Trypanosoma brucei plasma membrane-type Ca(2+)-ATPase 1 (TbPMC1) and 2 (TbPMC2) genes encode functional Ca(2+)-ATPases localized to the acidocalcisomes and plasma membrane, and essential for Ca(2+) homeostasis and growth.
Trypanosoma brucei adaptation and survival in its host involve integrated regulation of Ca(2+) pumps (Ca(2+)-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 Ca(2+)-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 Ca(2+) hypersensitivity of a mutant of S. cerevisiae that has a defect in vacuolar Ca(2+) accumulation. T. brucei Ca(2+)-ATPase genes were functionally characterized by using double-stranded RNA interference (RNAi) methodology to produce inducible Ca(2+)-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 Ca(2+) and decreased mobilization of Ca(2+) from intracellular stores following ionophore addition. These results establish that T. brucei PMCA-Ca(2+)-ATPases are essential for parasite viability and validate them as targets for drug development.
Luo
Shuhong
S
Department of Pathobiology, and Center for Zoonoses Research, University of Illinois at Urbana-Champaign, Urbana, Illinois 61802, USA.
Rohloff
Peter
P
Cox
Joanna
J
Uyemura
Sergio A
SA
Docampo
Roberto
R
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000235:genetics* / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D003001:Cloning, Molecular; D006706:Homeostasis; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D025262:Vacuolar Proton-Translocating ATPases / Q000378:metabolism
D015800:Protozoan Proteins; D029701:Saccharomyces cerevisiae Proteins; D025262:Vacuolar Proton-Translocating ATPases; D000252:Calcium-Transporting ATPases; D002118:Calcium