A novel receptor for platelet-activating factor and lysophosphatidylcholine in Trypanosoma cruzi.

The lipid mediators, platelet-activating factor (PAF) and lysophosphatidylcholine (LPC), play relevant pathophysiological roles in Trypanosoma cruzi infection. Several species of LPC, including C18:1 LPC, which mimics the effects of PAF, are synthesized by T. cruzi. The present study identified a receptor in T. cruzi, which was predicted to bind to PAF, and found it to be homologous to members of the progestin and adiponectin family of receptors (PAQRs). We constructed a three-dimensional model of the T. cruzi PAQR (TcPAQR) and performed molecular docking to predict the interactions of the TcPAQR model with C16:0 PAF and C18:1 LPC. We knocked out T. cruzi PAQR (TcPAQR) gene and confirmed the identity of the expressed protein through immunoblotting and immunofluorescence assays using an anti-human PAQR antibody. Wild-type and knockout (KO) parasites were also used to investigate the in vitro cell differentiation and interactions with peritoneal mouse macrophages; TcPAQR KO parasites were unable to react to C16:0 PAF or C18:1 LPC. Our data are highly suggestive that PAF and LPC act through TcPAQR in T. cruzi, triggering its cellular differentiation and ability to infect macrophages.

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.

Interplay Between EGFR and the Platelet-Activating Factor/PAF Receptor Signaling Axis Mediates Aggressive Behavior of Cervical Cancer.

Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase widely expressed in cervical tumors, being correlated with adverse clinical outcomes. EGFR may be activated by a diversity of mechanisms, including transactivation by G-protein coupled receptors (GPCRs). Studies have also shown that platelet-activating factor (PAF), a pro-inflammatory phospholipid mediator, plays an important role in the cancer progression either by modulating the cancer cells or the tumor microenvironment. Most of the PAF effects seem to be mediated by the interaction with its receptor (PAFR), a member of the GPCRs family. PAFR- and EGFR-evoked signaling pathways contribute to tumor biology; however, the interplay between them remains uninvestigated in cervical cancer. In this study, we employed The Cancer Genome Atlas (TCGA) and cancer cell lines to evaluate possible cooperation between EGFR, PAFR, and lysophosphatidylcholine acyltransferases (LPCATs), enzymes involved in the PAF biosynthesis, in the context of cervical cancer. It was observed a strong positive correlation between the expression of EGFR × PAFR and EGFR × LPCAT2 in 306 cervical cancer samples. The increased expression of LPCAT2 was significantly correlated with poor overall survival. Activation of EGFR upregulated the expression of PAFR and LPCAT2 in a MAPK-dependent fashion. At the same time, PAF showed the ability to transactivate EGFR leading to ERK/MAPK activation, cyclooxygenase-2 (COX-2) induction, and cell migration. The positive crosstalk between the PAF-PAFR axis and EGFR demonstrates a relevant linkage between inflammatory and growth factor signaling in cervical cancer cells. Finally, combined PAFR and EGFR targeting treatment impaired clonogenic capacity and viability of aggressive cervical cancer cells more strongly than each treatment separately. Collectively, we proposed that EGFR, LPCAT2, and PAFR emerge as novel targets for cervical cancer therapy.

Lysophosphatidylcholine triggers cell differentiation in the protozoan parasite Herpetomonas samuelpessoai through the CK2 pathway.

BACKGROUND: Protozoa are distantly related to vertebrates but present some features of higher eukaryotes, making them good model systems for studying the evolution of basic processes such as the cell cycle. Herpetomonas samuelpessoai is a trypanosomatid parasite isolated from the hemipteran insect Zelus leucogrammus. Lysophosphatidylcholine (LPC) is implicated in the transmission and establishment of Chagas disease, whose etiological agent is Trypanosoma cruzi. LPC is synthesized by T. cruzi and its vectors, the hemipteran Rhodnius prolixus and Triatoma infestans. Platelet-activating factor (PAF), a phospholipid with potent and diverse physiological and pathophysiological actions, is a powerful inducer of cell differentiation in Herpetomonas muscarum muscarum and T. cruzi. The enzyme phospholipase A2 (PLA2) catalyzes the hydrolysis of the 2-ester bond of 3-sn-phosphoglyceride, transforming phosphatidylcholine (PC) into LPC. METHODS: In this study, we evaluated cellular differentiation, PLA2 activity and protein kinase CK2 activity of H. samuelpessoai in the absence and in the presence of LPC and PAF. RESULTS: We demonstrate that both PC and LPC promoted a twofold increase in the cellular differentiation of H. samuelpessoai, through CK2, with a concomitant inhibition of its cell growth. Intrinsic PLA2 most likely directs this process by converting PC into LPC. CONCLUSIONS: Our results suggest that the actions of LPC on H. samuelpessoai occur upon binding to a putative PAF receptor and that the protein kinase CK2 plays a major role in this process. Cartoon depicting a model for the synthesis and functions of LPC in Herpetomonas samuelpessoai, based upon our results regarding the role of LPC on the cell biology of Trypanosoma cruzi [28-32]. N nucleus, k kinetoplast, PC phosphatidylcholine, LPC lysophosphatidylcholine, PLA2 phospholipase A2, PAFR putative PAF receptor in trypanosomatids [65], CK2 protein kinase CK2 [16].

Natural infection by the protozoan Leptomonas wallacei impacts the morphology, physiology, reproduction, and lifespan of the insect Oncopeltus fasciatus.

Trypanosomatids are protozoan parasites that infect thousands of globally dispersed hosts, potentially affecting their physiology. Several species of trypanosomatids are commonly found in phytophagous insects. Leptomonas wallacei is a gut-restricted insect trypanosomatid only retrieved from Oncopeltus fasciatus. The insects get infected by coprophagy and transovum transmission of L. wallacei cysts. The main goal of the present study was to investigate the effects of a natural infection by L. wallacei on the hemipteran insect O. fasciatus, by comparing infected and uninfected individuals in a controlled environment. The L. wallacei-infected individuals showed reduced lifespan and morphological alterations. Also, we demonstrated a higher infection burden in females than in males. The infection caused by L. wallacei reduced host reproductive fitness by negatively impacting egg load, oviposition, and eclosion, and promoting an increase in egg reabsorption. Moreover, we associated the egg reabsorption observed in infected females, with a decrease in the intersex gene expression. Finally, we suggest alterations in population dynamics induced by L. wallacei infection using a mathematical model. Collectively, our findings demonstrated that L. wallacei infection negatively affected the physiology of O. fasciatus, which suggests that L. wallacei potentially has a vast ecological impact on host population growth.

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.

Effects of linalool and eugenol on the survival of Leishmania (L.) infantum chagasi within macrophages.

The most commonly used drugs against visceral leishmaniasis are based on pentavalent antimonial compounds, which have played a fundamental role in therapy for over 70 years. However, the treatment is painful and has severe toxic side effects that can be fatal. Antimonial resistance is spreading and reaching alarming proportions. Linalool and eugenol have been shown to kill Leishmania (L.) amazonensis and Trypanosoma cruzi at low doses. In the present study, we demonstrate the effects of linalool and eugenol, components of essential oils, on Leishmania (L.) infantum chagasi, one of the causative agents of visceral leishmaniasis. We compared the effects of those compounds to the effects of glucantime, a positive control. In L. infantum chagasi killing assays, the LD50 for eugenol was 220µg/ml, and that for linalool was 550µg/ml. L. infantum chagasi was added to cultures of peritoneal mouse macrophages for four hours prior to drug treatment. Eugenol and linalool significantly decreased the number of parasites within the macrophages. Eugenol and linalool enhanced the activities of the L. infantum chagasi protein kinases PKA and PKC. Linalool also decreased L. infantum chagasi oxygen consumption. In conclusion, both linalool and eugenol promoted a decrease in the proliferation and viability of L. infantum chagasi. These effects were more pronounced during the interaction between the parasites and peritoneal mouse macrophages.

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.

Here, There, and Everywhere: The Ubiquitous Distribution of the Immunosignaling Molecule Lysophosphatidylcholine and Its Role on Chagas Disease.

Chagas disease is a severe illness, which can lead to death if the patients are not promptly treated. The disease is caused by the protozoan parasite Trypanosoma cruzi, which is mostly transmitted by a triatomine insect vector. There are 8-10 million people infected with T. cruzi in the world, but the transmission of such disease by bugs occurs only in the Americas, especially Latin America. Chronically infected patients will develop cardiac diseases (30%) and up digestive, neurological, or mixed disorders (10%). Lysophosphatidylcholine (LPC) is the major phospholipid component of oxidized low-density lipoproteins associated with atherosclerosis-related tissue damage. Insect-derived LPC powerfully attracts inflammatory cells to the site of the insect bite, enhances parasite invasion, and inhibits the production of nitric oxide by T. cruzi-stimulated macrophages. The recognition of the ubiquitous presence of LPC on the vector saliva, its production by the parasite itself and its presence both on patient plasma and its role on diverse host × parasite interaction systems lead us to compare its distribution in nature with the title of the famous Beatles song "Here, There and Everywhere" recorded exactly 50 years ago in 1966. Here, we review the major findings pointing out the role of such molecule as an immunosignaling modulator of Chagas disease transmission. Also, we believe that future investigation of the connection of this ubiquity and the immune role of such molecule may lead in the future to novel methods to control parasite transmission, infection, and pathogenesis.

Structural and functional analysis of a platelet-activating lysophosphatidylcholine of Trypanosoma cruzi.

BACKGROUND: Trypanosoma cruzi is the causative agent of the life-threatening Chagas disease, in which increased platelet aggregation related to myocarditis is observed. Platelet-activating factor (PAF) is a potent intercellular lipid mediator and second messenger that exerts its activity through a PAF-specific receptor (PAFR). Previous data from our group suggested that T. cruzi synthesizes a phospholipid with PAF-like activity. The structure of T. cruzi PAF-like molecule, however, remains elusive. METHODOLOGY/PRINCIPAL FINDINGS: Here, we have purified and structurally characterized the putative T. cruzi PAF-like molecule by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). Our ESI-MS/MS data demonstrated that the T. cruzi PAF-like molecule is actually a lysophosphatidylcholine (LPC), namely sn-1 C18:1(delta 9)-LPC. Similar to PAF, the platelet-aggregating activity of C18:1-LPC was abrogated by the PAFR antagonist, WEB 2086. Other major LPC species, i.e., C16:0-, C18:0-, and C18:2-LPC, were also characterized in all T. cruzi stages. These LPC species, however, failed to induce platelet aggregation. Quantification of T. cruzi LPC species by ESI-MS revealed that intracellular amastigote and trypomastigote forms have much higher levels of C18:1-LPC than epimastigote and metacyclic trypomastigote forms. C18:1-LPC was also found to be secreted by the parasite in extracellular vesicles (EV) and an EV-free fraction. A three-dimensional model of PAFR was constructed and a molecular docking study was performed to predict the interactions between the PAFR model and PAF, and each LPC species. Molecular docking data suggested that, contrary to other LPC species analyzed, C18:1-LPC is predicted to interact with the PAFR model in a fashion similar to PAF. CONCLUSIONS/SIGNIFICANCE: Taken together, our data indicate that T. cruzi synthesizes a bioactive C18:1-LPC, which aggregates platelets via PAFR. We propose that C18:1-LPC might be an important lipid mediator in the progression of Chagas disease and its biosynthesis could eventually be exploited as a potential target for new therapeutic interventions.

The immune response of hemocytes of the insect Oncopeltus fasciatus against the flagellate Phytomonas serpens.

The genus Phytomonas includes parasites that are etiological agents of important plant diseases, especially in Central and South America. These parasites are transmitted to plants via the bite of an infected phytophagous hemipteran. Despite the economic impact of these parasites, many basic questions regarding the genus Phytomonas remain unanswered, such as the mechanism by which the parasites cope with the immune response of the insect vector. In this report, using a model of systemic infection, we describe the function of Oncopeltus fasciatus hemocytes in the immune response towards the tomato parasite Phytomonas serpens. Hemocytes respond to infection by trapping parasites in nodular structures and phagocytizing the parasites. In electron microscopy of hemocytes, parasites were located inside vacuoles, which appear fused with lysosomes. The parasites reached the O. fasciatus salivary glands at least six hours post-infection. After 72 hours post-infection, many parasites were attached to the salivary gland outer surface. Thus, the cellular responses did not kill all the parasites.

Leishmania amazonensis: characterization of an ecto-3'-nucleotidase activity and its possible role in virulence.

Ecto-3'-nucleotidase/nuclease (3'NT/NU) is a membrane-bound enzyme that plays a key role in the nutrition of Leishmania sp. protozoan parasites. This enzyme generates nucleosides via hydrolyzes of 3'mononucleotides and nucleic acids, which enter the cell by specific transporters. In this work, we identify and characterize Leishmania amazonensis ecto-3'-nucleotidase activity (La3'-nucleotidase), report ammonium tetrathiomolybdate (TTM) as a novel La3'-nucleotidase inhibitor and approach the possible involvement of ecto-3'-nucleotidase in cellular adhesion. La3'-nucleotidase presented characteristics similar to those reported for the class I single-strand nuclease family; a molecular weight of approximately 40 kDa and optimum activity in an alkaline pH range were observed. Although it is conserved among the genus, La3'-nucleotidase displays different kinetic properties; it can be inhibited by vanadate, molybdate and Cu(2+) ions. Interestingly, ecto-3'-nucleotidase activity is 60-fold higher than that of ecto-5'-nucleotidase in L. amazonensis. Additionally, ecto-3'-nucleotidase activity is two-fold higher in virulent L. amazonensis cells than in avirulent ones. Notably, macrophage-parasite attachment/invasion was increased by 400% in the presence of adenosine 3'-monophosphate (3'AMP); however, this effect was reverted by TTM treatment. We believe that La3'-nucleotidase may play a significant role in the generation of adenosine, which may contribute to mammalian host immune response impairment and establishment of infection.

Possible roles of ectophosphatases in host-parasite interactions.

The interaction and survival of pathogens in hostile environments and in confrontation with host immune responses are important mechanisms for the establishment of infection. Ectophosphatases are enzymes localized at the plasma membrane of cells, and their active sites face the external medium rather than the cytoplasm. Once activated, these enzymes are able to hydrolyze phosphorylated substrates in the extracellular milieu. Several studies demonstrated the presence of surface-located ecto-phosphatases in a vast number of pathogenic organisms, including bacteria, protozoa, and fungi. Little is known about the role of ecto-phosphatases in host-pathogen interactions. The present paper provides an overview of recent findings related to the virulence induced by these surface molecules in protozoa and fungi.

Platelet activating factor blocks interkinetic nuclear migration in retinal progenitors through an arrest of the cell cycle at the S/G2 transition.

Nuclear migration is regulated by the LIS1 protein, which is the regulatory subunit of platelet activating factor (PAF) acetyl-hydrolase, an enzyme complex that inactivates the lipid mediator PAF. Among other functions, PAF modulates cell proliferation, but its effects upon mechanisms of the cell cycle are unknown. Here we show that PAF inhibited interkinetic nuclear migration (IKNM) in retinal proliferating progenitors. The lipid did not, however, affect the velocity of nuclear migration in cells that escaped IKNM blockade. The effect depended on the PAF receptor, Erk and p38 pathways and Chk1. PAF induced no cell death, nor a reduction in nucleotide incorporation, which rules out an intra-S checkpoint. Notwithstanding, the expected increase in cyclin B1 content during G2-phase was prevented in the proliferating cells. We conclude that PAF blocks interkinetic nuclear migration in retinal progenitor cells through an unusual arrest of the cell cycle at the transition from S to G2 phases. These data suggest the operation, in the developing retina, of a checkpoint that monitors the transition from S to G2 phases of the cell cycle.

Biochemical properties of Candida parapsilosis ecto-5'-nucleotidase and the possible role of adenosine in macrophage interaction.

Candida parapsilosis is considered to be an emerging fungal pathogen because it is associated with an increasing range of infections. In this work, we biochemically characterized ecto-5'-nucleotidase activity on the surface of living, intact C. parapsilosis cells. At a pH of 4.5, intact cells were able to hydrolyze 5'-AMP at a rate of 52.44 ± 7.01 nmol Pi h(-1) 10(-7) cells. 5'-AMP, 5'-IMP and 5'-UMP were hydrolyzed at similar rates, whereas 5'-GMP and 5'-CMP hydrolyzed at lower rates. Enzyme activity was increased by about 42% with addition of Mg(2+) or Ca(2+), and the optimum pH was in the acidic range. An inhibitor of phosphatase activities, sodium orthovanadate, showed no effect on AMP hydrolysis; however, as expected, ammonium molybdate, a classical nucleotidase inhibitor, inhibited the activity in a dose-dependent manner. The results indicated that the existence of an ecto-5'-nucleotidase could play a role in the control of extracellular nucleotide concentrations.

Effects of platelet-activating factor on the interaction of Trypanosoma cruzi with Rhodnius prolixus.

We investigated the effects of platelet-activating factor (PAF) on the interaction of Trypanosoma cruzi with Rhodnius prolixus. The parasites (epimastigotes) were treated with PAF and/or WEB 2086 (PAF antagonist) for 1 h prior to the interaction experiments. PAF stimulated both in vivo and ex vivo interactions between T. cruzi and R. prolixus while WEB 2086 abrogated these effects. PAF-treated epimastigotes also showed an increase in surface negativity and in the amount of surface sialic acid. Neither of these effects was observed when the epimastigotes were treated with neuraminidase following PAF treatment. In the ex vivo interaction experiments, the number of epimastigotes bound to the midguts of the insects was reduced when the epimastigotes had been treated with neuraminidase. We conclude that PAF modulates the interaction of T. cruzi with R. prolixus by altering the amount of sialyl residues at the surface of the parasite.

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.

Stimulation of Leishmania tropica protein kinase CK2 activities by platelet-activating factor (PAF).

Leishmania tropica is one of the causative agents of cutaneous leishmaniasis. Platelet-activating factor (PAF) is a phospholipid mediator in diverse biological and pathophysiological processes. Here we show that PAF promoted a three-fold increase on ecto-protein kinase and a three-fold increase on the secreted kinase activity of L. tropica live promastigotes. When casein was added to the reaction medium, along with PAF, there was a four-fold increase on the ecto-kinase activity. When live L. tropica promastigotes were pre-incubated for 30 min in the presence of PAF-plus casein, a six-fold increase on the secreted kinase activity was observed. Also, a protein released from L. tropica promastigotes reacted with polyclonal antibodies for the mammalian CK2 alpha catalytic subunit. Furthermore, in vitro mouse macrophage infection by L. tropica was doubled when promastigotes were pre-treated for 2 h with PAF. Similar results were obtained when the interaction was performed in the presence of purified CK2 or casein. TBB and DRB, CK2 inhibitors, reversed PAF enhancement of macrophage infection by L. tropica. WEB 2086, a competitive PAF antagonist, reversed all PAF effects here described. This study shows for the first time that PAF promotes the activation of two isoforms of CK2, secreted and membrane-bound, correlating these activities to infection of mouse macrophages.

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.

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.