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.

Tartrate-resistant phosphatase type 5 in Trypanosoma cruzi is important for resistance to oxidative stress promoted by hydrogen peroxide.

Trypanosoma cruzi (the causative agent of Chagas disease) presents a complex life cycle that involves adaptations in vertebrate and invertebrate hosts. As a protozoan parasite of hematophagous insects and mammalian hosts, T. cruzi is exposed to reactive oxygen species (ROS). To investigate the functionality of T. cruzi tartrate-resistant acid phosphatase type 5 (TcACP5), we cloned, superexpressed and purified the enzyme. Purified TcACP5 exhibited a Vmax and apparent Km for pNPP hydrolysis of 7.7 ±â€¯0.2 nmol pNP × µg-1 × h-1 and 169.3 ±â€¯22.6 µM, respectively. The pH dependence was characterized by sharp maximal activity at pH 5.0, and inhibition assays demonstrated its sensitivity to acid phosphatase inhibitors. Similar activities were obtained with saturating concentrations of P-Ser and P-Thr as substrates. The enzyme metabolizes hydrogen peroxide (H2O2) in vitro, and parasites superexpressing this enzyme were more resistant to oxidative stress promoted by H2O2. Taken together, these results suggest that TcACP5 plays a central role in phosphoryl transfer and redox reactions.

Cloning, expression and purification of 3'-nucleotidase/nuclease, an enzyme responsible for the Leishmania escape from neutrophil extracellular traps.

Leishmaniasis is one of the most significant of the neglected tropical diseases, with 350 million people in 98 countries worldwide living at risk of developing one of the many forms of the disease. During the transmission of the parasite from its vector to the vertebrate host, neutrophils are rapidly recruited to the site of the sandfly bite. Using different strategies, neutrophils can often kill a large number of parasites. However, some parasites can resist neutrophil-killing mechanisms and survive until macrophage arrival at the infection site. One of the strategies for neutrophil-mediated killing is the production of neutrophil extracellular traps (NETs). Because of its ecto-localized nuclease activity, the enzyme 3'-nucleotidase/nuclease (3'NT/NU), present in different Leishmania species, was recently identified as part of a possible parasite escape mechanism from NET-mediated death. Previous studies showed that 3'NT/NU also plays an important role in the establishment of Leishmania infection by generating extracellular adenosine that favors the parasite and macrophage interaction. This study aims to deepen the knowledge about 3'NT/NU, mainly with respect to its nuclease activity that is little studied in the current literature. For this, we cloned, expressed and purified the recombinant La3'NT/NU and have confirmed its contribution to the parasite escape from NET-mediated killing.

Ectophosphatase activity in the early-diverging fungus Blastocladiella emersonii: Biochemical characterization and possible role on cell differentiation.

Blastocladiella emersonii is an interesting model for studding the evolution of cell differentiation in eukaryotic cell because of its taxonomic position towards the base of the fungal phylogenetic tree and because it undergoes radical morphological and biochemical changes throughout its life cycle. In this work, we biochemically characterized a high alkaline phosphotyrosine phosphatase activity present on the cell surface (ectophosphatase) of B. emersonii. The ectophosphatase activity was strongly inhibited at acidic pH values as well as by specific phosphatase inhibitors, such as sodium orthovanadate and bpv-PHEN. In addition, the enzyme activity was modulated by the extracellular concentration of inorganic phosphate (Pi) present in both reaction mixture and culture medium. Phosphotyrosine was hydrolysed at the same extent of its analog, p-NPP, while the hydrolysis of phosphothreonine was 2-fold lower, suggesting that a phosphotyrosine ectophosphatase activity is present on the cell surface of B. emersonii. The ectophosphatase activity was also strongly inhibited by EGTA, indicating the participation of Ca2+ ions on catalysis. The hydrolysis of p-NPP was differentially regulated throughout the B. emersonii life cycle, suggesting that the ectophosphatase activity could be involved in cell differentiation processes. In support of this, the addition of bpv-PHEN or vanadate at the beginning of germination inhibited the differentiation of zoospores to germ cells, compared to control or tartrate-treated cells. On the other hand, if the inhibitors are added 15 or 30 min after initiation of germination the inhibitory effect on zoospore germination decreases significantly, suggesting that the phosphotyrosine ectophosphatase activity is important at the first minutes of germination. The addition of vanadate, molybdate and bpv-PHEN during vegetative growth inhibited the enlargement of the cells compared to control or tartrate-treated cells. Finally, vanadate or bpv-PHEN added during sporulation strongly inhibited zoospore biogenesis, indicating an important role of such ectophosphatases in this differentiation process. Taken together, these data show the existence of a high alkaline ectophosphotyrosine phosphatase activity in B. emersonii that is probably tied to cell differentiation processes of the fungus.

Driving mosquito refractoriness to Plasmodium falciparum with engineered symbiotic bacteria.

The huge burden of malaria in developing countries urgently demands the development of novel approaches to fight this deadly disease. Although engineered symbiotic bacteria have been shown to render mosquitoes resistant to the parasite, the challenge remains to effectively introduce such bacteria into mosquito populations. We describe a Serratia bacterium strain (AS1) isolated from Anopheles ovaries that stably colonizes the mosquito midgut, female ovaries, and male accessory glands and spreads rapidly throughout mosquito populations. Serratia AS1 was genetically engineered for secretion of anti-Plasmodium effector proteins, and the recombinant strains inhibit development of Plasmodium falciparum in mosquitoes.

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.

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.

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

The salivary glands of insect's 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.