Anaerobic energy metabolism in human microaerophile parasites.

Mucosal-associated parasites, such as Giardia intestinalis, Entamoeba histolytica, and Trichomonas vaginalis, have significant clinical relevance. The pathologies associated with infection by these parasites are among those with the highest incidence of gastroenteritis (giardiasis and amoebiasis) and sexually transmitted infections (trichomoniasis). The treatment of these diseases is based on drugs that act on the anaerobic metabolism of these parasites, such as nitroimidazole and benzimidazole derivatives. One interesting feature of parasites is their ability to produce ATP under anaerobic conditions. Due to the absence of enzymes capable of producing ATP under anaerobic conditions in the vertebrate host, they have become interesting therapeutic targets. This review discusses anaerobic energy metabolism in mucosal-associated parasites, focusing on the anaerobic metabolism of pyruvate, the importance of these enzymes as therapeutic targets, and the importance of treating their infections.

Inorganic phosphate transporter in Giardia duodenalis and its possible role in ATP synthesis.

Giardia duodenalis is a flagellated protozoan that inhabits vertebrate host intestines, causing the disease known as giardiasis. Similar to other parasites, G. duodenalis must take advantage of environmental resources to survive, such as inorganic phosphate (Pi) availability. Pi is an anionic molecule and an essential nutrient for all organisms because it participates in the biosynthesis of biomolecules, energy storage, and cellular structure formation. The first step in Pi metabolism is its uptake through specific transporters on the plasma membrane. We identified a symporter H+:Pi-type ORF sequence in the G. duodenalis genome (GenBank ID: GL50803_5164), named GdPho84, which is homologous to Saccharomyces cerevisiae PHO84. In trophozoites, Pi transport was linear for up to 15 min, and the cell density was 3 × 107 cells/ml. Physiological variations in pH (6.4-8.0) did not influence Pi uptake. This Pi transporter had a high affinity, with K0.5 = 67.7 ± 7.1 µM Pi. SCH28080 (inhibitor of H+, K+-ATPase), bafilomycin A1 (inhibitor of vacuolar H+-ATPase), and FCCP (H+ ionophore) were able to inhibit Pi transport, indicating that an H+ gradient in the cell powered uphill Pi movement. PAA, an H+-dependent Pi transport inhibitor, reduced cell proliferation, Pi transport activity, and GdPHO48 mRNA levels. Pi starvation stimulated membrane potential-sensitive Pi uptake coupled to H+ fluxes, increased GdPho84 expression, and reduced intracellular ATP levels. These events indicate that these cells had an increased capacity to internalize Pi as a compensatory mechanism compared to cells maintained in control medium conditions. Internalized Pi can be used in glycolytic metabolism once iodoacetamide (GAPDH inhibitor) inhibits Pi influx. Together, these results reinforce the hypothesis that Pi is a crucial nutrient for G. duodenalis energy metabolism.

Acanthamoeba castellanii phosphate transporter (AcPHS) is important to maintain inorganic phosphate influx and is related to trophozoite metabolic processes.

Acanthamoeba castellanii is a free-living amoeba and the etiological agent of granulomatous amoebic encephalitis and amoebic keratitis. A. castellanii can be present as trophozoites or cysts. The trophozoite is the vegetative form of the cell and has great infective capacity compared to the cysts, which are the dormant form that protect the cell from environmental changes. Phosphate transporters are a group of proteins that are able to internalize inorganic phosphate from the extracellular to intracellular medium. Plasma membrane phosphate transporters are responsible for maintaining phosphate homeostasis, and in some organisms, regulating cellular growth. The aim of this work was to biochemically characterize the plasma membrane phosphate transporter in A. castellanii and its role in cellular growth and metabolism. To measure inorganic phosphate (Pi) uptake, trophozoites were grown in liquid PYG medium at 28 °C for 2 days. The phosphate uptake was measured by the rapid filtration of intact cells incubated with 0.5 µCi of 32Pi for 1 h. The Pi transport was linear as a function of time and exhibited Michaelis-Menten kinetics with a Km = 88.78 ± 6.86 µM Pi and Vmax = 547.5 ± 16.9 Pi × h-1 × 10-6 cells. A. castellanii presented linear phosphate uptake up to 1 h with a cell density ranging from 1 × 105 to 2 × 106 amoeba × ml-1. The Pi uptake was higher in the acidic pH range than in the alkaline range. The oxygen consumption of living trophozoites increased according to Pi addition to the extracellular medium. When the cells were treated with FCCP, no effect from Pi on the oxygen flow was observed. The addition of increasing Pi concentrations not only increased oxygen consumption but also increased the intracellular ATP pool. These phenomena were abolished when the cells were treated with FCCP or exposed to hypoxia. Together, these results reinforce the hypothesis that Pi is a key nutrient for Acanthamoeba castellanii metabolism.

Characterization of inorganic phosphate transport in the triple-negative breast cancer cell line, MDA-MB-231.

BACKGROUND: Recent studies demonstrate that interstitial inorganic phosphate is significantly elevated in the breast cancer microenvironment as compared to normal tissue. In addition it has been shown that breast cancer cells express high levels of the NaPi-IIb carrier (SLC34A2), suggesting that this carrier may play a role in breast cancer progression. However, the biochemical behavior of inorganic phosphate (Pi) transporter in this cancer type remains elusive. METHODS: In this work, we characterize the kinetic parameters of Pi transport in the aggressive human breast cancer cell line, MDA-MB-231, and correlated Pi transport with cell migration and adhesion. RESULTS: We determined the influence of sodium concentration, pH, metabolic inhibitors, as well as the affinity for inorganic phosphate in Pi transport. We observed that the inorganic phosphate is dependent on sodium transport (K0,5 value = 21.98 mM for NaCl). Furthermore, the transport is modulated by different pH values and increasing concentrations of Pi, following the Michaelis-Menten kinetics (K0,5 = 0.08 mM Pi). PFA, monensin, furosemide and ouabain inhibited Pi transport, cell migration and adhesion. CONCLUSIONS: Taken together, these results showed that the uptake of Pi in MDA-MB-231 cells is modulated by sodium and by regulatory mechanisms of intracellular sodium gradient. General Significance: Pi transport might be regarded as a potential target for therapy against tumor progression.