ABSTRACT
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.
Subject(s)
Leishmania infantum/enzymology , Phosphates/metabolism , Protozoan Proteins/metabolism , Amino Acid Sequence , Biological Transport , Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone/pharmacology , Culture Media , Enzyme Inhibitors/pharmacology , Gene Expression Regulation/drug effects , Imidazoles/pharmacology , Kinetics , Leishmania infantum/genetics , Macrolides/pharmacology , Molecular Sequence Data , Nigericin/pharmacology , Phosphates/pharmacology , Phosphorus Radioisotopes , Phylogeny , Proton Ionophores/pharmacology , Proton-Phosphate Symporters/antagonists & inhibitors , Proton-Phosphate Symporters/genetics , Proton-Phosphate Symporters/metabolism , Protozoan Proteins/genetics , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Sodium-Phosphate Cotransporter Proteins/antagonists & inhibitors , Sodium-Phosphate Cotransporter Proteins/metabolism , Sodium-Potassium-Exchanging ATPase/antagonists & inhibitors , Sodium-Potassium-Exchanging ATPase/metabolism , Vacuolar Proton-Translocating ATPases/antagonists & inhibitors , Vacuolar Proton-Translocating ATPases/metabolismABSTRACT
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.
ABSTRACT
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.
