correspondingly reduced. Down-regulating 14-3-3 also prevented enlargement of the flagellar pocket by clathrin depletion. These results suggest that there is a remarkably specific requirement for 14-3-3 in normal functioning of the Rab11-positive recycling endosome compartment.

Membrane domains and flagellar pocket boundaries are influenced by the cytoskeleton in African trypanosomes

A key feature of immune evasion for African trypanosomes is the functional specialization of their surface membrane in an invagination known as the flagellar pocket (FP), the cell's sole site of endocytosis and exocytosis. The FP membrane is biochemically distinct yet continuous with those of the cell body and the flagellum. The structural features maintaining this individuality are not known, and we lack a clear understanding of how extracellular components gain access to the FP. Here, we have defined domains and boundaries on these surface membranes and identified their association with internal cytoskeletal features. The FP membrane appears largely homogeneous and uniformly involved in endocytosis. However, when endocytosis is blocked, receptor-mediated and fluid-phase endocytic markers accumulate specifically on membrane associated with four specialized microtubules in the FP region. These microtubules traverse a distinct boundary and associate with a channel that connects the FP lumen to the extracellular space, suggesting that the channel is the major transport route into the FP.

The trypanosome flagellar pocket

Trypanosomes are important disease agents and excellent models for the study of evolutionary cell biology. The trypanosome flagellar pocket is a small invagination of the plasma membrane where the flagellum exits the cytoplasm and participates in many cellular processes. It is the only site of exocytosis and endocytosis and part of a multiorganelle complex that is involved in cell polarity and cell division. Several flagellar pocket-associated proteins have been identified and found to contribute to trafficking and virulence. In this Review we discuss the contribution of the flagellar pocket to protein trafficking, immune evasion and other processes.

Myristoyl-CoA:protein N-myristoyltransferase depletion in trypanosomes causes avirulence and endocytic defects

The enzyme myristoyl-CoA:protein N-myristoyltransferase (NMT) catalyses the co-translational covalent attachment of the fatty acid myristate to the N-terminus of target proteins. NMT is known to be essential for viability in Trypanosoma brucei and Leishmania major. Here we describe phenotypic analysis of T. brucei bloodstream form cells following knockdown of NMT expression by tetracycline-inducible RNA interference. Cell death occurs from 72h post-induction, with approximately 50% of cells displaying a defect in endocytic uptake by this time. The majority of these induced cells do not have an enlarged flagellar pocket typical of a block in endocytosis but vesicle accumulation around the flagellar pocket indicates a defect in vesicular progression following endocytic fusion. Induced parasites have a wild-type or slightly enlarged Golgi apparatus, unlike the phenotype of cells with reduced expression of a major N-myristoylated protein, ARL1. Critically we show that following NMT knockdown, T. brucei bloodstream form cells are unable to establish an infection in a mouse model, therefore providing further validation of this enzyme as a target for drug development.

Nitric oxide hinders antibody clearance from the surface of Trypanoplasma borreli and increases susceptibility to complement-mediated lysis

Trypanoplasma borreli is an extracellular blood parasite of carp belonging to the same Order (Kinetoplastida) as African trypanosomes. These mammalian parasites have developed different strategies to evade the host immune system including antigenic variation, immunosuppression and clearance of surface-bound antibodies. The latter mechanism allows trypanosomes to use their swimming movement to cause surface-bound antibodies to ‘sail’ and accumulate at the posterior end of the parasite, to be internalized via the flagellar pocket and be degraded. There is no evidence that T. borreli shows antigenic variation, but during the late phases of infection NO-mediated immunosuppression is observed. High levels of nitric oxide (NO) lead to extensive tissue nitration whereas the parasite itself is not affected. Therefore, the induction of NO has thus far been considered a parasite-driven response with immunosuppressive effects. In the present study, we show that the induction of NO, particularly during the early phase of T. borreli infections, should be re-considered an effective part of the host immune response. We show that T. borreli rapidly removes surface-bound IgM. In addition, moderate concentrations of NO, by hindering surface antibody clearance, maintain high the concentrations of membrane-bound IgM, thereby favoring antibody-dependent complement-mediated parasite lysis. We performed a comprehensive quantitative gene expression analysis of in total seven different complement factors involved in all three activation pathways, differentiating between 1 and 4 isoforms for each complement gene. Our gene expression analysis supports an important role for antibody-dependent complement-mediated lysis of T. borreli in vivo. To our knowledge, NO-dependent inhibition of antibody clearance from the surface of kinetoplastid parasites has not been investigated. Our data support a role for NO as an important player in host–parasite interactions, not only as immune suppressor (late response) but also as immune effector (early response) in infections with bloodstream parasites such as T. borreli.

Immunolocalization and Challenge Studies Using a Recombinant Vibrio cholerae Ghost Expressing Trypanosoma brucei Ca2+ ATPase (TBCA2) Antigen

Human African trypanosomiasis is a neglected disease caused by Trypanosoma brucei spp. A parasite cation pump (Ca(2+) ATPase; TBCA2) essential for survival and cation homeostasis was identified and characterized. It was hypothesized that targeting this pump using a Vibrio cholerae ghost (VCG)-based vaccine could protect against murine T. brucei infection. mRNA and protein expression of TBCA2 was differentially expressed in blood and insect stages of parasites and immunolocalized in the pericellular membrane and the flagellar pocket of bloodstream forms. Antigen-specific antibodies and Th1 cytokines, interleukin-2, interferon-gamma, and tumor necrosis factor-alpha were induced in rVCG-TBCA2-immunized mice and in vitro on antigen stimulation of splenic immune T cells, but the corresponding Th2-type response was unremarkable. Despite an increased median survival of 6 days in vaccinated mice, the mice were not protected against infection. Thus, immunization of mice produced robust parasite-specific antibodies but failed to protect mice against parasite challenge.

Role of AP-1 in Developmentally Regulated Lysosomal Trafficking in Trypanosoma brucei

African trypanosomes are the causative agents of human trypanosomiasis (sleeping sickness). The pathogenic stage of the parasite has unique adaptations to life in the bloodstream of the mammalian host, including upregulation of endocytic and lysosomal activities. We investigated stage-specific requirements for cytoplasmic adaptor/clathrin machinery in post-Golgi apparatus biosynthetic sorting to the lysosome using RNA interference silencing of the Tbmu1 subunit of adaptor complex 1 (AP-1), in conjunction with immunolocalization, kinetic analyses of reporter transport, and quantitative endocytosis assays. Tbmu1 silencing was lethal in both stages, indicating a critical function(s) for the AP-1 machinery. Transport of soluble and membrane-bound secretory cargoes was Tbmu1 independent in both stages. In procyclic parasites, trafficking of the lysosomal membrane protein, p67, was disrupted, leading to cell surface mislocalization. The lysosomal protease trypanopain was also secreted, suggesting a transmembrane-sorting receptor for this soluble hydrolase. In bloodstream trypanosomes, both p67 and trypanopain trafficking were unaffected by Tbmu1 silencing, suggesting that AP-1 is not necessary for biosynthetic lysosomal trafficking. Endocytosis in bloodstream cells was also unaffected, indicating that AP-1 does not function at the flagellar pocket. These results indicate that post-Golgi apparatus sorting to the lysosome is critically dependent on the AP-1/clathrin machinery in procyclic trypanosomes but that this machinery is not necessary in bloodstream parasites. We propose a simple model for stage-specific default secretory trafficking in trypanosomes that is consistent with the behavior of other soluble and glycosylphosphatidylinositol-anchored cargos and which is influenced by upregulation of endocytosis in bloodstream parasites as an adaptation to life in the mammalian bloodstream.

CDNA cloning and partial characterization of amastigote specific surface protein from Trypanosoma cruzi

Trypanosoma cruzi amastigote surface proteins are the target of both humoral and cell-mediated immune responses; however, few such molecules have been thoroughly studied. In order to study a T. cruzi amastigote-specific protein (SSP4), we used antibodies against the deglycosylated form of this molecule to clone cDNA. The selected cDNA clone (2070 bp) encodes for a 64 kDa protein product whose sequence analysis revealed no N-glycosylation signal. The DNA sequence showed high homology with a member of a previously reported dispersed repetitive gene family of T. cruzi. Antibodies against the recombinant protein reacted strongly with a 66 kDa protein and weakly with an 84 kDa protein in amastigote extracts. Immunoelectron microscopy studies showed that intracellular amastigotes express the native protein on their surfaces and flagellar pockets. The antibody label was also associated with an amorphous material present in the parasitic cavity and in direct contact with the parasite surface, which suggest that amastigotes are releasing this material. On cell-free amastigotes, the antibody showed strong decoration of the cell surface and labeling of intracellular vesicles. Immunofluorescence analysis showed that the superficial protein is expressed shortly after trypomastigotes begin to transform into amastigotes. Anti-recombinant protein antibodies recognized proteins of 100 kDa and 50–60 kDa in protein extracts of rat heart and skeletal muscle, respectively.

The bilobe structure of Trypanosoma brucei contains a MORN-repeat protein

The Golgi of the kinetoplastid parasite Trypanosoma brucei is closely apposed to a bilobe structure containing TbCentrin2 and TbCentrin4 in procyclic cells. However, both are additionally localized to the basal bodies. Here we report the characterization of a membrane occupation and recognition nexus (MORN)-repeat protein, TbMORN1, present at the bilobe but not at the basal body. The anterior part of the TbMORN1 structure partially overlapped with the flagellar attachment zone while the posterior part overlapped with the flagellar pocket. Depletion studies using RNAi showed that there was a modest growth inhibition in procyclic cells but lethality in bloodstream cells, showing that it is an essential protein in the bloodstream form of the organism. TbMORN1 appears to be a useful marker for the bilobe in T. brucei.

Three-dimensional cellular architecture of the flagellar pocket and associated cytoskeleton in trypanosomes revealed by electron microscope tomography

This study uses electron tomography linked to a variety of other EM methods to provide an integrated view of the flagellar pocket and basal body area of the African trypanosome procyclic trypomastigote. We reveal the pocket as an asymmetric membranous 'balloon' with two boundary structures. One of these - the collar - defines the flagellum exit point. The other defines the entry point of the flagellum into the pocket and consists of both an internal transitional fibre array and an external membrane collarette. A novel set of nine radial fibres is described in the basal body proximal zone. The pocket asymmetry is invariably correlated with the position of the probasal body and Golgi. The neck region, just distal to the flagellum exit site, is a specialised area of membrane associated with the start of the flagellum attachment zone and signifies the point where a special set of four microtubules, nucleated close to the basal bodies, joins the subpellicular array. The neck region is also associated with the single Golgi apparatus of the cell. The flagellar exit point interrupts the subpellicular microtubule array with discrete endings of microtubules at the posterior side. Overall, our studies reveal a highly organised, yet dynamic, area of cytoplasm and will be informative in understanding its function.

Characterization of unusual families of ATG8-like proteins and ATG12 in the protozoan parasite Leishmania major

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 - suggesting 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.

Structural Modeling, Evolution and Ligand Interaction of KMP11 Protein of Different Leishmania Strains

The kinetoplastid-specific KMP11 protein was first described for Leishmania donovani associated with the lypophosphoglycan molecule and is localized mainly around the flagellum and flagellar pocket. This protein is well conserved among kinetoplastids and plays an analogous role in all the flagellates, irrespective of their pathogenicity in humans. The structural elucidation of this important protein may bring about information required to target KMP11 to find valid drug candidates. The atomic-resolution model of KMP11 protein of six different Leishmania strains has been determined from its amino acid sequence by using homology modeling. The stereochemical validation of modeled protein has been done by PROCHECK and Profiles-3D scores. The ligand protein interaction of the KMP11 protein models were carried out with several anti-leishmanial drugs i.e. miltefosine, sitamaquine, pentamidine, amphotericin B, SAG (sodium antimony gluconate), leishmanial peptide, paromomycin and vinblastine and an anticancer compound, sulforaphane. Glutamic acid (E) and lysine (K) of KMP11 are the key amino acids during ligand-receptor interaction. From structural and docking analyses, it is hypothesized that KMP11 of a specific Leishmania strain interacts with a specific anti-leishmanial drug candidate i.e. miltefosine interacts only with KMP11 of L. braziliensis but not with KMP11 of any other Leishmania strain. Highest docking score was found in case of pentamidine. Anticarcinogenic compound, sulphoraphane has shown comparable docking scores and H-bonds with KMP11 protein of six Leishmania strains.

Flagellum elongation is required for correct structure, orientation and function of the flagellar pocket in Trypanosoma brucei

In trypanosomes, the flagellum is rooted in the flagellar pocket, a surface micro-domain that is the sole site for endocytosis and exocytosis. By analysis of anterograde or retrograde intraflagellar transport in IFT88RNAi or IFT140RNAi mutant cells, we show that elongation of the new flagellum is not required for flagellar pocket formation but is essential for its organisation, orientation and function. Transmission electron microscopy revealed that the flagellar pocket exhibited a modified shape (smaller, distorted and/or deeper) in cells with abnormally short or no flagella. Scanning electron microscopy analysis of intact and detergent-extracted cells demonstrated that the orientation of the flagellar pocket collar was more variable in trypanosomes with short flagella. The structural protein BILBO1 was present but its localisation and abundance was altered. The membrane flagellar pocket protein CRAM leaked out of the pocket and reached the short flagella. CRAM also accumulated in intracellular compartments, indicating defects in routing of resident flagellar pocket proteins. Perturbations of vesicular trafficking were obvious; vesicles were observed in the lumen of the flagellar pocket or in the short flagella, and fluid-phase endocytosis was drastically diminished in non-flagellated cells. We propose a model to explain the role of flagellum elongation in correct flagellar pocket organisation and function.

A Parasite Cysteine Protease Is Key to Host Protein Degradation and Iron Acquisition

Cysteine proteases of the Clan CA (papain) family are the predominant protease group in primitive invertebrates. Cysteine protease inhibitors arrest infection by the protozoan parasite, Trypanosoma brucei. RNA interference studies implicated a cathepsin B-like protease, tbcatB, as a key inhibitor target. Utilizing parasites in which one of the two alleles of tbcatb has been deleted, the key role of this protease in degradation of endocytosed host proteins is delineated. TbcatB deficiency results in a decreased growth rate and dysmorphism of the flagellar pocket and the subjacent endocytic compartment. Western blot and microscopic analysis indicate that deficiency in tbcatB results in accumulation of both host and parasite proteins, including the lysosomal marker p67. A critical function for parasitism is the degradation of host transferrin, which is necessary for iron acquisition. Substrate specificity analysis of recombinant tbcatB revealed the optimal peptide cleavage sequences for the enzyme and these were confirmed experimentally using FRET-based substrates. Degradation of transferrin was validated by SDS-PAGE and the specific cleavage sites identified by N-terminal sequencing. Because even a modest deficiency in tbcatB is lethal for the parasite, tbcatB is a logical target for the development of new anti-trypanosomal chemotherapy.

An Unconventional Form of Actin in Protozoan Hemoflagellate, Leishmania

Leishmania actin was cloned, overexpressed in baculovirus-insect cell system, and purified to homogeneity. The purified protein polymerized optimally in the presence of Mg2+ and ATP, but differed from conventional actins in its following properties: (i) it did not polymerize in the presence of Mg2+ alone, (ii) it polymerized in a restricted range of pH 7.0-8.5, (iii) its critical concentration for polymerization was found to be 3-4-fold lower than of muscle actin, (iv) it predominantly formed bundles rather than single filaments at pH 8.0, (v) it displayed considerably higher ATPase activity during polymerization, (vi) it did not inhibit DNase-I activity, and (vii) it did not bind the F-actin-binding toxin phalloidin or the actin polymerization disrupting agent Latrunculin B. Computational and molecular modeling studies revealed that the observed unconventional behavior of Leishmania actin is related to the diverged amino acid stretches in its sequence, which may lead to changes in the overall charge distribution on its solvent-exposed surface, ATP binding cleft, Mg2+ binding sites, and the hydrophobic loop that is involved in monomer-monomer interactions. Phylogenetically, it is related to ciliate actins, but to the best of our knowledge, no other actin with such unconventional properties has been reported to date. It is therefore suggested that actin in Leishmania may serve as a novel target for design of new antileishmanial drugs.

The C-terminal extension of Leishmania pifanoi amastigote-specific cysteine proteinase Lpcys2: A putative function in macrophage infection

Cysteine proteinases have been implicated in many aspects of protozoan parasite pathogenesis. These hydrolases are normally found as zymogens, and some classes in trypanosomatids possess a long C-terminal extension (CTE), for which no function has been assigned. In this paper we hypothesize that the CTE domain of Lpcys2, the abundant lysosomal cysteine proteinase of Leishmania pifanoi amastigotes, is involved in host cell infection. Confirming previous reports that this peptide is highly immunogenic in Trypanosoma cruzi, we detected antibodies against CTE in sera of leishmaniasis patients. We produced a polyclonal antibody specific to Lpcys2 CTE and determined that this antibody was capable of recognizing both L. pifanoi and Leishmania amazonensis cysteine proteinases. Using this antibody, we detected a predominant localization of Lpcys2 CTE in the lysosome and flagellar pocket of cultured axenic amastigotes of both parasite species; however, its location was shifted towards the surface of the parasites during macrophage infection. We examined the role of Lpcys2 CTE in macrophage infection and found a significant reduction in the percentage of infected cells when macrophages were infected with L. pifanoi and L. amazonensis in the presence of anti-CTE antibody. This study suggests a role for leishmanial cysteine proteinases CTE at early stages of infection.