Elevated extracellular inorganic phosphate inhibits ecto-phosphatase activity in breast cancer cells: Regulation by hydrogen peroxide.

For cells to obtain inorganic phosphate, ectoenzymes in the plasma membrane, which contain a catalytic site facing the extracellular environment, hydrolyze phosphorylated molecules. In this study, we show that increased Pi levels in the extracellular environment promote a decrease in ecto-phosphatase activity, which is associated with Pi-induced oxidative stress. High levels of Pi inhibit ecto-phosphatase because Pi generates H2 O2 . Ecto-phosphatase activity is inhibited by H2 O2 , and this inhibition is selective for phospho-tyrosine hydrolysis. Additionally, it is shown that the mechanism of inhibition of ecto-phosphatase activity involves lipid peroxidation. In addition, the inhibition of ecto-phosphatase activity by H2 O2 is irreversible. These findings have new implications for understanding ecto-phosphatase regulation in the tumor microenvironment. H2 O2 stimulated by high Pi inhibits ecto-phosphatase activity to prevent excessive accumulation of extracellular Pi, functioning as a regulatory mechanism of Pi variations in the tumor microenvironment.

Iron Uptake Controls Trypanosoma cruzi Metabolic Shift and Cell Proliferation.

(1) Background: Ionic transport in Trypanosoma cruzi is the object of intense studies. T. cruzi expresses a Fe-reductase (TcFR) and a Fe transporter (TcIT). We investigated the effect of Fe depletion and Fe supplementation on different structures and functions of T. cruzi epimastigotes in culture. (2) Methods: We investigated growth and metacyclogenesis, variations of intracellular Fe, endocytosis of transferrin, hemoglobin, and albumin by cell cytometry, structural changes of organelles by transmission electron microscopy, O2 consumption by oximetry, mitochondrial membrane potential measuring JC-1 fluorescence at different wavelengths, intracellular ATP by bioluminescence, succinate-cytochrome c oxidoreductase following reduction of ferricytochrome c, production of H2O2 following oxidation of the Amplex® red probe, superoxide dismutase (SOD) activity following the reduction of nitroblue tetrazolium, expression of SOD, elements of the protein kinase A (PKA) signaling, TcFR and TcIT by quantitative PCR, PKA activity by luminescence, glyceraldehyde-3-phosphate dehydrogenase abundance and activity by Western blotting and NAD+ reduction, and glucokinase activity recording NADP+ reduction. (3) Results: Fe depletion increased oxidative stress, inhibited mitochondrial function and ATP formation, increased lipid accumulation in the reservosomes, and inhibited differentiation toward trypomastigotes, with the simultaneous metabolic shift from respiration to glycolysis. (4) Conclusion: The processes modulated for ionic Fe provide energy for the T. cruzi life cycle and the propagation of Chagas disease.

Functional characterization of maternally accumulated hydrolases in the mature oocytes of the vector Rhodnius prolixus reveals a new protein phosphatase essential for the activation of the yolk mobilization and embryo development.

Yolk biogenesis and consumption have been well conserved in oviparous animals throughout evolution. Most egg-laying animals store yolk proteins within the oocytes' yolk granules (Ygs). Following fertilization, the Ygs participate in controlled pathways of yolk breakdown to support the developing embryo's anabolic metabolism. While the unfolding of the yolk degradation program is a crucial process for successful development in many species, the molecular mechanisms responsible for yolk mobilization are still mysterious and have mostly not been explored. Here, we investigate the functional role of the oocyte maternally accumulated mRNAs of a protein phosphatase (PP501) and two aspartic proteases (cathepsin-D 405, CD405 and cathepsin-D 352, CD352) in the yolk degradation and reproduction of the insect vector of Chagas disease Rhodnius prolixus. We found that PP501 and CD352 are highly expressed in the vitellogenic ovary when compared to the other organs of the adult insect. Parental RNAi silencing of PP501 resulted in a drastic reduction in oviposition and increased embryo lethality whereas the silencing of CD352 resulted only in a slight decrease in oviposition and embryo viability. To further investigate the PP501-caused high reproduction impairment, we investigated the Ygs biogenesis during oocyte maturation and the activation of the yolk degradation program at early development. We found that the Ygs biogenesis was deficient during oogenesis, as seen by flow cytometry, and that, although the PP501-silenced unviable eggs were fertilized, the Ygs acidification and acid phosphatase activity were affected, culminating in a full impairment of the yolk proteins degradation at early embryogenesis. Altogether we found that PP501 is required for the oocyte maturation and the activation of the yolk degradation, being, therefore, essential for this vector reproduction.

Hypoxia Effects on Trypanosoma cruzi Epimastigotes Proliferation, Differentiation, and Energy Metabolism.

Trypanosoma cruzi, the causative agent of Chagas disease, faces changes in redox status and nutritional availability during its life cycle. However, the influence of oxygen fluctuation upon the biology of T. cruzi is unclear. The present work investigated the response of T. cruzi epimastigotes to hypoxia. The parasites showed an adaptation to the hypoxic condition, presenting an increase in proliferation and a reduction in metacyclogenesis. Additionally, parasites cultured in hypoxia produced more reactive oxygen species (ROS) compared to parasites cultured in normoxia. The analyses of the mitochondrial physiology demonstrated that hypoxic condition induced a decrease in both oxidative phosphorylation and mitochondrial membrane potential (ΔΨm) in epimastigotes. In spite of that, ATP levels of parasites cultivated in hypoxia increased. The hypoxic condition also increased the expression of the hexokinase and NADH fumarate reductase genes and reduced NAD(P)H, suggesting that this increase in ATP levels of hypoxia-challenged parasites was a consequence of increased glycolysis and fermentation pathways. Taken together, our results suggest that decreased oxygen levels trigger a shift in the bioenergetic metabolism of T. cruzi epimastigotes, favoring ROS production and fermentation to sustain ATP production, allowing the parasite to survive and proliferate in the insect vector.

An Iron Transporter Is Involved in Iron Homeostasis, Energy Metabolism, Oxidative Stress, and Metacyclogenesis in Trypanosoma cruzi.

The parasite Trypanosoma cruzi causes Chagas' disease; both heme and ionic Fe are required for its optimal growth, differentiation, and invasion. Fe is an essential cofactor in many metabolic pathways. Fe is also harmful due to catalyzing the formation of reactive O2 species; for this reason, all living systems develop mechanisms to control the uptake, metabolism, and storage of Fe. However, there is limited information available on Fe uptake by T. cruzi. Here, we identified a putative 39-kDa Fe transporter in T. cruzi genome, TcIT, homologous to the Fe transporter in Leishmania amazonensis and Arabidopsis thaliana. Epimastigotes grown in Fe-depleted medium have increased TcIT transcription compared with controls grown in regular medium. Intracellular Fe concentration in cells maintained in Fe-depleted medium is lower than in controls, and there is a lower O2 consumption. Epimastigotes overexpressing TcIT, which was encountered in the parasite plasma membrane, have high intracellular Fe content, high O2 consumption-especially in phosphorylating conditions, high intracellular ATP, very high H2O2 production, and stimulated transition to trypomastigotes. The investigation of the mechanisms of Fe transport at the cellular and molecular levels will assist in elucidating Fe metabolism in T. cruzi and the involvement of its transport in the differentiation from epimastigotes to trypomastigotes, virulence, and maintenance/progression of the infection.

Ectophosphatase activity in the triple-negative breast cancer cell line MDA-MB-231.

Breast cancer is one of the most common cancers in the female population worldwide, and its development is thought to be associated with genetic mutations that lead to uncontrolled and accelerated growth of breast cells. This abnormal behavior requires extra energy, and indeed, tumor cells display a rewired energy metabolism compared to normal breast cells. Inorganic phosphate (Pi) is a glycolytic substrate of glyceraldehyde-3-phosphate dehydrogenase and has an important role in cancer cell proliferation. For cells to obtain Pi, ectoenzymes in the plasma membrane with their catalytic site facing the extracellular environment can hydrolyze phosphorylated molecules, and this is an initial and possibly limiting step for the uptake of Pi by carriers that behave as adjuvants in the process of energy harvesting and thus partially contributes to tumor energy requirements. In this study, the activity of an ectophosphatase in MDA-MB-231 cells was biochemically characterized, and the results showed that the activity of this enzyme was higher in the acidic pH range and that the enzyme had a Km = 4.5 ± 0.5 mM para-nitrophenylphosphate and a Vmax = 2280 ± 158 nM × h-1 × mg protein-1 . In addition, classical acid phosphatase inhibitors, including sodium orthovanadate, decreased enzymatic activity. Sodium orthovanadate was able to inhibit ectophosphatase activity while also inhibiting cell proliferation, adhesion, and migration, which are important processes in tumor progression, especially in metastatic breast cancer MDA-MB-231 cells that have higher ectophosphatase activity than MCF-7 and MCF-10 breast cells.

Endocytosis and Exocytosis in Leishmania amazonensis Are Modulated by Bromoenol Lactone.

In the protozoan pathogen Leishmania, endocytosis, and exocytosis occur mainly in the small area of the flagellar pocket membrane, which makes this parasite an interesting model of strikingly polarized internalization and secretion. Moreover, little is known about vesicle recognition and fusion mechanisms, which are essential for both endo/exocytosis in this parasite. In other cell types, vesicle fusion events require the activity of phospholipase A2 (PLA2), including Ca2+-independent iPLA2 and soluble, Ca2+-dependent sPLA2. Here, we studied the role of bromoenol lactone (BEL) inhibition of endo/exocytosis in promastigotes of Leishmania amazonensis. PLA2 activities were assayed in intact parasites, in whole conditioned media, and in soluble and extracellular vesicles (EVs) conditioned media fractions. BEL did not affect the viability of promastigotes, but reduced the differentiation into metacyclic forms. Intact parasites and EVs had BEL-sensitive iPLA2 activity. BEL treatment reduced total EVs secretion, as evidenced by reduced total protein concentration, as well as its size distribution and vesicles in the flagellar pocket of treated parasites as observed by TEM. Membrane proteins, such as acid phosphatases and GP63, became concentrated in the cytoplasm, mainly in multivesicular tubules of the endocytic pathway. BEL also prevented the endocytosis of BSA, transferrin and ConA, with the accumulation of these markers in the flagellar pocket. These results suggested that the activity inhibited by BEL, which is one of the irreversible inhibitors of iPLA2, is required for both endocytosis and exocytosis in promastigotes of L. amazonensis.

Resveratrol Reverses Functional Chagas Heart Disease in Mice.

Chronic chagasic cardiomyopathy (CCC) develops years after acute infection by Trypanosoma cruzi and does not improve after trypanocidal therapy, despite reduction of parasite burden. During disease, the heart undergoes oxidative stress, a potential causative factor for arrhythmias and contractile dysfunction. Here we tested whether antioxidants/ cardioprotective drugs could improve cardiac function in established Chagas heart disease. We chose a model that resembles B1-B2 stage of human CCC, treated mice with resveratrol and performed electrocardiography and echocardiography studies. Resveratrol reduced the prolonged PR and QTc intervals, increased heart rates and reversed sinus arrhythmia, atrial and atrioventricular conduction disorders; restored a normal left ventricular ejection fraction, improved stroke volume and cardiac output. Resveratrol activated the AMPK-pathway and reduced both ROS production and heart parasite burden, without interfering with vascularization or myocarditis intensity. Resveratrol was even capable of improving heart function of infected mice when treatment was started late after infection, while trypanocidal drug benznidazole failed. We attempted to mimic resveratrol's actions using metformin (AMPK-activator) or tempol (SOD-mimetic). Metformin and tempol mimicked the beneficial effects of resveratrol on heart function and decreased lipid peroxidation, but did not alter parasite burden. These results indicate that AMPK activation and ROS neutralization are key strategies to induce tolerance to Chagas heart disease. Despite all tissue damage observed in established Chagas heart disease, we found that a physiological dysfunction can still be reversed by treatment with resveratrol, metformin and tempol, resulting in improved heart function and representing a starting point to develop innovative therapies in CCC.

Increased expression of NTPDases 2 and 3 in mesenteric endothelial cells during schistosomiasis favors leukocyte adhesion through P2Y1 receptors.

Schistosomiasis is caused by an intravascular parasite and linked to phenotypic changes in endothelial cells that favor inflammation. Endothelial cells express P2Y1 receptors (P2Y1R), and their activation by ADP favors leukocyte adhesion to the endothelial monolayer. We aimed to evaluate the influence of schistosomiasis upon endothelial purinergic signaling-mediated leukocyte adhesion. Mesenteric endothelial cells and mononuclear cells from control and Schistosoma mansoni-infected mice were used in co-culture. P2Y1R levels were similar in both groups. Basal leukocyte adhesion was higher in the infected than in the control group; leukocyte adhesion increased after treatment with the P2Y1R agonist 2-MeSATP in both groups, though it only marginally increased in the infected group. Pre-incubation with the selective P2Y1R antagonist MRS2179 (0.3µM) prevented the agonist effect. However, in the infected group it also reduced the basal leukocyte adhesion, suggesting endothelial cell pre-activation. The endothelial expressions of NTPDases 2 and 3 were significantly increased in the infected group, increasing extracellular ATP hydrolysis and ADP formation by endothelial cells. Therefore, mesenteric endothelial cells are primed by schistosomiasis to a pro-inflammatory phenotype characterized by an increased expression of NTPDases 2 and 3, favoring ADP accumulation and mononuclear cell adhesion, possibly contributing to mesenteric inflammation and schistosomiasis morbidity.

Trypanosoma cruzi-secreted vesicles have acid and alkaline phosphatase activities capable of increasing parasite adhesion and infection.

Trypanosoma cruzi virulence factors include molecules expressed on the cell surface as well as those secreted or shed into the extracellular medium. Phosphatase activities modulate different aspects of T. cruzi infection, although no studies to date addressed the presence and activity of phosphatases in vesicles secreted by this parasite. Here, we characterized acidic and alkaline secreted phosphatase activities of human-infective trypomastigote forms of T. cruzi from the Y strain and the CL-Brener clone. These are widely studied T. cruzi strains that represent "opposite ends of the spectrum" regarding both in vitro and in vivo behavior. Ecto-phosphatase activities were determined in live parasites, and secreted phosphatase activities were analyzed in soluble protein (SP) and vesicular membrane fractions (VFs) of parasite-conditioned medium. Our analysis using different phosphatase inhibitors strongly suggests that vesicles secreted by Y strain (VF(Y)) and CL-Brener (VF(CLB)) trypomastigotes are derived mostly from the cell surface and from exosome secretion, respectively. Importantly, our results show that the acid phosphatase activities in vesicles secreted by trypomastigotes are largely responsible for the VF-induced increase in adhesion of Y strain parasites to host cells and also for the VF-induced increase in host cell infection by CL-Brener trypomastigotes.

Identification of uncoupling protein 4 from the blood-sucking insect Rhodnius prolixus and its possible role on protection against oxidative stress.

Uncoupling proteins (UCPs) play a critical role in the control of the mitochondrial membrane potential (ΔΨm) due to their ability to dissipate the proton gradient, which results in the uncoupling of mitochondrial respiration from ATP production. Most reactive oxygen species generation in mitochondria occurs in complex III, due to an increase of semiquinone (Q(-)) half-life. When active, UCPs can account as a potential antioxidant system by decreasing ΔΨm and increasing mitochondrial respiration, thus reducing Q(-) life time. The hematophagous insect Rhodnius prolixus, a vector of Chagas disease, is exposed to a huge increase in oxidative stress after a blood meal because of the hydrolysis of hemoglobin and the release of the cytotoxic heme molecule. Although some protective mechanisms were already described for this insect and other hematophagous arthropods, the putative role of UCP proteins as antioxidants in this context has not been explored. In this report, two genes encoding UCP proteins (RpUcp4 and RpUcp5) were identified in the R. prolixus genome. RpUcp4 is the predominant transcript in most analyzed organs, and both mRNA and protein expression are upregulated (13- and 3-fold increase, respectively) in enterocytes the first day after the blood feeding. The increase in UCP4 expression is coincident with the decrease in hydrogen peroxide (H2O2) generation by midgut cells. Furthermore, in mitochondria isolated from enterocytes, the modulation of UCP activity by palmitic acid and GDP resulted in altered ΔΨm, as well as modulation of H2O2 generation rates. These results indicate that R. prolixus UCP4 may function in an antioxidation mechanism to protect the midgut cells against oxidative damage caused by blood digestion.

Inorganic phosphate uptake in unicellular eukaryotes.

BACKGROUND: Inorganic phosphate (Pi) is an essential nutrient for all organisms. The route of Pi utilization begins with Pi transport across the plasma membrane. SCOPE OF REVIEW: Here, we analyzed the gene sequences and compared the biochemical profiles, including kinetic and modulator parameters, of Pi transporters in unicellular eukaryotes. The objective of this review is to evaluate the recent findings regarding Pi uptake mechanisms in microorganisms, such as the fungi Neurospora crassa and Saccharomyces cerevisiae and the parasite protozoans Trypanosoma cruzi, Trypanosoma rangeli, Leishmania infantum and Plasmodium falciparum. MAJOR CONCLUSION: Pi uptake is the key step of Pi homeostasis and in the subsequent signaling event in eukaryotic microorganisms. GENERAL SIGNIFICANCE: Biochemical and structural studies are important for clarifying mechanisms of Pi homeostasis, as well as Pi sensor and downstream pathways, and raise possibilities for future studies in this field.

Trypanosoma rangeli: an alkaline ecto-phosphatase activity is involved with survival and growth of the parasite.

The aim of this work was to investigate whether an alkaline ecto-phosphatase activity is present in the surface of Trypanosoma rangeli. Intact short epimastigote forms were assayed for ecto-phosphatase activity to study kinetics and modulators using ß-glycerophosphate (ß-GP) and p-nitrophenyl phosphate (pNPP) as substrates. Its role in parasite development and differentiation was also studied. Competition assays using different proportions of ß-GP and pNPP evidenced the existence of independent and non-interacting alkaline and acid phosphatases. Hydrolysis of ß-GP increased progressively with pH, whereas the opposite was evident using pNPP. The alkaline enzyme was inhibited by levamisole in a non-competitive fashion. The Ca(2+) present in the reaction medium was enough for full activity. Pretreatment with PI-PLC decreased the alkaline but not the acid phosphatase evidence that the former is catalyzed by a GPI-anchored enzyme, with potential intracellular signaling ability. ß-GP supported the growth and differentiation of T. rangeli to the same extent as high orthophosphate (Pi). Levamisole at the IC50 spared significantly parasite growth when ß-GP was the sole source of Pi and stopped it in the absence of ß-GP, indicating that the alkaline enzyme can utilize phosphate monoesters present in serum. These results demonstrate the existence of an alkaline ecto-phosphatase in T. rangeli with selective requirements and sensitivity to inhibitors that participates in key metabolic processes in the parasite life cycle.

Rhodnius prolixus lipophorin: lipid composition and effect of high temperature on physiological role.

Lipophorin is a major lipoprotein that transports lipids in insects. In Rhodnius prolixus, it transports lipids from midgut and fat body to the oocytes. Analysis by thin-layer chromatography and densitometry identified the major lipid classes present in the lipoprotein as diacylglycerol, hydrocarbons, cholesterol, and phospholipids (PLs), mainly phosphatidylethanolamine and phosphatidylcholine. The effect of preincubation at elevated temperatures on lipophorin capacity to deliver or receive lipids was studied. Transfer of PLs to the ovaries was only inhibited after preincubation of lipophorin at temperatures higher than 55 °C. When it was pretreated at 75 °C, maximal inhibition of phospholipid transfer was observed after 3-min heating and no difference was observed after longer times, up to 60 min. The same activity was also obtained when lipophorin was heated for 20 min at 75 °C at protein concentrations from 0.2 to 10 mg/ml. After preincubation at 55 °C, the same rate of lipophorin loading with PLs at the fat body was still present, and 30% of the activity was observed at 75 °C. The effect of temperature on lipophorin was also analyzed by turbidity and intrinsic fluorescence determinations. Turbidity of a lipophorin solution started to increase after preincubations at temperatures higher than 65 °C. Emission fluorescence spectra were obtained for lipophorin, and the spectral area decreased after preincubations at 85 °C or above. These data indicated no difference in the spectral center of mass at any tested temperature. Altogether, these results demonstrate that lipophorin from R. prolixus is very resistant to high temperatures.

Different secreted phosphatase activities in Leishmania amazonensis.

Leishmania has strong acid phosphatase activity on the external surface of the plasma membrane and secreted into the extracellular milieu. Secreted acid phosphatase (sAcP), which is the most abundant secreted protein of Leishmania, is also a virulence factor that plays a role in vertebrate infection and survival in sand flies. In this study, we characterized the secreted phosphatase activities in Leishmania amazonensis. Both acidic and alkaline secreted phosphatase activities were observed with ß-glycerophosphate and p-nitrophenyl phosphate (p-NPP) hydrolysis and were inhibited with sodium tartrate and sodium orthovanadate. Cytochemical labeling revealed a significant difference in the localization of the electron-dense precipitates depending on the substrate. ß-Glycerophosphate electron-dense precipitates were concentrated on both the cell surface and flagellar pocket, whereas p-NPP labeling occurred primarily within intracellular organelles. Orthovanadate-treated metacyclic promastigotes were less infective and were confined to a tight parasitophorous vacuole (PV), which is not characteristic of this Leishmania species. Based on the results, we characterized the presence of different secreted phosphatase activities in L. amazonensis, the influence of the substrate in cytochemical labeling, and the potential involvement of secreted phosphatase activity in both PV maturation and amastigote survival.

Gene identification and enzymatic properties of a membrane-bound trehalase from the ovary of Rhodnius prolixus.

Trehalose represents the main hemolymph sugar in most insects and its metabolic availability is regulated by trehalase. In this study, trehalase activity associated with the reproductive system was investigated in the insect Rhodnius prolixus, a hematophagous hemipteran vector of Chagas' disease. A single-copy gene that encodes a membrane-bound trehalase (RpTre-2) was identified in the genome of R. prolixus. RpTre-2 deduced amino acid sequence is closely related to other insect membrane-bound trehalases. The expression of this gene was detected in all analyzed organs, including ovary, where total trehalase enzymatic activity was determined, and was highest at day 7 after blood meal. Ovary membranes showed a major trehalase specific activity, which confirmed the presence of a membrane-bound trehalase in this insect. This trehalase activity seemed not to be regulated at transcriptional level, as the expression of RpTre-2 gene in the ovary did not change over the days after feeding. Similarly, ovarian follicles at different developmental stages did not show any variation in the transcription level of this gene. The RpTre-2 kinetic parameters were also investigated. Activity was highest at pH 5.5 and followed Michaelis-Menten kinetics, with an apparent K(m) = 1.42 ± 0.36 mM and Vmax = 167.90 ± 12.91 nmol/mg protein/h. These data reveal the presence of a membrane-bound trehalase in R. prolixus that is active in ovary and probably takes part in the insect carbohydrate metabolism associated with the reproductive process.

Interaction between Trypanosoma rangeli and the Rhodnius prolixus salivary gland depends on the phosphotyrosine ecto-phosphatase activity of the parasite.

Trypanosoma rangeli is the trypanosomatid that colonizes the salivary gland of its insect vector, with a profound impact on the feeding capacity of the insect. In this study we investigated the role of the phosphotyrosine (P-Tyr) ecto-phosphatase activity of T. rangeli in its interaction with Rhodnius prolixus salivary glands. Long but not short epimastigotes adhered to the gland cells and the strength of interaction correlated with the enzyme activity levels in different strains. Differential interference contrast microscopy demonstrated that clusters of parasites are formed in most cases, suggesting cooperative interaction in the adhesion process. The tightness of the correlation was evidenced by modulating the P-Tyr ecto-phosphatase activity with various concentrations of inhibitors. Sodium orthovanadate, ammonium molybdate and zinc chloride decreased the interaction between T. rangeli and R. prolixus salivary glands in parallel. Levamisole, an inhibitor of alkaline phosphatases, affected neither process. EDTA strongly inhibited adhesion and P-Tyr ecto-phosphatase activity to the same extent, an effect that was no longer seen if the parasites were pre-incubated with the chelator and then washed. When the P-Tyr ecto-phosphatase of living T. rangeli epimastigotes was irreversibly inactivated with sodium orthovanadate and the parasite cells were then injected into the insect thorax, colonization of the salivary glands was greatly depressed for several days after blood feeding. Addition of P-Tyr ecto-phosphatase substrates such as p-nitrophenyl phosphate (pNPP) and P-Tyr inhibited the adhesion of T. rangeli to salivary glands, but P-Ser, P-Thr and ß-glycerophosphate were completely ineffective. Immunoassays using anti-P-Tyr-residues revealed a large number of P-Tyr-proteins in extracts of R. prolixus salivary glands, which could be potentially targeted by T. rangeli during adhesion. These results indicate that dephosphorylation of structural P-Tyr residues on the gland cell surfaces, mediated by a P-Tyr ecto-phosphatase of the parasite, is a key event in the interaction between T. rangeli and R. prolixus salivary glands.

Leishmania amazonensis impairs DC function by inhibiting CD40 expression via A2B adenosine receptor activation.

Dendritic cells (DCs) play an essential role in the modulation of immune responses and several studies have evaluated the interactions between Leishmania parasites and DCs. While extracellular ATP exhibits proinflammatory properties, adenosine is an important anti-inflammatory mediator. Here we investigated the effects of Leishmania infection on DC responses and the participation of purinergic signalling in this process. Bone marrow-derived dendritic cells (BMDCs) from C57BL/6J mice infected with Leishmania amazonensis, Leishmania braziliensis or Leishmania major metacyclic promastigotes showed decreased major histocompatibility complex (MHC) class II and CD86 expression and increased ectonucleotidase expression as compared with uninfected cells. In addition, L. amazonensis-infected DCs, which had lower CD40 expression, exhibited a decreased ability to induce T-cell proliferation. The presence of MRS1754, a highly selective A(2B) adenosine receptor antagonist at the time of infection increased MHC class II, CD86 and CD40 expression in L. amazonensis-infected DCs and restored the ability of the infected DCs to induce T-cell proliferation. Similar results were obtained through the inhibition of extracellular ATP hydrolysis using suramin. In conclusion, we propose that A(2B) receptor activation may be used by L. amazonensis to inhibit DC function and evade the immune response.

Surface phosphatase in Rhinocladiella aquaspersa: biochemical properties and its involvement with adhesion.

Rhinocladiella aquaspersa is an etiologic agent of chromoblastomycosis, a subcutaneous chronic infectious disease. In the present work, we found that the three morphological forms of this fungus (conidia, mycelia and sclerotic bodies) expressed different levels of ecto-phosphatase activity. Our results demonstrated that surface conidial enzyme is an acid phosphatase, inhibited by sodium salts of molybdate, orthovanadate and fluoride and that the inhibition caused by orthovanadate and molybdate was irreversible. The conidial ecto-phosphatase efficiently released phosphate groups from different phosphorylated substrates, causing a higher rate of phosphate removal when p-nitrophenylphosphate was used as substrate. This ecto-enzyme of R. aquaspersa is modulated by Co(2 +) ions and inorganic phosphate (Pi). Accordingly, removal of Pi from the culture medium resulted in a marked (121-fold) increase of ecto-phosphatase activity. Surface phosphatase activity is apparently involved in fungal adhesive properties, since the attachment of R. aquaspersa to epithelial cells was reversed by the pre-treatment of the conidia with orthovanadate, molybdate and anti-phosphatase antibody. Corroborating this finding, conidia with greater ecto-phosphatase activity (grown in Pi-depleted medium) showed higher adherence to epithelial cells than fungi cultivated in the presence of Pi.

Immune and inflammatory responses to Leishmania amazonensis isolated from different clinical forms of human leishmaniasis in CBA mice.

Leishmania amazonensis causes different diseases depending on the host and parasitic virulence factors. In this study, CBA mice were infected with L. amazonensis isolates from patients with localized (Ba125), diffuse cutaneous (Ba276) or visceral leishmaniasis (Ba109). Mice infected with Ba125 and Ba276 progressed rapidly and lesions displayed an infiltrate rich in parasitized macrophages and were necrotic and ulcerated. Ba109 induced smaller lesions and a mixed inflammatory infiltrate without necrosis or ulceration. Ba109 induced an insidious disease with lower parasite load in CBA mice, similar to human disease. Levels of IFN-γ, IL-4 and IL-10 did not differ among the groups. Because all groups were unable to control the infection, expression of IL-4 associated with low production of IFN-γ in the early phase of infection may account for susceptibility, but others factors may contribute to the differences observed in inflammatory responses and infection progression. Evaluation of some parasitic virulence factors revealed that Ba276 exhibits higher ecto-ADPase and 5'-nucleotidase activities compared to the Ba109 and Ba125 strains. Both Ba276 and Ba125 had higher arginase activity in comparison to Ba109. Finally, these data suggest that the differences in enzyme activities among parasites can account for differences in host inflammatory responses and infection progression.