Trypanosoma brucei plasma membrane-type Ca2+-ATPase 1 (TbPMC1) and 2 (TbPMC2) genes encode functional Ca2+-ATPases localized to the acidocalcisomes and plasma membrane, and essential for Ca2+ homeostasis and growth

Trypanosoma brucei adaptation and survival in its host involve integrated regulation of Ca2+ pumps (Ca2+-ATPases), which are essential in calcium ion homeostasis. Here we report the cloning and sequencing of two genes (TbPMC1 and TbPMC2) encoding plasma membrane-type Ca2+-ATPases (PMCAs) of T. brucei, an agent of African trypanosomiasis. Indirect immunofluorescence analysis using antibodies against the proteins and against epitope tags introduced into each protein showed that TbPMC1 co-localized with the vacuolar H+-pyrophosphatase to the acidocalcisomes while TbPMC2 localized to the plasma membrane. Northern and Western blot analyses revealed that TbPMC1 and TbPMC2 are up-regulated during blood stages. TbPMC1 and TbPMC2 suppressed the Ca2+ hypersensitivity of a mutant of S. cerevisiae that has a defect in vacuolar Ca2+ accumulation. T. brucei Ca2+-ATPase genes were functionally characterized by using double-stranded RNA interference (RNAi) methodology to produce inducible Ca2+-ATPase-deficient procyclic forms. Similar results were obtained with bloodstream form trypomastigotes, except that the RNAi system was leaky and mRNA and protein levels recovered with time. The induction of dsRNA ( RNAi) caused gross morphological alterations, and growth inhibition of procyclic forms. Induction of RNAi against TbPMC1 but not against TbPMC2 caused elevated levels of cytosolic Ca2+ and decreased mobilization of Ca2+ from intracellular stores following ionophore addition. These results establish that T. brucei PMCA-Ca2+-ATPases are essential for parasite viability and validate them as targets for drug development.