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.

Inhibitory effects promoted by 5'-nucleotides on the ecto-3'-nucleotidase activity of Leishmania amazonensis.

The protozoan parasite Leishmania amazonensis is the etiological agent of cutaneous leishmaniasis. During its life cycle, the flagellated metacyclic promastigote forms are transmitted to vertebrate hosts by sandfly bites, and they develop into amastigotes inside macrophages, where they multiply. L. amazonensis possesses a bifunctional enzyme, called 3'-nucleotidase/nuclease (3'NT/NU), which is able to hydrolyze extracellular 3'-monophosphorylated nucleosides and nucleic acids. 3'NT/NU plays an important role in the generation of extracellular adenosine and has been described as a key enzyme in the acquisition of purines by trypanosomatids. Furthermore, it has been observed that 3'NT/NU also plays a valuable role in the establishment of parasitic infection. In this context, this study aimed to investigate the modulation of the 3'-nucleotidase (3'NT) activity of L. amazonensis by several nucleotides. It was observed that 3'NT activity is inhibited by micromolar concentrations of guanosine and guanine nucleotides. The inhibition promoted by 5'-GMP on the 3'NT activity of L. amazonensis is reversible and uncompetitive because the addition of the inhibitor decreased the kinetic parameters Km and Vmax. Finally, we found that the addition of 5'-GMP is able to reverse the stimulation promoted by 3'-AMP in a macrophage-parasite interaction assay. The determination of compounds that can inhibit the 3'NT activity of Leishmania is very important because this enzyme does not occur in mammals, making it a potential therapeutic target.

Leishmania chagasi: an ecto-3'-nucleotidase activity modulated by inorganic phosphate and its possible involvement in parasite-macrophage interaction.

In this work we showed that living cells of Leishmania chagasi was able to hydrolyse 3'AMP 10 times more than 5'AMP. When parasites were grown in a low phosphate concentration (2 mM) the cellular proliferation decreased by 50% compared to cells grown in the presence of a high phosphate concentration (80 mM). However, the ecto-3'nucleotidase activity was 2-fold higher when L. chagasi was grown in a low phosphate concentration. This modulation observed on ecto-3'nucleotidase activity was not observed on ecto-5'nucleotidase activity. These results suggest that low concentration of Pi in the culture medium modulates ecto-3'nucleotidase activity that may lead to modulation of important processes for the cell. Interestingly, the macrophage-parasite interaction increased by 45% when L. chagasi were grown at low phosphate concentration compared to the parasites grown in the presence of high phosphate source. Altogether, the results described here suggest that 3'nucleotidase activity modulated by external stimuli, Pi concentration, could be involved on parasite-macrophage interaction.

Trypanosoma cruzi infection is enhanced by vector saliva through immunosuppressant mechanisms mediated by lysophosphatidylcholine.

Trypanosoma cruzi, the etiological agent of Chagas disease, is transmitted by bug feces deposited on human skin during a blood meal. However, parasite infection occurs through the wound produced by insect mouthparts. Saliva of the Triatominae bug Rhodnius prolixus is a source of lysophosphatidylcholine (LPC). Here, we tested the role of both triatomine saliva and LPC on parasite transmission. We show that vector saliva is a powerful inducer of cell chemotaxis. A massive number of inflammatory cells were found at the sites where LPC or saliva was inoculated into the skin of mice. LPC is a known chemoattractant for monocytes, but neutrophil recruitment induced by saliva is LPC independent. The preincubation of peritoneal macrophages with saliva or LPC increased fivefold the association of T. cruzi with these cells. Moreover, saliva and LPC block nitric oxide production by T. cruzi-exposed macrophages. The injection of saliva or LPC into mouse skin in the presence of the parasite induces an up-to-sixfold increase in blood parasitemia. Together, our data suggest that saliva of the Triatominae enhances T. cruzi transmission and that some of its biological effects are attributed to LPC. This is a demonstration that a vector-derived lysophospholipid may act as an enhancing factor of Chagas disease.

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.

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.

Leishmanicidal activity of polyphenolic-rich extract from husk fiber of Cocos nucifera Linn. (Palmae).

The available therapy for leishmaniasis, which affects 2 million people per annum, still causes serious side effects. The polyphenolic-rich extract from the husk fiber of Cocos nucifera Linn. (Palmae) presents antibacterial and antiviral activities, also inhibiting lymphocyte proliferation, as shown by our group in previous works. In the present study, the in vitro leishmanicidal effects of C. nucifera on Leishmania amazonensis were evaluated. The minimal inhibitory concentration of the polyphenolic-rich extract from C. nucifera to completely abrogate parasite growth was 10 microg/ml. Pretreatment of peritoneal mouse macrophages with 10 microg/ml of C. nucifera polyphenolic-rich extract reduced approximately 44% the association index between these macrophages and L. amazonensis promastigotes, with a concomitant increase of 182% in nitric oxide production by the infected macrophage in comparison to nontreated macrophages. These results provide new perspectives on drug development against leishmaniasis, since the extract of C. nucifera at 10 microg/ml is a strikingly potent leishmanicidal substance which inhibited the growth of both promastigote and amastigote developmental stages of L. amazonensis after 60 min, presenting no in vivo allergenic reactions or in vitro cytotoxic effects in mammalian systems.

Characterization of an ecto-ATPase of Tritrichomonas foetus.

In this work, we describe the ability of living Tritrichomonas foetus to hydrolyze extracellular ATP. The addition of MgCl(2) to the assay medium increased the ecto-ATPase activity in a dose-dependent manner. At 5mM ATP, half maximal stimulation of ATP hydrolysis was obtained with 0.46mM MgCl(2). The ecto-ATPase activity was also stimulated by MnCl(2) and CaCl(2), but not by SrCl(2). The Mg(2+)-dependent ATPase presents two apparent K(m) values for Mg-ATP(2-) (K(m1)=0.03 mM and K(m2)=2.01 mM). ATP was the best substrate for this enzyme, although other nucleotides such as ITP, CTP, UTP also produced high reaction rates. GTP produced a low reaction rate and ADP was not a substrate for this enzyme. The Mg(2+)-dependent ecto-ATPase activity was insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A(1), ouabain, furosemide, vanadate, molybdate, sodium fluoride and levamizole. The acid phosphatase inhibitors (vanadate and molybdate) inhibited about 60-70% of the Mg(2+)-independent ecto-ATPase activity, suggesting that the ATP hydrolysis measured in the absence of any metal divalent could, at least in part, also be catalyzed by an ecto-phosphatase present in this cell. In order to confirm the observed Mg(2+)-dependent activity as an ecto-ATPase, we used an impermeant inhibitor, 4,4'-diisothiocyanostylbene-2',2'-disulfonic acid (DIDS) as well as suramin, an antagonist of P(2) purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg(2+)-dependent ATPase activity in a dose-dependent manner. This ecto-ATPase was stimulated by more than 90% by 50mM D-galactose. Since previous results showed that D-galactose exposed on the surface of host cells is involved with T. foetus adhesion, the Mg(2+)-dependent ecto-ATPase may be involved with cellular adhesion and possible pathogenicity.

An ectonucleotide ATP-diphosphohydrolase activity in Trichomonas vaginalis stimulated by galactose and its possible role in virulence.

This work describes the ability of living Trichomonas vaginalis to hydrolyze extracellular ATP (164.0 +/- 13.9 nmol Pi/h x 10(7) cells). This ecto-enzyme was stimulated by ZnCl2, CaCl2 and MgCl2, was insensitive to several ATPase and phosphatase inhibitors and was able to hydrolyze several nucleotides besides ATP. The activity was linear with cell density and with time for at least 60 min. The optimum pH for the T. vaginalis ecto-ATPase lies in the alkaline range. D-galactose, known to be involved in adhesion of T. vaginalis to host cells, stimulated this enzyme by more than 90%. A comparison between two strains of T. vaginalis showed that the ecto-ATPase activity of a fresh isolate was twice as much as that of a strain axenically maintained in culture, through daily passages, for several years. The results suggest a possible role for this ecto-ATPase in adhesion of T. vaginalis to host cells and in its pathogenicity.

Crithidia deanei: influence of parasite gp63 homologue on the interaction of endosymbiont-harboring and aposymbiotic strains with Aedes aegypti midgut.

The present study demonstrates that the endosymbiont of Crithidia deanei influences the expression of surface gp63 molecules. Ultrastructural immunocytochemical analysis shows the presence of the gp63-like protein in the protozoan flagellum and flagellar pocket, either attached to shed membranes or in a free form. This molecule is glycosylphosphatidylinositol (GPI) anchored to the plasma membrane as demonstrated by phospholipase C (PLC) treatment and cross-reacting determinant detection by immunoblotting. The gp63 molecule mediates the adhesive process of the protozoan to Aedes aegypti explanted guts, since the binding was reduced by pre-incubating the C. deanei parasites (wild and aposymbiotic strains) with anti-gp63 antibodies, PLC or PLC followed by anti-gp63 antibodies incubation. In addition, the number of wild C. deanei bound to A. aegypti explanted guts was twice as that of aposymbiotic parasites. Flow cytometry assays revealed that the reactivity of the wild strain with anti-gp63 antibodies was approximately twice as that of the aposymbiotic strain. We may conclude that higher expression of surface gp63 by the wild strain of C. deanei may positively influence this interaction, posing a prominent advantage for the endosymbiont-containing trypanosomatids.

Leishmania amazonensis: Biological and biochemical characterization of ecto-nucleoside triphosphate diphosphohydrolase activities.

The presence of Leishmania amazonensis ecto-nucleoside triphosphate triphosphohydrolase activities was demonstrated using antibodies against different NTPDase members by Western blotting, flow cytometry, and immunoelectron microscopy analysis. Living promastigote cells sequentially hydrolyzed the ATP molecule generating ADP, AMP, and adenosine, indicating that this surface enzyme may play a role in the salvage of purines from the extracellular medium. The L. amazonensis ecto-NTPDase activities were insensitive to Triton X-100, but they were enhanced by divalent cations, such as Mg(2+). In addition, the ecto-NTPDase activities decreased with time for 96 h when promastigotes were grown in vitro. On the other hand, these activities increased considerably when measured in living amastigote forms. Furthermore, the treatment with adenosine, a mediator of several relevant biological phenomena, induced a decrease in the reactivity with anti-CD39 antibody, raised against mammalian E-NTPDase, probably because of down regulation in the L. amazonensis ecto-NTPDase expression. Also, adenosine and anti-NTPDase antibodies induced a significant diminishing in the interaction between promastigotes of L. amazonensis and mouse peritoneal macrophages.

Platelet-activating factor (PAF) activates casein kinase 2 in the protozoan parasite Herpetomonas muscarum muscarum.

Herpetomonas muscarum muscarum is a flagellate parasite of the family Trypanosomatidae, whose cell differentiation can be triggered by the lipid mediator, PAF. In this study we demonstrate for the first time that PAF effect relies on the activation of casein kinase 2 (CK2). The classical antagonist of PAF receptor, WEB 2086, abrogated PAF-enhanced CK2 activity. CK2 activation by PAF was also inhibited when parasite extracts were assayed in the presence of modulators of PKC, MAPK, and both Ser/Thr and Tyr phosphatases. Finally, a cell permeable inhibitor of CK2 (DRB) suppressed PAF-induced cell differentiation in a dose-dependent manner.

Trypanosoma rangeli uptakes the main lipoprotein from the hemolymph of its invertebrate host.

During its life cycle Trypanosoma rangeli crosses the hemolymph of its invertebrate host. In the present study, we demonstrate for the first time the uptake of lipophorin (Lp), the main lipid-transporting particle of insect hemolymph. We observed that living T. rangeli parasites uptake lipids from both 32P- and 3H-, or 125I-labeled Lp. However, the parasites do not uptake any other hemolymphatic protein such as 32P-labeled vitellogenin. The presence of a specific receptor to Lp in the parasite surface is suggested based on experiments using 125I-Lp. We also investigated the intracellular fate of lipids using Texas Red-labeled phosphatidylethanolamine-Lp. Parasites were observed under confocal microscope and displayed fluorescent-labeled lipids close to the flagellar pocket and in vesicles at the posterior region. In conclusion, this study raises a novel set of molecular events which takes place during vector-parasite interaction.