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.
Article: Induction of immunogenicity by live attenuated Leishmania donovani centrin deleted parasites in dogs.[show abstract] [hide abstract]
ABSTRACT: Zoonotic visceral leishmaniasis, caused by the intracellular protozoan parasite Leishmania infantum, is a neglected tropical disease that is often fatal when untreated. Dogs are considered the main reservoir of L. infantum in zoonotic VL as the presence of infected dogs may increase the risk for human infection. Canine visceral leishmaniasis (CVL) is a major veterinary and public health problem in Southern Europe, Middle East and South America. Control of animal reservoirs relies on elimination of seropositive dogs in endemic areas. However, treatment of infected dogs is not considered a favorable approach as this can lead to emergence of drug resistance since the same drugs are used to treat human infections. Therefore, vaccination against CVL remains the best alternative in control of the animal reservoirs. In this study, we present data on the immunogenicity profile of a live attenuated parasite LdCen(-/-) in a canine infection model and compared it to that of Leishmune(®), a commercially available recombinant vaccine. The immunogenicity of the LdCen(-/-) parasites was evaluated by antibody secretion, production of intracytoplasmic and secreted cytokines, activation and proliferation of T cells. Vaccination with LdCen(-/-) resulted in high immunogenicity as revealed by the higher IgGTotal, IgG1, and IgG2 production and higher lymphoproliferative response. Further, LdCen(-/-) vaccinated dogs showed higher frequencies of activated CD4+ and CD8+ T cells, IFN-γ production by CD8+ T cells, increased secretion of TNF-α and IL-12/IL-23p40 and decreased secretion of IL-4. These results contribute to the understanding of immunogenicity elicited by live attenuated L. donovani parasites and, consequently, to the development of effective vaccines against visceral leishmaniasis.Vaccine 02/2013; · 3.77 Impact Factor
Article: Targeting essential pathways in trypanosomatids gives insights into protozoan mechanisms of cell death.[show abstract] [hide abstract]
ABSTRACT: Apoptosis is a normal component of the development and health of multicellular organisms. However, apoptosis is now considered a prerogative of unicellular organisms, including the trypanosomatids of the genera Trypanosoma spp. and Leishmania spp., causative agents of some of the most important neglected human diseases. Trypanosomatids show typical hallmarks of apoptosis, although they lack some of the key molecules contributing to this process in metazoans, like caspase genes, Bcl-2 family genes and the TNF-related family of receptors. Despite the lack of these molecules, trypanosomatids appear to have the basic machinery to commit suicide. The components of the apoptotic execution machinery of these parasites are slowly coming into light, by targeting essential processes and pathways with different apoptogenic agents and inhibitors. This review will be confined to the events known to drive trypanosomatid parasites to apoptosis.Parasites & Vectors 11/2010; 3:107. · 2.94 Impact Factor