Claudia Fernanda Dick

Inorganic Phosphate as an Important Regulator of Phosphatases

Cellular metabolism depends on the appropriate concentration of intracellular inorganic phosphate (Pi). Pi starvation-responsive genes appear to be involved in multiple metabolic pathways, implying a complex Pi regulation system in microorganisms and plants. A group of enzymes is required for absorption and maintenance of adequate phosphate levels, which is released from phosphate esters and anhydrides. The phosphatase system is particularly suited for the study of regulatory mechanisms because phosphatase activity is easily measured using specific methods and the difference between the repressed and derepressed levels of phosphatase activity is easily detected. This paper analyzes the protein phosphatase system induced during phosphate starvation in different organisms.

Trypanosoma rangeli: A possible role for ecto-phosphatase activity on cell proliferation

Here we demonstrate for the first time that growth of Trypanosoma rangeli, a protozoa parasite, is strongly dependent on the presence of inorganic phosphate (Pi) in the culture medium and that the replacement of the inorganic phosphate in the culture medium by beta-glycerophosphate, a substrate for phosphatases lead the cells to achieve its maximal growth. The ecto-phosphatase activity present on the external surface of T. rangeli decreased during the growth phase of the parasite, suggesting that this enzyme could be important for the development. Accordingly, the inhibition of this ecto-phosphatase activity by sodium orthovanadate also inhibited the proliferation of T. rangeli. Parasites maintained in a Pi-starved culture medium (2 mM Pi) had 4-fold more ecto-phosphatase activity as compared to parasites maintained in a Pi-supplemented culture medium (50 mM Pi). Altogether, these results presented here suggest that this ecto-phosphatase activity leads to hydrolysis of phosphorylated compounds present in the extracellular medium, which could contribute to the acquisition of inorganic phosphate during the development of T. rangeli epimastigotes.

Trypanosoma rangeli: differential expression of ecto-phosphatase activities in response to inorganic phosphate starvation.

In this work, we showed that living cells of Trypanosoma rangeli express different ecto-phosphatase activities in response to different inorganic phosphate (Pi) concentrations in the culture medium. The ecto-phosphatase activity from T. rangeli grown at low-Pi concentration was inhibited by the increase of the pH, while the ecto-phosphatase of the cells grown at high Pi concentration was not modulated by the change of the pH of the medium. Okadaic acid inhibited only the ecto-phosphatase activity from cells grown at low-Pi concentration but not the ecto-phosphatase activity from cells grown at high-Pi concentration. Accordingly, phosphatase activity from T. rangeli grown at low Pi concentration was able to hydrolyze P-serine and P-threonine at high rate but not P-tyrosine. The phosphatase activity from T. rangeli grown at high-Pi concentration was able to hydrolyze P-serine, P-threonine and P-tyrosine with the same rate. The addition of anterior midgut homogenate of Rhodnius prolixus on the epimastigotes suspension inhibited the enzyme activity of T. rangeli grown at low-Pi concentration. On the other hand, anterior midgut homogenate had no effect in the ecto-phosphatase of T. rangeli maintained at high-Pi concentration. Altogether, the results described here indicate that ecto-phosphatase activities hydrolyzing phosphorylated compounds present in the extracellular medium of T. rangeli are regulated by the external Pi concentration.

A Mg2+-dependent ecto-phosphatase activity on the external surface of Trypanosoma rangeli modulated by exogenous inorganic phosphate

In this work, we characterized a Mg(2+)-dependent ecto-phosphatase activity present in live Trypanosoma rangeli epimastigotes. This enzyme showed capacity to hydrolyze the artificial substrate for phosphatases, p-nitrophenylphosphate (p-NPP). At saturating concentration of p-NPP, half-maximal p-NPP hydrolysis was obtained with 0.23mM Mg(2+). Ca(2+) had no effect on the basal phosphatase activity, could not substitute Mg(2+) as an activator and in contrast inhibited the p-NPP hydrolysis stimulated by Mg(2+). The dependence on p-NPP concentration showed a normal Michaelis-Menten kinetics for this phosphatase activity with values of V(max) of 8.94+/-0.36 nmol p-NP x h(-1) x 10(-7) cells and apparent K(m) of 1.04+/-0.16 mM p-NPP. Mg(2+)-dependent ecto-phosphatase activity was stimulated by the alkaline pH range. Experiments using inhibitors, such as, sodium fluoride, sodium orthovanadate and ammonium molybdate, inhibited the Mg(2+)-dependent ecto-phosphatase activity. Inorganic phosphate (Pi), a product of phosphatases, inhibited reversibly in 50% this activity. Okadaic acid and microcystin-LR, specific phosphoserine/threonine phosphatase inhibitors, inhibited significantly the Mg(2+)-dependent ecto-phosphatase activity. In addition, this phosphatase activity was able to recognize as substrates only o-phosphoserine and o-phosphothreonine, while o-phosphotyrosine was not a good substrate for this phosphatase. Epimastigote forms of T. rangeli exhibit a typical growth curve, achieving the stationary phase around fifth or sixth day and the Mg(2+)-dependent ecto-phosphatase activity decreased around 10-fold with the cell growth progression. Cells maintained at Pi-deprived medium (2 mM Pi) present Mg(2+)-dependent ecto-phosphatase activity approximately threefold higher than that maintained at Pi-supplemented medium (50 mM Pi).

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.

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 β-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.

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.

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.

Ecto-phosphatase activity on the external surface of Rhodnius prolixus salivary glands: Modulation by carbohydrates and Trypanosoma rangeli

The salivary glands of insects vectors are target organs to study the vectors-pathogens interactions. Rhodnius prolixus an important vector of Trypanosoma cruzi can also transmit Trypanosoma rangeli by bite. In the present study we have investigated ecto-phosphatase activity on the surface of R. prolixus salivary glands. Ecto-phosphatases are able to hydrolyze phosphorylated substrates in the extracellular medium. We characterized these ecto-enzyme activities on the salivary glands external surface and employed it to investigate R. prolixus-T. rangeli interaction. Salivary glands present a low level of hydrolytic activity (4.30+/-0.35 nmol p-nitrophenol (p-NP)xh(-1)xgland pair(-1)). The salivary glands ecto-phosphatase activity was not affected by pH variation; and it was insensitive to alkaline inhibitor levamisole and inhibited approximately 50% by inorganic phosphate (Pi). MgCl2, CaCl2 and SrCl2 enhanced significantly the ecto-phosphatase activity detected on the surface of salivary glands. The ecto-phosphatase from salivary glands surface efficiently releases phosphate groups from different phosphorylated amino acids, giving a higher rate of phosphate release when phospho-tyrosine is used as a substrate. This ecto-phosphatase activity was inhibited by carbohydrates as d-galactose and d-mannose. Living short epimastigotes of T. rangeli inhibited salivary glands ecto-phosphatase activity at 75%, while boiled parasites did not. Living long epimastigote forms induced a lower, but significant inhibitory effect on the salivary glands phosphatase activity. Interestingly, boiled long epimastigote forms did not loose the ability to modulate salivary glands phosphatase activity. Taken together, these data suggest a possible role for ecto-phosphatase on the R. prolixus salivary glands-T. rangeli interaction.

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 β-glycerophosphate (β-GP) and p-nitrophenyl phosphate (pNPP) as substrates. Its role in parasite development and differentiation was also studied. Competition assays using different proportions of β-GP and pNPP evidenced the existence of independent and non-interacting alkaline and acid phosphatases. Hydrolysis of β-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. β-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 β-GP was the sole source of Pi and stopped it in the absence of β-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.