Centrin Gene Disruption Impairs Stage-specific Basal Body Duplication and Cell Cycle Progression in Leishmania

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.

Intracellular Replication-Deficient Leishmania donovani Induces Long Lasting Protective Immunity against Visceral Leishmaniasis

“…In pursuit of a candidate live attenuated vaccine, we generated a L. donovani parasite that had a complete deletion for a cell division gene, centrin1 (LdCen1 Ϫ/Ϫ ) (15). We have demonstrated that LdCen1 Ϫ/Ϫ attenuation is due to the failure of cytokinesis, resulting in multiplication of cellular organelles, cell enlargement, and eventual programmed cell death.…”

“…We demonstrated that deletion of LdCen1 (LdCen1 Ϫ/Ϫ ) did not affect the growth of the promastigote in vitro, however, the growth of the mammalian-infecting, amastigote form of the parasite, was blocked. These amastigotes showed failure of basal body duplication and cytokinesis, resulting in large multinucleated cells in culture and ex vivo in human macrophages (15). We tested this unique parasite line for its safety and protective efficacy against homologous and heterologous virulent Leishmania challenge and the immune response correlating with protection in rodent animal models.…”

“…Hence it is critical to develop attenuated lines through complete gene knockouts that generate avirulent organisms that persist for a brief duration but eventually are completely eliminated, thereby inducing effective immunity without clinical disease or the risk of reactivation. Recently, we have developed a L. donovani strain completely deleted for the centrin1 gene (15). Centrin1 is a calcium-binding basal body-associated protein involved in cell division in protozoan parasites like Leishmania, Trypanosoma, and Plasmodium (16 -18) and centrosome associated in higher eukaryotes (19).…”

Centrin4 coordinates cell and nuclear division inT. brucei

“…Centrins are key regulators for the normal function and duplication of these structures (Baum et al, 1986;Middendorp et al, 2000;Salisbury, 2002;Paoletti et al, 2003;Selvapandiyan et al, 2004), and are also required for a variety of other cellular processes including DNA repair, mRNA export and signal transduction (Araki et al, 2001;Wolfrum et al, 2002;Fischer et al, 2004). Although this variety of functions can be achieved by a single centrin gene in organisms such as Saccharomyces cerevisiae, multiple centrin genes have been found in many other organisms, including mammals.…”

AncientLeishmaniacoronin (CRN12) is involved in microtubule remodeling during cytokinesis

“…Recently, duplication of the basal body has been shown to be crucial for the survival of Leishmania (Selvapandiyan et al, 2004). We therefore analyzed whether CRN12 was associated with the basal body complex that could be marked by immunostaining centrin (Selvapandiyan et al, 2004).…”