Abstract
Centrin is a calcium-binding cytoskeletal protein involved in the duplication of centrosomes in higher eukaryotes. To explore the role of centrin in the protozoan parasite Leishmania, we created Leishmania deficient in the centrin gene (LdCEN). Remarkably, centrin null mutants (LdCEN(-/-)) showed selective growth arrest as axenic amastigotes but not as promastigotes. Flow cytometry analysis confirmed that the mutant axenic amastigotes have a cell cycle arrest at the G(2)/M stage. The axenic amastigotes also showed failure of basal body duplication and failure of cytokinesis resulting in multinucleated "large" cells. Increased terminal deoxy uridine triphosphate nick end labeling positivity was observed in centrin mutant axenic amastigotes compared with wild type cells, suggesting the activation of a programmed cell death pathway. Growth of LdCEN(-/-) amastigotes in infected macrophages in vitro was inhibited and also resulted in large multinucleated parasites. Normal basal body duplication and cell division in the LdCEN knockout promastigote is unique and surprising. Further, this is the first report where disruption of a centrin gene displays stage-specific/cell type-specific failure in cell division in a eukaryote. The centrin null mutant defective in amastigote growth could be useful as a vaccine candidate against leishmaniasis.
Highlights
Centrin is a calcium-binding cytoskeletal protein involved in the duplication of centrosomes in higher eukaryotes
Loss of centrin expression in the LdCENϪ/Ϫ parasite was confirmed by Western blot analysis using anti-LdCen antibody (Fig. 2C, lane 3)
LdCENϪ/Ϫ Parasites Do Not Survive inside Macrophages— Because centrin-deficient L. donovani do not grow as axenic amastigotes in vitro, we examined their survival in macrophages in vitro
Summary
Centrin is a calcium-binding cytoskeletal protein involved in the duplication of centrosomes in higher eukaryotes. Normal basal body duplication and cell division in the LdCEN knockout promastigote is unique and surprising This is the first report where disruption of a centrin gene displays stage-specific/cell type-specific failure in cell division in a eukaryote. The parasite L. donovani, a causative agent of human visceral leishmaniasis, has a digenic lifestyle with one form called the promastigote form that resides extracellularly in the mid-gut of the sand-fly vector and with another form called the amastigote form that multiplies intracellularly in vertebrate macrophages. These two forms have been adapted to grow under in vitro conditions [14, 15]. In this report we describe a stepwise disruption of the two alleles of LdCEN, using genes resistant to antibiotics hygromycin B and G418 (Geneticin) and
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