Leishmania amazonensis ferric iron reductase (LFR1) is a bifunctional enzyme: Unveiling a NADPH oxidase activity.

Leishmania amazonensis is one of leishmaniasis' causative agents, a disease that has no cure and leads to the appearance of cutaneous lesions. Recently, our group showed that heme activates a Na+/K+ ATPase in these parasites through a signaling cascade involving hydrogen peroxide (H2O2) generation. Heme has a pro-oxidant activity and signaling capacity, but the mechanism by which this molecule increases H2O2 levels in L. amazonensis has not been elucidated. Here we investigated the source of H2O2 stimulated by heme, ruling out the participation of mitochondria and raising the possibility of a role for a NADPH oxidase (Nox) activity. Despite the absence of a classical Nox sequence in trypanosomatid genomes, L. amazonensis expresses a surface ferric iron reductase (LFR1). Interestingly, Nox enzymes are thought to have evolved from ferric iron reductases because they share same core domain and are very similar in structure. The main difference is that Nox catalyses electron flow from NADPH to oxygen, generating reactive oxygen species (ROS), while ferric iron reductase promotes electron flow to ferric iron, generating ferrous iron. Using L. amazonensis overexpressing or knockout for LFR1 and heterologous expression of LFR1 in mammalian embryonic kidney (HEK 293) cells, we show that this enzyme is bifunctional, being able to generate both ferrous iron and H2O2. It was previously described that protozoans knockout for LFR1 have their differentiation to virulent forms (amastigote and metacyclic promastigote) impaired. In this work, we observed that LFR1 overexpression stimulates protozoan differentiation to amastigote forms, reinforcing the importance of this enzyme in L. amazonensis life cycle regulation. Thus, we not only identified a new source of ROS production in Leishmania, but also described, for the first time, an enzyme with both ferric iron reductase and Nox activities.

Anti-parasitic effect of the diuretic and Na+-ATPAse inhibitor furosemide in cutaneous leishmaniasis.

Leishmania amazonensis promastigotes are known to express furosemide (Lasix®)-sensitive P-type membrane Na+-ATPase. In the present study, furosemide activity was studied in intracellular amastigotes and infected BALB/c mice to investigate its efficacy in cutaneous leishmaniasis (CL). Intracellular parasites, but not macrophages, were found to be sensitive to killing by furosemide (IC50 = 87 µ m vs CC50 ≫ 1000 µ m, respectively). Although furosemide did not induce nitric oxide production or intracellular pH changes in infected macrophages, it led to a significant reactive oxygen species (ROS) burst. Freshly isolated tissue parasites expressed a high degree of Na+-ATPase activity that decreased with culture, indicative of a higher enzyme expression in amastigotes than in promastigotes. Both intraperitoneal and oral treatment of L. amazonensis-infected mice with furosemide dosages equivalent to that prescribed as a diuretic significantly reduced the parasite's growth compared with the situation in untreated mice. Combination with oral furosemide increased the efficacy and safety of intraperitoneal treatment with sodium stibogluconate (SSG). To summarize, furosemide control of intracellular leishmanial growth by means of parasite Na+-ATPase inhibition, and macrophage ROS activation may help explain its sole and SSG-combined therapeutic effect against murine CL.

Innate immunomodulation to trypanosomatid parasite infections.

The recognition of invading pathogens by the innate immune system is essential for host protection against human parasites and the initiation of an effective adaptive immune response. Innate immune cells such as macrophages and dendritic cells (DCs) are involved in the first line of defense against protozoan parasites via sensing the invaders through pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs). Activation of macrophages and dendritic cells starts with the interaction between microbial ligands (pathogen-associated molecular patterns - PAMPs) and PRRs, and these activated cells influence the overall immune response. Trypanosomatid PAMPs are sensed by TLRs; for example, TLR2 recognizes alkylacylglycerol and lipophosphoglycan in Trypanosoma cruzi and Leishmania, respectively; TLR2/TLR4 recognize glycoisnositolphospholipids and glycosylphosphatidyl inositol in Trypanosoma species; and TLR9 recognizes genomic DNA in Trypanosoma. TLR signaling includes the recruitment of different adaptor molecules that activate various transcription factors, such as NF-kB, IRF3/7, and MAP kinases, to induce the production of pro-inflammatory cytokines and type I interferons. Moreover, activated macrophages and dendritic cells produce ROS and NOS, which limit pathogen survival, and large amounts of cytokines; additionally, antigen presentation enhances the adaptive immune response. In this review, we highlight the recent findings on PAMP recognition in trypanosomatid infections and the signaling pathways activated by PRRs.

Periodate-oxidized ATP modulates macrophage functions during infection with Leishmania amazonensis.

Previously, we showed that treating macrophages with ATP impairs the intracellular growth of Leishmania amazonensis, and that the P2X7 purinergic receptor is overexpressed during leishmaniasis. In the present study, we directly evaluated the effect of periodate-oxidized ATP (oATP) on parasite control in Leishmania-infected macrophages. We found that oATP impaired the attachment/entrance of L. amazonensis promastigotes to C57BL/6 mouse macrophages in a P2X7 receptor-independent manner, as macrophages from P2X7(-/-) mice were similarly affected. Although oATP directly inhibited the growth of axenic promastigotes in culture, promoted rapid ultrastructural alterations, and impaired Leishmania internalization by macrophages, it did not affect intracellular parasite multiplication. Upon infection, phagosomal acidification was diminished in oATP-treated macrophages, accompanied by reduced endosomal proteolysis. Likewise, MHC class II molecules expression and ectoATPase activity was decreased by oATP added to macrophages at the time of parasite infection. These inhibitory effects were not due to a cytotoxic effect, as no additional release of lactate dehydrogenase was detected in culture supernatants. Moreover, the capacity of macrophages to produce nitric oxide and reactive oxygen species was not affected by the presence of oATP during infection. We conclude that oATP directly affects extracellular parasite integrity and macrophage functioning.

Transport of inorganic phosphate in Leishmania infantum and compensatory regulation at low inorganic phosphate concentration.

BACKGROUND: Proliferation of Leishmania infantum depends on exogenous inorganic phosphate (P(i)) but little is known about energy metabolism and transport of P(i) across the plasma membrane in Leishmania sp. METHODS: We investigated the kinetics of 32P(i) transport, the influence of H+ and K+ ionophores and inhibitors, and expression of the genes for the Na+:P(i) and H+:P(i) cotransporters. RESULTS: The proton ionophore FCCP, bafilomycin A1 (vacuolar ATPase inhibitor), nigericin (K+ ionophore) and SCH28080 (an inhibitor of H+, K(+)-ATPase) all inhibited the transport of P(i). This transport showed Michaelis-Menten kinetics with K0.5 and V(max) values of 0.016 +/- 0.002 mM and 564.9 +/- 18.06 pmol x h(-1) x 10(-7) cells, respectively. These values classify the P(i) transporter of L. infantum among the high-affinity transporters, a group that includes Pho84 of Saccharomyces cerevisiae. Two sequences were identified in the L. infantum genome that code for phosphate transporters. However, transcription of the PHO84 transporter was 10-fold higher than the PHO89 transporter in this parasite. Accordingly, P(i) transport and LiPho84 gene expression were modulated by environmental P(i) variations. CONCLUSIONS: These findings confirm the presence of a P(i) transporter in L. infantum, similar to PHO84 in S. cerevisiae, that contributes to the acquisition of inorganic phosphate and could be involved in growth and survival of the promastigote forms of L. infantum. GENERAL SIGNIFICANCE: This work provides the first description of a PHO84-like P(i) transporter in a Trypanosomatide parasite of the genus Leishmania, responsible for many infections worldwide.

Inorganic phosphate uptake in Trypanosoma cruzi is coupled to K(+) cycling and to active Na(+) extrusion.

BACKGROUND: Orthophosphate (Pi) is a central compound in the metabolism of all organisms, including parasites. There are no reports regarding the mechanisms of Pi acquisition by Trypanosoma cruzi. METHODS: (32)Pi influx was measured in T. cruzi epimastigotes. The expression of Pi transporter genes and the coupling of the uptake to Na(+), H(+) and K(+) fluxes were also investigated. The transport capacities of different evolutive forms were compared. RESULTS: Epimastigotes grew significantly more slowly in 2mM than in 50mM Pi. Influx of Pi into parasites grown under low Pi conditions took place in the absence and presence of Na(+). We found that the parasites express TcPho84, a H(+):Pi-symporter, and TcPho89, a Na(+):Pi-symporter. Both Pi influx mechanisms showed Michaelis-Menten kinetics, with a one-order of magnitude higher affinity for the Na(+)-dependent system. Collapsing the membrane potential with carbonylcyanide-p-trifluoromethoxyphenylhydrazone strongly impaired the influx of Pi. Valinomycin (K(+) ionophore) or SCH28028 (inhibitor of (H(+)+K(+))ATPase) significantly inhibited Pi uptake, indicating that an inwardly-directed H(+) gradient energizes uphill Pi entry and that K(+) recycling plays a key role in Pi influx. Furosemide, an inhibitor of the ouabain-insensitive Na(+)-ATPase, decreased only the Na(+)-dependent Pi uptake, indicating that this Na(+) pump generates the Na(+) gradient utilized by the symporter. Trypomastigote forms take up Pi inefficiently. CONCLUSIONS: Pi starvation stimulates membrane potential-sensitive Pi uptake through different pathways coupled to Na(+) or H(+)/K(+) fluxes. GENERAL SIGNIFICANCE: This study unravels the mechanisms of Pi acquisition by T. cruzi, a key process in epimastigote development and differentiation to trypomastigote forms.

The presence of a symbiotic bacterium in Strigomonas culicis is related to differential ecto-phosphatase activity and influences the mosquito-protozoa interaction.

Strigomonas culicis is a monoxenous trypanosomatid that co-evolves with a symbiotic bacterium in a mutualistic relationship that is characterized by intense metabolic exchanges between both partners. S. culicis infects and colonizes the Aedes aegypti mosquito midgut, reaches its hemocoel and then invades the salivary glands. An artificial aposymbiotic strain is unable to colonize insects, reinforcing the idea that the bacterium influences the protozoan surface composition and cell interaction. Here, we report the characterization of the hydrolytic activity of ecto-phosphatases evaluated in symbiont-bearing and aposymbiotic strains of S. culicis by incubating the protozoa with p-nitrophenyl phosphate (pNPP) at different pH levels, in the presence of phosphatase inhibitors, and with several divalent metals. The symbiont-bearing and aposymbiotic cells differ in their ecto-phosphatase enzymes, based on their activities and specificities. Furthermore, the ability of the protozoan to bind to the mosquito midgut and salivary glands was impaired by ecto-phosphatase inhibition. Taken together, our data suggest that the symbiont influences the host protozoan ecto-phosphatase activity and indicate a possible role of this enzyme during mosquito tissue colonization by S. culicis.

Transport of inorganic phosphate in Leishmania infantum and compensatory regulation at low inorganic phosphate concentration.

BACKGROUND: Proliferation of Leishmania infantum depends on exogenous inorganic phosphate (Pi) but little is known about energy metabolism and transport of Pi across the plasma membrane in Leishmania sp. METHODS: We investigated the kinetics of 32Pi transport, the influence of H+ and K+ ionophores and inhibitors, and expression of the genes for the Na+:Pi and H+:Pi cotransporters. RESULTS: The proton ionophore FCCP, bafilomycin A1 (vacuolar ATPase inhibitor), nigericin (K+ ionophore) and SCH28080 (an inhibitor of H+, K+-ATPase) all inhibited the transport of Pi. This transport showed Michaelis-Menten kinetics with K0.5 and Vmax values of 0.016±0.002mM and 564.9±18.06pmol×h-1×10-7cells, respectively. These values classify the Pi transporter of L. infantum among the high-affinity transporters, a group that includes Pho84 of Saccharomyces cerevisiae. Two sequences were identified in the L. infantum genome that code for phosphate transporters. However, transcription of the PHO84 transporter was 10-fold higher than the PHO89 transporter in this parasite. Accordingly, Pi transport and LiPho84 gene expression were modulated by environmental Pi variations. CONCLUSIONS: These findings confirm the presence of a Pi transporter in L. infantum, similar to PHO84 in S. cerevisiae, that contributes to the acquisition of inorganic phosphate and could be involved in growth and survival of the promastigote forms of L. infantum. GENERAL SIGNIFICANCE: This work provides the first description of a PHO84-like Pi transporter in a Trypanosomatide parasite of the genus Leishmania, responsible for many infections worldwide.

Characterization of ecto-ATPase activity in the surface of LLC-PK1 cells and its modulation by ischemic conditions.

BACKGROUND: The concentration of extracellular nucleotides is regulated by enzymes that have their catalytic site facing the extracellular space, the so-called ecto-enzymes. METHODS: We used LLC-PK1 cells, a well-characterized porcine renal proximal tubule cell line, to biochemically characterize ecto-ATPase activity in the luminal surface. The [γ-(32)P]Pi released after reaction was measured in aliquots of the supernatant by liquid scintillation. RESULTS: This activity was linear with time up to 20min of reaction and stimulated by divalent metals. The ecto-ATPase activity measured in the presence of 5mM MgCl(2) was (1) optimum at pH 8, (2) insensitive to different inhibitors of intracellular ATPases, (3) inhibited by 1mM suramin, an inhibitor of ecto-ATPases, (4) sensitive to high concentrations of sodium azide (NaN(3)) and (5) also able to hydrolyze ADP in the extracellular medium. The ATP:ADP hydrolysis ratio calculated was 4:1. The ecto-ADPase activity was also inhibited by suramin and NaN(3). The dose-response of ATP revealed a hyperbolic profile with maximal velocity of 25.2±1.2nmol Pixmg(-1)xmin(-1) and K(0.5) of 0.07±0.01mM. When cells were submitted to ischemia, the E-NTPDase activity was reduced with time, achieving 71% inhibition at 60min of ischemia. CONCLUSION: Our results suggest that the ecto-ATPase activity of LLC-PK1 cells has the characteristics of a type 3 E-NTPDase which is inhibited by ischemia. GENERAL SIGNIFICANCE: This could represent an important pathophysiologic mechanism that explains the increase in ATP concentration in the extracellular milieu in the proximal tubule during ischemia.

Na+-dependent and Na+-independent mechanisms for inorganic phosphate uptake in Trypanosoma rangeli.

BACKGROUND: Trypanosoma rangeli is dependent on the presence of exogenous orthophosphate (Pi) for maximal growth and ecto-phosphatase activity is responsible for Pi supply under low Pi. Here we investigated the mechanisms of Pi uptake. METHODS: We investigated the kinetics of 32Pi transport, its Na+ and H+ dependence, its correlation with the Na+-ATPase and H+-ATPase, and gene expression of the Na+:Pi cotransporter and Na+-ATPase. RESULTS: T. rangeli grown under limiting Pi transports this anion to the cytosol in the absence and presence of Na+, suggesting that influx is mediated by both Na+-independent and Na+-dependent transporters. Cloning studies demonstrated that this parasite expresses a Pi transporter not previously studied in trypanosomatids. The H+ ionophore, carbonylcyanide-p-trifluoromethoxyphenylhydrazone, decreased both components of 32Pi influx by 80-95%. The H+-ATPase inhibitor, bafilomycin A1, inhibited the Na+-independent mechanism. Furosemide, an inhibitor of ouabain-insensitive Na+-ATPase, decreased both uptake mechanisms of 32Pi to the same extent, whereas ouabain had no effect, indicating that the former is the pump responsible for inwardly directed Na+ and the electric gradients required by the transporters. Parasite growth in high Pi had a lower Pi influx than that found in those grown in low Pi, without alteration in TrPho89 expression, showing that turnover of the transporters is stimulated by Pi starvation. CONCLUSIONS: Two modes of Pi transport, one coupled to Na+-ATPase and other coupled to H+-ATPase seem to be responsible for Pi acquisition during development of T. rangeli. GENERAL SIGNIFICANCE: This study provides the first description of the mechanism of Pi transport across the plasma membrane of trypanosomatids.

Looking for reference genes for real-time quantitative PCR experiments in Rhodnius prolixus (Hemiptera: Reduviidae).

Quantitative real-time PCR (qPCR) has become one of the most used techniques to measure gene expression. However, normalization of gene expression data against reference genes is essential, although these are usually used without any kind of validation. The expression of seven genes was compared in organs of Rhodnius prolixus under diverse conditions, using published software to test gene expression stability. Rp18S and elongation factor 1 (RpEF -1) were the most reliable genes for normalization in qPCR when gene expression in different organs was compared. Moreover, both genes were found to be the best references when transcript levels were compared in the posterior midgut of insects infected with Trypanosoma cruzi. Rp18S was also the best reference gene in the fat bodies of unfed and fed insects. By contrast, RpEF-1 was found to be the best reference gene for comparison between posterior midguts, and RpMIP or RpActin should be used to compare gene expression in the ovaries. Although Rp18S is indicated here as the best reference in most cases, reports from the literature show that it is difficult to find an optimum reference gene. Nevertheless, validation of candidate genes to be taken as references is important when new experimental conditions are tested to avoid incorrect data interpretation.

Ectophosphatase activity in Candida albicans influences fungal adhesion: study between HIV-positive and HIV-negative isolates.

OBJECTIVE: This study describes the expression of acidic ectophosphatase activity on twenty isolates of C. albicans from oral cavities of HIV-infected children (HIV+) and compares them with fifteen isolates from HIV-negative children (HIV-), as well as the fungal adhesion to epithelial cells and medical records. METHODS: The activities were measured in intact cells grown in BHI medium for 48 h at 37 degrees C. Phosphatase activity was assayed at pH 5.5 using 4-methylumbelliferyl phosphate. Yeast adhesion was measured using the MA 104 epithelial cell line. RESULTS: Mean values of ectophosphatase activity were 610.27 +/- 166.36 and 241.25 +/- 78.96 picomoles 4-methylumbelliferone/h/10(7) cells for HIV+ and HIV- group, respectively (P = 0.049). No correlation between C. albicans enzyme activity from HIV children with viral load and CD4 percentual was observed. Yeasts with high enzyme activity, isolated from HIV+ children showed greater adherence than yeasts with basal levels of ectophosphatases from HIV- (Spearman correlation, r = 0.8). Surface phosphatase activity was apparently involved in the adhesion to host cells, as the enhanced attachment of C. albicans to host epithelial cells was reversed by pretreatment of yeast with sodium orthovanadate (1 mM), an acid phosphatase inhibitor. CONCLUSION: These results show that C. albicans from HIV+ has an ectophosphatase activity significantly higher than the other isolates. Yeasts expressing higher levels of surface phosphatase activity showed greater adhesion to epithelial cells. So, the activity of acidic surface phosphatases on these cells may contribute to the early mechanisms required for disease establishment.

Characterization of Ca2+ uptake in a subcellular membrane fraction of Herpetomonas sp. promastigotes.

ATP-dependent Ca2+ uptake was studied in a subcellular fraction from Herpetomonas sp. prepared by mechanical disruption and using 45Ca2+ as a tracer. The uptake was stimulated by Ca2+ with a K0.5 of 0.1 microm and a Hill number (nH)=2.8+/-0.4. The Ca2+-dependent ATP hydrolysis was optimal at pH 7.0 and had a Ca2+ dependence identical to uptake. The uptake was highly stimulated by oxalate whereas calmodulin had no activating effect. ATP stimulated Ca2+ uptake with a biphasic pattern that resembled the curves described for the purified preparations of rabbit sarcoplasmic reticulum. The ATP stimulation is described as the sum of two Michaelis-Menten curves with Km1=0.25+/-0.19 microm and Km2=29.6+/-6.8 microm. GTP or UTP could also promote Ca2+ uptake, but with less efficiency than ATP. Vanadate inhibited the uptake with low apparent affinity. Thapsigargin and cyclopiazonic acid were almost ineffective. The Ca2+ uptake was insensitive to H+ ionophores and to bafilomycin suggesting no participation of acidocalcisomes. The results are comparable to those obtained using cells permeabilized with digitonin and using arsenaze III as Ca2+ indicator. The Ca2+ uptake activity described here seems to belong to the endoplasmic reticulum of Herpetomonas sp. and is suitable for further studies on the mechanisms of calcium homeostasis in parasites.

CrATP as a new inhibitor of ecto-ATPases of trypanosomatids.

Trypanosomatid protozoa include heteroxenic species some of them pathogenic for men, animals and plants. Parasite membrane contains ecto-enzymes whose active sites face the external medium rather than the cytoplasm. Herpetomonas sp. displayed a Mg2+-dependent ecto-ATPase activity, a Mg-independent ecto-ADPase and an ecto-phosphatase activity. Both, the ecto-ADPase and phosphatase activities were insensitive to CrATP (chromium(III) adenosine 5'-triphosphate complex). Ecto-ATPase activity was reversibly inhibited. At 2 mm ATP the apparent Ki was 4 x 7+/-1 x 0 microm but a fraction of about 40-50% was insensitive to CrATP. Remarkably, at low substrate concentration (0 x 2 mm) more than 90% of the ecto-ATPase was inhibited with Ki=0 x 33+/-0 x 10 microm. These parameter dependences are interpreted as the presence of 2 ecto-ATPases activities, one of them with high ATP apparent affinity and sensitivity to CrATP. DIDS (4,4 diisothiocyanatostilbene 2,2' disulfonic acid), suramin and ADP were also effective as inhibitors. Only ADP presented no additive inhibition with CrATP. The pattern of partial inhibition by CrATP was also observed for the ecto-ATPase activities of Leishmania amazonensis, Trypanosoma cruzi and Trypanosoma rangeli. CrATP emerges as a new inhibitor of ecto-ATPases and as a tool for a better understanding of properties and role of ecto-ATPases in the biology of parasites.

Characterization of ecto-phosphatase activities of Trypanosoma cruzi: a comparative study between Colombiana and Y strains.

The etiological agent of Chagas disease, Trypanosoma cruzi, is consisted of two phylogenetic lineages. Using live epimastigotes, in this study we have characterized ecto-phosphatase activities of two strains of T. cruzi, one (Y strain) is a member of group T. cruzi I and the other (Colombiana) is a member of group T. cruzi II. About one-third of the total ecto-phosphatase activity from the Y strain was Mg(2+)-dependent, but no such activity was observed with Colombiana. The level of Mg(2+)-independent activity was dramatically different in the two strains, with Colombiana showing more than 15-fold higher activity. Experiments using classical inhibitors of acid phosphatases, as well as inhibitors of phosphotyrosine phosphatase, showed a decrease in these phosphatase activities, with different patterns of inhibition. The Mg(2+)-independent activities of the Colombiana and Y strains decreased inversely with pH, varying from 6.5 to 8.0. On the other hand, the Mg(2+)-dependent activity of the Y strain increased concomitantly with the increase in pH in the same range.

Trichomonas vaginalis virulence against epithelial cells and morphological variability: the comparison between a well-established strain and a fresh isolate.

The FMVI strain of Trichomonas vaginalis was freshly isolated from an asymptomatic patient, and its morphological properties and virulence in vitro compared with the well-established JT strain. The morphological variability of the parasites was assessed by differential interference microscopy and both scanning and transmission electron microscopy. The FMV1 strain presented nearly 20% amoeboid cells whereas the JT strain presented high percentages of ellipsoid but no amoeboid cells. The FMV1 morphotype population was unaltered after at least 1 year of subculturing. Electron microscopy revealed that this strain produced numerous pseudopod structures which mediated intimate contact and interdigitation among trophozoites. Dead FMV1 parasites were often phagocytosed by conspecific cells. We also compared the cytolytic capacity of these two populations against epithelial MDCK cells and its contact dependence. The FMV1 strain rapidly adhered to plastic or glass surfaces and to MDCK monolayers. This strain destroyed about 93% of the epithelial cells in 90 min whereas the cytolytic activity of the JT parasites was very much lower (about 41%). Parasite supernatants displayed no cytolytic activity, indicating contact-mediated lysis. The protozoan virulence in vitro did not correlate well with the clinical observations. The implications of these results are discussed.

Magnesium-dependent ecto-ATP diphosphohydrolase activity in Herpetomonas muscarum muscarum.

In the present work we characterized the ecto-ATP diphosphohydrolase activity of the trypanosomatid parasite Herpetomonas muscarum muscarum. This parasite hydrolyzed ATP at a rate of 15.52 nmol Pi/mg protein/min and this activity reached a maximum at pH 7.5. Classical inhibitors of acid phosphatases, such as sodium orthovanadate (NaVO3), sodium fluoride (NaF), and ammonium molybdate presented no effect on this activity. MgCl2, ZnCl2, and MnCl2 stimulated the ATP hydrolysis by H. m. muscarum. The ecto-ATPase activity was insensitive to oligomycin and sodium azide, two inhibitors of mitochondrial Mg-ATPase, bafilomycin A1, a V-ATPase inhibitor, ouabain, a Na(+)+K+-ATPase inhibitor and to levamizole, an inhibitor of alkaline phosphatase. An extracellular impermeant inhibitor 4,4'-diisothiocyanostylbene 2',2'-disulfonic acid (DIDS) and a inhibitor of some ecto-ATPases, suramin, which is also a competitive antagonist of P2-purinergic receptors, promoted a great inhibition on the ATP hydrolysis. This enzyme is able to hydrolysis ATP, ADP, UTP, and UDP, but not GTP, GDP, CTP, or CDP. ADP inhibited the enzymatic activity in a concentration dependent manner, reaching 70% inhibition.

Phosphatase activity on the cell wall of Fonsecaea pedrosoi.

The activity of a phosphatase was characterized in intact mycelial forms of Fonsecaea pedrosoi, a pathogenic fungus that causes chromoblastomycosis. At pH 5.5, this fungus hydrolyzed p-nitrophenylphosphate (p-NPP) to p-nitrophenol (p-NP) at a rate of 12.78 +/- 0.53 nmol p-NP per h per mg hyphal dry weight. The values of Vmax and apparent Km for p-NPP hydrolyses were measured as 17.89 +/- 0.92 nmol p-NP per h per mg hyphal dry weight and 1.57 +/- 0.26 mmol/l, respectively. This activity was inhibited at increased pH, a finding compatible with an acid phosphatase. The enzymatic activity was strongly inhibited by classical inhibitors of acid phosphatases such as sodium orthovanadate (Ki = 4.23 micromol/l), sodium molybdate (Ki = 7.53 micromol/l) and sodium fluoride (Ki = 126.78 micromol/l) in a dose-dependent manner. Levamizole (1 mmol/l) and sodium tartrate (10 mmol/l), had no effect on the enzyme activity. Cytochemical localization of the acid phosphatase showed electrondense cerium phosphate deposits on the cell wall, as visualized by transmission electron microscopy. Phosphatase activity in F. pedrosoi seems to be associated with parasitism, as sclerotic cells, which are the fungal forms mainly detected in chromoblastomycosis lesions, showed much higher activities than conidia and mycelia did. A strain of F. pedrosoi recently isolated from a human case of chromoblastomycosis also showed increased enzyme activity, suggesting that the expression of surface phosphatases may be stimulated by interaction with the host.

Characterization of an ecto-phosphatase activity in the human parasite Trichomonas vaginalis.

We have characterized phosphatase activity present on the external surface of Trichomonas vaginalis, using intact living parasites. This enzyme hydrolyzes the substrate p-nitrophenylphosphate (p-NPP) at a rate of 134.3+/-14.8 nmol Pi/h per 10(7) cells. This phosphatase activity decreased by increasing the pH from 6.8 to 8.4, a pH range in which cell viability was maintained for at least 1 h. Experiments using classical inhibitors of acid phosphatases, such as ammonium molybdate and sodium fluoride, as well as inhibitors of phosphotyrosine phosphatase, such as sodium orthovanadate, [monoperoxo(picolinato)oxovanadate(V)] (mpV-PIC) and [potassiumbisperoxo(1,10-phenanthroline)oxovanadate(V)] (bpV-PHEN), showed a decrease in this phosphatase activity, with different patterns of inhibition. Cytochemical analysis showed the localization of this enzyme on the parasite surface (cell body and flagellum) and in intracellular vacuoles. Phosphatase reaction products were also observed in exocytosed membrane-bound material.

Suramin inhibits respiration and induces membrane permeability transition in isolated rat liver mitochondria.

Suramin, a polysulfonated naphthylamine, caused a dose dependent inhibition of carbonyl cyanide p-(tri-fluoromethoxy)phenylhydrazone-stimulated respiration supported either by succinate or a cocktail of alphaketoglutarate, malate and isocitrate in isolated rat liver mitochondria. The half-maximum effect was obtained at 40 and 140 microM suramin for NADH- or FADH(2)-linked substrates, respectively. The respiration supported by N,N,N'N'-tetramethyl-p-phenylenediamine oxidation was unaffected by suramin (