H⁺-dependent inorganic phosphate uptake in Trypanosoma brucei is influenced by myo-inositol transporter

Trypanosoma brucei is an extracellular protozoan parasite that causes human African trypanosomiasis or "sleeping sickness". During the different phases of its life cycle, T. brucei depends on exogenous inorganic phosphate (P_i), but little is known about the transport of P_i in this organism. In the present study, we showed that the transport of ³²P_i across the plasma membrane follows Michaelis-Menten kinetics and is modulated by pH variation, with higher activity at acidic pH. Bloodstream forms presented lower P_i transport in comparison to procyclic forms, that displayed an apparent K_0.5 = 0.093 ± 0.008 mM. Additionally, FCCP (H⁺-ionophore), valinomycin (K⁺-ionophore) and SCH28080 (H⁺, K⁺-ATPase inhibitor) inhibited the P_i transport. Gene Tb11.02.3020, previously described to encode the parasite H⁺:myo-inositol transporter (TbHMIT), was hypothesized to be potentially involved in the H⁺:P_i cotransport because of its similarity with the Pho84 transporter described in S. cerevisiae and other trypanosomatids. Indeed, the RNAi mediated knockdown remarkably reduced TbHMIT gene expression, compromised cell growth and decreased P_i transport by half. In addition, P_i transport was inhibited when parasites were incubated in the presence of concentrations of myo-inositol that are above 300 μM. However, when expressed in Xenopus laevis oocytes, two-electrode voltage clamp experiments provided direct electrophysiological evidence that the protein encoded by TbHMIT is definitely a myo-inositol transporter that may be only marginally affected by the presence of P_i. These results confirmed the presence of a P_i carrier in T. brucei, similar to the H⁺-dependent inorganic phosphate system described in S. cerevisiae and other trypanosomatids. This transport system contributes to the acquisition of P_i and may be involved in the growth and survival of procyclic forms. In summary, this work presents the first description of a P_i transport system in T. brucei.