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

Abstract

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_i) availability. P_i 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_i-type ORF sequence in the G. duodenalis genome (GenBank ID: GL50803_5164), named GdPho84, which is homologous to Saccharomyces cerevisiae PHO84. In trophozoites, P_i 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 P_i uptake. This P_i transporter had a high affinity, with K_0.5 = 67.7 ± 7.1 µM P_i. SCH28080 (inhibitor of H⁺, K⁺-ATPase), bafilomycin A₁ (inhibitor of vacuolar H⁺-ATPase), and FCCP (H⁺ ionophore) were able to inhibit P_i transport, indicating that an H⁺ gradient in the cell powered uphill P_i movement. PAA, an H⁺-dependent P_i transport inhibitor, reduced cell proliferation, P_i transport activity, and GdPHO48 mRNA levels. P_i starvation stimulated membrane potential-sensitive P_i 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_i as a compensatory mechanism compared to cells maintained in control medium conditions. Internalized P_i can be used in glycolytic metabolism once iodoacetamide (GAPDH inhibitor) inhibits P_i influx. Together, these results reinforce the hypothesis that P_i is a crucial nutrient for G. duodenalis energy metabolism.