The Enhanced Expression of Cruzipain-Like Molecules in the Phytoflagellate Phytomonas serpens Recovered From the Invertebrate and Plant Hosts.

Phytomonas serpens is a protozoan parasite that alternates its life cycle between two hosts: an invertebrate vector and the tomato fruit. This phytoflagellate is able to synthesize proteins displaying similarity to the cysteine peptidase named cruzipain, an important virulence factor from Trypanosoma cruzi, the etiologic agent of Chagas disease. Herein, the growth of P. serpens in complex medium (BHI) supplemented with natural tomato extract (NTE) resulted in the increased expression of cysteine peptidases, as verified by the hydrolysis of the fluorogenic substrate Z-Phe-Arg-AMC and by gelatin-SDS-PAGE. Phytoflagellates showed no changes in morphology, morphometry and viability, but the proliferation was slightly reduced when cultivated in the presence of NTE. The enhanced proteolytic activity was accompanied by a significant increase in the expression of cruzipain-like molecules, as verified by flow cytometry using anti-cruzipain antibodies. In parallel, parasites incubated under chemically defined conditions (PBS supplemented with glucose) and added of different concentration of NTE revealed an augmentation in the production of cruzipain-like molecules in a typically dose-dependent way. Similarly, P. serpens recovered from the infection of mature tomatoes showed an increase in the expression of molecules homologous to cruzipain; however, cells showed a smaller size compared to parasites grown in BHI medium. Furthermore, phytoflagellates incubated with dissected salivary glands from Oncopeltus fasciatus or recovered from the hemolymph of infected insects also showed a strong enhance in the expression of cruzipain-like molecules that is more relevant in the hemolymph. Collectively, our results showed that cysteine peptidases displaying similarities to cruzipain are more expressed during the life cycle of the phytoflagellate P. serpens both in the invertebrate and plant hosts.

Docking simulation between HIV peptidase inhibitors and Trypanosoma cruzi aspartyl peptidase.

OBJECTIVE: The low investment in research, diagnosis and treatment are factors that contribute to the continuity of Chagas' disease as a neglected tropical diseases (NTDs). In this context, the repositioning of drugs represents a useful strategy, in the search for new chemotherapeutic approaches for NTDs. HIV aspartic peptidase inhibitors (HIV IPs) are good candidates for drug repurposing. Here, we modeled the three dimensional structure of an aspartyl peptidase of Trypanosoma cruzi, the causative agent of Chagas' disease, aligned it to the HIV aspartyl peptidase and performed docking binding assays with the HIV PIs. RESULTS: The 3D structure confirmed the presence of acid aspartic residues, which are critical to enzyme activity. The docking experiment revealed that HIV IPs bind to the active site of the enzyme, being ritonavir and lopinavir the ones with greater affinity. Benznidazole presented the worst binding affinity, this drug is currently used in Chagas' disease treatment and was included as negative control. These results together with previous data on the trypanocidal effect of the HIV PIs support the hypothesis that a T. cruzi aspartyl peptidase can be the intracellular target of these inhibitors. However, the direct demonstration of the inhibition of T. cruzi aspartyl peptidase activity by HIV PIs is still a goal to be persuaded.

In vitro selection of Phytomonas serpens cells resistant to the calpain inhibitor MDL28170: alterations in fitness and expression of the major peptidases and efflux pumps.

The species Phytomonas serpens is known to express some molecules displaying similarity to those described in trypanosomatids pathogenic to humans, such as peptidases from Trypanosoma cruzi (cruzipain) and Leishmania spp. (gp63). In this work, a population of P. serpens resistant to the calpain inhibitor MDL28170 at 70 µ m (MDLR population) was selected by culturing promastigotes in increasing concentrations of the drug. The only relevant ultrastructural difference between wild-type (WT) and MDLR promastigotes was the presence of microvesicles within the flagellar pocket of the latter. MDLR population also showed an increased reactivity to anti-cruzipain antibody as well as a higher papain-like proteolytic activity, while the expression of calpain-like molecules cross-reactive to anti-Dm-calpain (from Drosophila melanogaster) antibody and calcium-dependent cysteine peptidase activity were decreased. Gp63-like molecules also presented a diminished expression in MDLR population, which is probably correlated to the reduction in the parasite adhesion to the salivary glands of the insect vector Oncopeltus fasciatus. A lower accumulation of Rhodamine 123 was detected in MDLR cells when compared with the WT population, a phenotype that was reversed when MDLR cells were treated with cyclosporin A and verapamil. Collectively, our results may help in the understanding of the roles of calpain inhibitors in trypanosomatids.

RNA viruses in trypanosomatid parasites: a historical overview

Viruses of trypanosomatids are now being extensively studied because of their diversity and the roles they play in flagellates' biology. Among the most prominent examples are leishmaniaviruses implicated in pathogenesis of Leishmania parasites. Here, we present a historical overview of this field, starting with early reports of virus-like particles on electron microphotographs, and culminating in detailed molecular descriptions of viruses obtained using modern next generation sequencing-based techniques. Because of their diversity, different life cycle strategies and host specificity, we believe that trypanosomatids are a fertile ground for further explorations to better understand viral evolution, routes of transitions, and molecular mechanisms of adaptation to different hosts.

The potent cell permeable calpain inhibitor MDL28170 affects the interaction of Leishmania amazonensis with macrophages and shows anti-amastigote activity.

Since the discovery of the28 first drugs used in leishmaniasis treatment up to now, the search for compounds with anti-Leishmania activity without toxic effects and able to overcome the emergency of resistant strains remains a major goal to combat this neglected disease. With this in mind, in the present work, we evaluated the effects of the calpain inhibitor MDL28170 on the interaction process of Leishmania amazonensis promastigote forms with murine peritoneal macrophages and on the intracellular amastigotes. Our results showed that the calpain inhibitor MDL28170 at 15 and 30µM significantly reduced the interaction process of promastigotes with macrophages by 16% and 41%, respectively. The inhibitor was also able to drastically reduce the number of infected macrophages in a time- and dose-dependent manner: after only 24h, MDL28170 was able to significantly diminish the infection rate, presenting an IC50 value of 18.2µM for amastigotes. The treatment with MDL28170 did not alter the nitric oxide production, but the production of TNF-α was significantly raised. Altogether, the results presented here contribute to the search of new proteolytic inhibitors able to act in a selective and effective manner against the diseases caused by trypanosomatids.

The calpain inhibitor MDL28170 induces the expression of apoptotic markers in Leishmania amazonensis promastigotes.

BACKGROUND: Human cutaneous leishmaniasis is caused by distinct species, including Leishmania amazonensis. Treatment of cutaneous leishmaniasis is far from satisfactory due to increases in drug resistance and relapses, and toxicity of compounds to the host. As a consequence for this situation, the development of new leishmanicidal drugs and the search of new targets in the parasite biology are important goals. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we investigated the mechanism of death pathway induced by the calpain inhibitor MDL28170 on Leishmania amazonensis promastigote forms. The combined use of different techniques was applied to contemplate this goal. MDL28170 treatment with IC50 (15 µM) and two times the IC50 doses induced loss of parasite viability, as verified by resazurin assay, as well as depolarization of the mitochondrial membrane, which was quantified by JC-1 staining. Scanning and transmission electron microscopic images revealed drastic alterations on the parasite morphology, some of them resembling apoptotic-like death, including cell shrinking, surface membrane blebs and altered chromatin condensation pattern. The lipid rearrangement of the plasma membrane was detected by Annexin-V labeling. The inhibitor also induced a significant increase in the proportion of cells in the sub-G0/G1 phase, as quantified by propidium iodide staining, as well as genomic DNA fragmentation, detected by TUNEL assay. In cells treated with MDL28170 at two times the IC50 dose, it was also possible to observe an oligonucleossomal DNA fragmentation by agarose gel electrophoresis. CONCLUSIONS/SIGNIFICANCE: The data presented in the current study suggest that MDL28170 induces apoptotic marker expression in promastigotes of L. amazonensis. Altogether, the results described in the present work not only provide a rationale for further exploration of the mechanism of action of calpain inhibitors against trypanosomatids, but may also widen the investigation of the potential clinical utility of calpain inhibitors in the chemotherapy of leishmaniases.

Differential expression of cruzipain- and gp63-like molecules in the phytoflagellate trypanosomatid Phytomonas serpens induced by exogenous proteins.

Phytomonas serpens synthesizes metallo- and cysteine-proteases that are related to gp63 and cruzipain, respectively, two virulence factors produced by pathogenic trypanosomatids. Here, we described the cellular distribution of gp63- and cruzipain-like molecules in P. serpens through immunocytochemistry and confocal fluorescence microscopy. Both proteases were detected in distinct cellular compartments, presenting co-localization in membrane domains and intracellular regions. Subsequently, we showed that exogenous proteins modulated the production of both protease classes, but in different ways. Regarding the metalloprotease, only fetal bovine serum (FBS) influenced the gp63 expression, reducing its surface exposition (≈30%). Conversely, the cruzipain-like molecule was differentially modulated according to the proteins: human and bovine albumins reduced its expression around 50% and 35%, respectively; mucin and FBS did not alter its production, while IgG and hemoglobin drastically enhanced its surface exposition around 7- and 11-fold, respectively. Additionally, hemoglobin induced an augmentation in the cell-associated cruzipain-like activity in a dose-dependent manner. A twofold increase of the secreted cruzipain-like protein was detected after parasite incubation with 1% hemoglobin compared to the parasites incubated in PBS-glucose. The results showed the ability of P. serpens in modulating the expression and the activity of proteolytic enzymes after exposition to exogenous proteins, with emphasis in its cruzipain-like molecules.

Cysteine proteinases from promastigotes of Leishmania (Viannia) braziliensis.

Leishmania (Viannia) braziliensis is the major causative agent of American tegumentary leishmaniasis, a disease that has a wide geographical distribution and is a severe public health problem. The cysteine proteinase B (CPB) from Leishmania spp. represents an important virulence factor. In this study, we characterized and localized cysteine proteinases in L. (V.) braziliensis promastigotes. By a combination of triton X-114 extraction, concanavalin A-affinity, and ion exchange chromatographies, we obtained an enriched fraction of hydrophobic proteins rich in mannose residues. This fraction contained two proteinases of 63 and 43 kDa, which were recognized by a CPB antiserum, and were partially sensitive to E-64 in enzymatic assays with the peptide Glu-Phe-Leu. In confocal microscopy, the CPB homologues localized in the peripheral region of the parasite. This data together with direct agglutination and flow cytometry assays suggest a surface localization of the CPB homologues. The incubation of intact promastigotes with phospholipase C reduced the number of CPB-positive cells, while anti-cross-reacting determinant and anti-CPB antisera recognized two polypeptides (63 and 43 kDa) derived from phospholipase C treatment, suggesting that some CPB isoforms may be glycosylphosphatidylinositol-anchored. Collectively, our results suggest the presence of CPB homologues in L. braziliensis surface and highlight the need for further studies on L. braziliensis cysteine proteinases, which require enrichment methods for enzymatic detection.

Cysteine peptidases from Phytomonas 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.

Sialoglycoconjugates in Herpetomonas megaseliae: role in the adhesion to insect host epithelial cells.

Herpetomonas megaseliae is a monoxenic trypanosomatid isolated from the phorid fly Megaselia scalaris. In the present report, the expression of cell surface sialoglycoconjugates in this parasite was analyzed by Western blotting, flow cytometry and fluorescence microscopy analyses using lectins that specifically recognize sialic acid residues. A strong reaction was detected when parasites were treated with Limax flavus, Maackia amurensis and Sambucus nigra lectins. Analysis of crude protein extracts by Western blotting revealed that bands with molecular masses ranging from 19 to 80 kDa were reactive to these lectins, which showed a sugar-inhibited recognition with the parasite extract. These results indicated that molecules containing alpha2,3- and alpha2,6-sialylgalactosyl sequences are present in this protozoan. The role of the surface sialomolecules in the interaction with explanted guts from Aedes aegypti was assessed. The interaction of H. megaseliae with the insect gut was strongly inhibited in the presence of mucin (71%), fetuin (68%) and sialyllactose (68%). Collectively, our results suggest a possible involvement of sialomolecules in the interaction between this insect trypanosomatid and the invertebrate host.

HIV aspartyl peptidase inhibitors interfere with cellular proliferation, ultrastructure and macrophage infection of Leishmania amazonensis.

BACKGROUND: Leishmania is the etiologic agent of leishmanisais, a protozoan disease whose pathogenic events are not well understood. Current therapy is suboptimal due to toxicity of the available therapeutic agents and the emergence of drug resistance. Compounding these problems is the increase in the number of cases of Leishmania-HIV coinfection, due to the overlap between the AIDS epidemic and leishmaniasis. METHODOLOGY/PRINCIPAL FINDINGS: In the present report, we have investigated the effect of HIV aspartyl peptidase inhibitors (PIs) on the Leishmania amazonensis proliferation, ultrastructure, interaction with macrophage cells and expression of classical peptidases which are directly involved in the Leishmania pathogenesis. All the HIV PIs impaired parasite growth in a dose-dependent fashion, especially nelfinavir and lopinavir. HIV PIs treatment caused profound changes in the leishmania ultrastructure as shown by transmission electron microscopy, including cytoplasm shrinking, increase in the number of lipid inclusions and some cells presenting the nucleus closely wrapped by endoplasmic reticulum resembling an autophagic process, as well as chromatin condensation which is suggestive of apoptotic death. The hydrolysis of HIV peptidase substrate by L. amazonensis extract was inhibited by pepstatin and HIV PIs, suggesting that an aspartyl peptidase may be the intracellular target of the inhibitors. The treatment with HIV PIs of either the promastigote forms preceding the interaction with macrophage cells or the amastigote forms inside macrophages drastically reduced the association indexes. Despite all these beneficial effects, the HIV PIs induced an increase in the expression of cysteine peptidase b (cpb) and the metallopeptidase gp63, two well-known virulence factors expressed by Leishmania spp. CONCLUSIONS/SIGNIFICANCE: In the face of leishmaniasis/HIV overlap, it is critical to further comprehend the sophisticated interplays among Leishmania, HIV and macrophages. In addition, there are many unresolved questions related to the management of Leishmania-HIV-coinfected patients. For instance, the efficacy of therapy aimed at controlling each pathogen in coinfected individuals remains largely undefined. The results presented herein add new in vitro insight into the wide spectrum efficacy of HIV PIs and suggest that additional studies about the synergistic effects of classical antileishmanial compounds and HIV PIs in macrophages coinfected with Leishmania and HIV-1 should be performed.

Cysteine peptidases in the tomato trypanosomatid Phytomonas serpens: influence of growth conditions, similarities with cruzipain and secretion to the extracellular environment.

We have characterized the cysteine peptidase production by Phytomonas serpens, a tomato trypanosomatid. The parasites were cultivated in four distinct media, since growth conditions could modulate the synthesis of bioactive molecules. The proteolytic profile has not changed qualitatively regardless the media, showing two peptidases of 38 and 40kDa; however, few quantitative changes were observed including a drastic reduction (around 70%) on the 40 and 38kDa peptidase activities when parasites were grown in yeast extract and liver infusion trypticase medium, respectively, in comparison with parasites cultured in Warren medium. The time-span of growth did not significantly alter the protein and peptidase expression. The proteolytic activities were blocked by classical cysteine peptidase inhibitors (E-64, leupeptin, and cystatin), being more active at pH 5.0 and showing complete dependence to reducing agents (dithiothreitol and l-cysteine) for full activity. The cysteine peptidases were able to hydrolyze several proteinaceous substrates, including salivary gland proteins from Oncopeltus fasciatus, suggesting broad substrate utilization. By means of agglutination, fluorescence microscopy, flow cytometry and Western blotting analyses we showed that both cysteine peptidases produced by P. serpens share common epitopes with cruzipain, the major cysteine peptidase of Trypanosoma cruzi. Moreover, our data suggest that the 40kDa cysteine peptidase was located at the P. serpens cell surface, attached to membrane domains via a glycosylphosphatidylinositol anchor. The 40kDa peptidase was also detected in the cell-free culture supernatant, in an active form, which suggests secretion of this peptidase to the extracellular environment.

Phytomonas serpens: immunological similarities with the human trypanosomatid pathogens.

The present review provides an overview of recent discoveries concerning the immunological similarities between Phytomonas serpens, a tomato parasite, and human trypanosomatid pathogens, with special emphasis on peptidases. Leishmania spp. and Trypanosoma cruzi express peptidases that are well-known virulence factors, named leishmanolysin and cruzipain. P. serpens synthesizes two distinct classes of proteolytic enzymes, metallo- and cysteine-type peptidases, that share common epitopes with leishmanolysin and cruzipain, respectively. The leishmanolysin-like and cruzipain-like molecules from P. serpens participate in several biological processes including cellular growth and adhesion to the salivary glands of Oncopeltus fasciatus, a phytophagous insect experimental model. Since previous reports demonstrated that immunization of mice with P. serpens induced a partial protective immune response against T. cruzi, this plant trypanosomatid may be a suitable candidate for vaccine studies. Moreover, comparative approaches in the Trypanosomatidae family may be useful to understand kinetoplastid biology, biochemistry and evolution.

The ubiquitous gp63-like metalloprotease from lower trypanosomatids: in the search for a function

Plant and insect trypanosomatids constitute the "lower trypanosomatids", which have been used routinely as laboratory models for biochemical and molecular studies because they are easily cultured under axenic conditions, and they contain homologues of virulence factors from the classic human trypanosomatid pathogens. Among the molecular factors that contribute to Leishmania spp. virulence and pathogenesis, the major surface protease, alternatively called MSP, PSP, leishmanolysin, EC 3.4.24.36 and gp63, is the most abundant surface protein of Leishmania promastigotes. A myriad of functions have been described for the gp63 from Leishmania spp. when the metacyclic promastigote is inside the mammalian host. However, less is known about the functions performed by this molecule in the invertebrate vector. Intriguingly, gp63 is predominantly expressed in the insect stage of Leishmania, and in all insect and plant trypanosomatids examined so far. The gp63 homologues found in lower trypanosomatids seem to play essential roles in the nutrition as well as in the interaction with the insect epithelial cells. Since excellent reviews were produced in the last decade regarding the roles played by proteases in the vertebrate hosts, we focused in the recent developments in our understanding of the biochemistry and cell biology of gp63-like proteins in lower trypanosomatids.

Tripanossomatídeos de insetos e de plantas são informalmente denominados de "tripanossomatídeos inferiores". Estes microrganismos são utilizados rotineiramente como modelos para estudos de bioquímica e de biologia molecular porque são facilmente cultivados sob condições axênicas e porque possuem homólogos aos fatores de virulência encontrados nos tripanossomatídeos que são patógenos humanos clássicos. Dentre os fatores moleculares que contribuem para a virulência e patogênese de Leishmania spp. destaca-se a principal protease de superfície, também conhecida como MSP, PSP, leishmanolisina, EC 3.4.24.36 e gp63, que é a proteína de superfície mais abundante encontrada nas formas promastigotas de Leishmania. Diversas funções foram descritas para a gp63 de Leishmania no hospedeiro vertebrado. Entretanto, pouco é conhecido sobre as funções desempenhadas por essa molécula no inseto vetor. Curiosamente, a gp63 é predominantemente expressa na forma evolutiva de Leishmania encontrada no inseto, e em todos os tripanossomatídeos de insetos e plantas analisados até o presente momento. Os homólogos da gp63 presentes nos tripanossomatídeos inferiores desempenham um papel essencial na nutrição assim como na interação com as células epiteliais do inseto. Uma vez que revisões de excelente qualidade foram produzidas na última década sobre a função de proteases nos hospedeiros vertebrados, nesta revisão nós abordamos os recentes progressos sobre os aspectos bioquímicos e as prováveis funções biológicas desempenhadas pelas proteínas homólogas à gp63 nos tripanossomatídeos inferiores.

Antileishmanial activity of MDL 28170, a potent calpain inhibitor.

Several calpain inhibitors are under development and some are useful agents against important human pathogens. We therefore investigated the effect of MDL 28170, a potent calpain inhibitor, on the growth of Leishmania amazonensis. After 48 h of treatment, the inhibitor exhibited a dose-dependent antileishmanial activity, with a 50% lethal dose (LD(50)) of 23.3 microM. The inhibitor promoted cellular alterations, such as the parasites becoming short and round. A calpain-like protein migrating at 80 kDa was identified by Western blotting. In addition, the calpain-like molecules were identified on the cell surface of the flagellate. These results add new in vitro insights into the exploitation of calpain inhibitors in treating parasitic infections and add this family of peptidases to the list of potential targets for development of more potent and specific inhibitors against trypanosomatids.

Gp63-like molecules in Phytomonas serpens: possible role in the insect interaction.

In this study, we demonstrated that metallopeptidase inhibitors (EDTA, EGTA, and 1,10-phenanthroline) were able to arrest Phytomonas serpens growth in distinct patterns. This parasite released exclusively metallopeptidases to the extracellular environment, whereas in cellular extracts only cysteine peptidases were detected. In addition, an extracellular polypeptide of 60 kDa reacted in Western blotting probed with polyclonal antibody raised against gp63 of Leishmania amazonensis. In the cellular parasite extract, this antibody recognized bands migrating at 63 and 52 kDa, which partitioned on both aqueous and membrane-rich fractions. Flow cytometry and fluorescence microscopy analyses showed that the gp63-like molecules have a surface location. Moreover, phospholipase C (PLC)-treated parasites reduced the number of gp63-positive cells. The anti-cross-reacting determinant (CRD) and anti-gp63 antibodies recognized the 60-kDa band in the supernatant from PLC-treated cells, suggesting that this protein is glycosylphosphatidylinositol-anchored to the plasma membrane. This is the first report on the presence of gp63-like molecules in members of the Phytomonas genus. The pretreatment of the parasites with anti-gp63 antibody significantly diminished their adhesion index to explanted salivary glands of the phytophagous insect Oncopeltus fasciatus, suggesting a potential involvement of the gp63-like molecules in the adhesive process of this plant trypanosomatid.

Peptidases and gp63-like proteins in Herpetomonas megaseliae: possible involvement in the adhesion to the invertebrate host.

The cell-associated and extracellular peptidases of Herpetomonas megaseliae grown in brain-heart infusion and in modified Roitman's complex media were analyzed by measuring peptidase activity on gelatin, casein and hemoglobin in zymograms. Casein was the best proteinaceous substrate for the peptidase detection on both growth conditions. However, no proteolytic activity was detected when hemoglobin was used. Our results showed that cellular cysteine peptidase (115-100, 40 and 35 kDa) and metallopeptidase (70 and 60 kDa) activities were detected on both media in casein and gelatin zymograms. Additionally, the use of casein in the gel revealed a distinct acidic metallopeptidase of 50 kDa when the parasite was cultured in the modified Roitman's complex medium. Irrespective of the culture medium composition, H. megaseliae released metallopeptidases exclusively in the extracellular environment. The presence of gp63-like molecules on the H. megaseliae surface was shown by flow cytometry using anti-gp63 antibody raised against recombinant gp63 from Leishmania mexicana. The pre-treatment of parasites with phospholipase C reduced the number of gp63-positive cells, suggesting that these molecules were glycosylphosphatidylinositol-anchored to the surface. Additionally, the supernatant obtained from phospholipase C-treated cells and probed with anti-cross-reacting determinant confirmed that at least a 52 kDa gp63-like molecule is glycosylphosphatidylinositol-anchored. Furthermore, we assessed a possible function for the gp63-like molecules in H. megaseliae on the interaction with explanted guts of its original host, Megaselia scalaris, and with an experimental model employing Aedes aegypti. Parasites pre-treated with either anti-gp63 antibody or phospholipase C showed a significant reduction in the adhesion to M. scalaris and A. aegypti guts. Similarly, the pre-treatment of the explanted guts with purified gp63 diminished the interaction process. Collectively, these results corroborate the ubiquitous existence of gp63 homologues in insect trypanosomatids and the potential adhesion of these molecules to invertebrate host tissues.

Phytomonas serpens: cysteine peptidase inhibitors interfere with growth, ultrastructure and host adhesion.

In this study, we report the ultrastructural and growth alterations caused by cysteine peptidase inhibitors on the plant trypanosomatid Phytomonas serpens. We showed that the cysteine peptidase inhibitors at 10 microM were able to arrest cellular growth as well as promote alterations in the cell morphology, including the parasites becoming short and round. Additionally, iodoacetamide induced ultrastructural alterations, such as disintegration of cytoplasmic organelles, swelling of the nucleus and kinetoplast-mitochondrion complex, which culminated in parasite death. Leupeptin and antipain induced the appearance of microvillar extensions and blebs on the cytoplasmic membrane, resembling a shedding process. A 40 kDa cysteine peptidase was detected in hydrophobic and hydrophilic phases of P. serpens cells after Triton X-114 extraction. Additionally, we have shown through immunoblotting that anti-cruzipain polyclonal antibodies recognised two major polypeptides in P. serpens, including a 40 kDa component. Flow cytometry analysis confirmed that this cruzipain-like protein has a location on the cell surface. Ultrastructural immunocytochemical analysis demonstrated the presence of the cruzipain-like protein on the surface and in small membrane fragments released from leupeptin-treated parasites. Furthermore, the involvement of cysteine peptidases of P. serpens in the interaction with explanted salivary glands of the phytophagous insect Oncopeltus fasciatus was also investigated. When P. serpens cells were pre-treated with either cysteine peptidase inhibitors or anti-cruzipain antibody, a significant reduction of the interaction process was observed. Collectively, these results suggest that cysteine peptidases participate in several biological processes in P. serpens including cell growth and interaction with the invertebrate vector.

A novel extracellular calcium-dependent cysteine proteinase from Crithidia deanei.

An extracellular cysteine proteinase from an aposymbiotic strain of Crithidia deanei was purified 39-fold by a combination of anion-exchange and gel filtration chromatographies. The native molecular mass of this proteinase was estimated to be 225 kDa by gel filtration chromatography and it migrates in SDS-PAGE as a single band of 80 kDa. The optimal enzymatic activity on gelatin was found to occur in the presence of calcium at a neutral pH and at 28 degrees C. The enzyme was completely blocked by E-64 and EGTA, and partially inhibited by iodoacetamide, leupeptin, and EDTA. Compounds such as PMSF, aprotinin, and pepstatin weakly inhibited the enzyme. The protein purified in the present work shares some features with those of the family of neutral calcium-dependent cysteine proteinases named calpains, previously detected in the family Trypanosomatidae as cell-associated enzymes in Leishmania donovani and Trypanosoma brucei. The cysteine proteinase from C. deanei is distinct from the well-characterized mammalian calpains, but some degree of similarity is displayed to invertebrate calpain-related enzymes.