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

Background: Orthophosphate (P-i) is a central compound in the metabolism of all organisms, including parasites. There are no reports regarding the mechanisms of P-i acquisition by Trypanosoma cruzi. Methods: P-32(i) influx was measured in T. cruzi epimastigotes. The expression of P-i 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 2 mM than in 50 mM P-i. Influx of P-i into parasites grown under low P-i conditions took place in the absence and presence of Na+. We found that the parasites express TcPho84, a H+:P-i-symporter, and TcPho89, a Na+:P-i-symporter. Both P-i 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 P-i. Valinomycin (K+ ionophore) or SCH28028 (inhibitor of (H+ + K+)ATPase) significantly inhibited P-i uptake, indicating that an inwardly-directed H+ gradient energizes uphill P-i entry and that K+ recycling plays a key role in P-i influx. Furosemide, an inhibitor of the ouabain-insensitive Na+-ATPase, decreased only the Na+-dependent P-i uptake, indicating that this Na+ pump generates the Na+ gradient utilized by the symporter. Trypomastigote forms take up P-i inefficiently. Conclusions: P-i starvation stimulates membrane potential-sensitive P-i uptake through different pathways coupled to Na+ or H+/K+ fluxes. General significance: This study unravels the mechanisms of P-i acquisition by T. cruzi, a key process in epimastigote development and differentiation to trypomastigote forms. (C) 2013 Elsevier B.V. All rights reserved.