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 (P) availability. P 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:P-type ORF sequence in the G. duodenalis genome (GenBank ID: GL50803_5164), named GdPho84, which is homologous to Saccharomyces cerevisiae PHO84. In trophozoites, P transport was linear for up to 15 min, and the cell density was 3 × 10 cells/ml. Physiological variations in pH (6.4-8.0) did not influence P uptake. This P transporter had a high affinity, with K = 67.7 ± 7.1 µM P. SCH28080 (inhibitor of H, K-ATPase), bafilomycin A (inhibitor of vacuolar H-ATPase), and FCCP (H ionophore) were able to inhibit P transport, indicating that an H gradient in the cell powered uphill P movement. PAA, an H-dependent P transport inhibitor, reduced cell proliferation, P transport activity, and GdPHO48 mRNA levels. P starvation stimulated membrane potential-sensitive P 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 P as a compensatory mechanism compared to cells maintained in control medium conditions. Internalized P can be used in glycolytic metabolism once iodoacetamide (GAPDH inhibitor) inhibits P influx. Together, these results reinforce the hypothesis that P is a crucial nutrient for G. duodenalis energy metabolism.