Expression of a Mutant Form of Leishmania donovani Centrin Reduces the Growth of the Parasite

Selvapandiyan, Angamuthu; Duncan, Robert C.; Debrabant, Alain; Bertholet, Sylvie; Gannavaram, Sreenivas; Negi, Narender Singh; Salotra, Poonam; Nakhasi, Hira L.

doi:10.1074/jbc.m106806200

Cited by 74 publications

(74 citation statements)

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“…L. donovani centrin protein (LdCenp) sequence similarity and immunoreactivity confirmed that Leishmania centrin (LdCEN) is a homolog of human centrin 2 (12). The high level expression of LdCenp during log phase growth and reduction of growth by overexpression of an N-terminally deleted centrin in both promastigote and amastigote forms indicate that centrin has an important role in Leishmania growth (12). To study the mechanism of centrin function in this lower eukaryote, we targeted the LdCEN gene for a null mutation.…”

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“…These two forms have been adapted to grow under in vitro conditions (14,15). To understand the molecular mechanisms of growth control, we cloned and characterized the gene encoding a basal body-associated protein centrin (LdCen) (12). L. donovani centrin protein (LdCenp) sequence similarity and immunoreactivity confirmed that Leishmania centrin (LdCEN) is a homolog of human centrin 2 (12).…”

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“…Abnormal centrosomes were noticed in human tumor cells because of the presence of excess levels of many centrosome proteins including centrin (11). We recently cloned several centrin genes 1 and characterized one such a gene, which is involved in the growth of the protozoan parasite, Leishmania donovani (12).…”

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confidence: 99%

“…To understand the molecular mechanisms of growth control, we cloned and characterized the gene encoding a basal body-associated protein centrin (LdCen) (12). L. donovani centrin protein (LdCenp) sequence similarity and immunoreactivity confirmed that Leishmania centrin (LdCEN) is a homolog of human centrin 2 (12). The high level expression of LdCenp during log phase growth and reduction of growth by overexpression of an N-terminally deleted centrin in both promastigote and amastigote forms indicate that centrin has an important role in Leishmania growth (12).…”

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

Selvapandiyan¹,

Debrabant²,

Duncan³

et al. 2004

Journal of Biological Chemistry

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129

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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.

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

Selvapandiyan¹,

Debrabant²,

Duncan³

et al. 2004

Journal of Biological Chemistry

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120

129

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“…La eliminación de genes en Leishmania ha permitido la caracterización de varias moléculas involucradas en el metabolismo celular y el transporte, así como de proteínas transductoras de señal, chaperonas y enzimas involucradas en la virulencia y resistencia a drogas (cuadro 2). También se han evaluado los efectos de la eliminación de dominios esenciales para la actividad enzimática (31,32).…”

Section: Eliminación De Genes En Leishmaniaunclassified

Manipulación genética y el estudio del parásito protozoario Leishmania.

Cortázar

Walker

2004

biomedica

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Durante los últimos 15 años se ha dado paso al entendimiento de muchos aspectos de la genómica funcional de Leishmania gracias a los avances en la metodología de transfección de ADN dentro de la célula de este protozoario, la eliminación y la complementación de genes por medio de recombinación homóloga y las estrategias para la selección de células transfectadas. Estos acercamientos tienen el potencial de brindar información sobre la expresión génica y la función de las proteínas en el contexto del parásito intacto. Dado que el genoma de Leishmania muestra una carencia acentuada de los factores conocidos de iniciación de la transcripción y que la expresión génica está regulada casi completamente a nivel postranscripcional (a través del empalme de los ARNm y los mecanismos que involucran el procesamiento diferencial de la región no traducida 3' del ARNm (3'UTR), la transfección génica representa una herramienta útil para la identificación y el análisis funcional de los genes de interés así como de los mecanismos que dirigen su regulación. El desarrollo de los sistemas de manipulación genética también ha abierto nuevos horizontes para la identificación de genes esenciales involucrados en la virulencia, la supervivencia intracelular y la resistencia a drogas de Leishmania, así como para la validación de proteínas específicas del parásito como nuevos blancos quimio e inmunoterapéuticos. En esta revisión presentamos los avances más recientes en el campo de la manipulación genética en Leishmania, los cuales permiten análisis estructurales, funcionales y de fenotipo, por medio de la eliminación y complementación génica a través de la transfección transitoria o permanente de genes en este parásito.Palabras clave: Leishmania, transfección de ADN, expresión génica, eliminación y complementación génica, genómica funcional. Genetic manipulation and the study of the protozoan parasite LeishmaniaDuring the last 15 years, many aspects of the functional genomics of Leishmania have been revealed due to advances in DNA transfection, gene disruption and complementation through homologous recombination, and efficient strategies for the selection of transfected cells. These strategies have provided information about gene expression and protein function in the context of the intact parasite. The genome of Leishmania shows a marked deficiency of known transcription initiation factors, and gene expression is regulated almost entirely at the posttranscriptional level through trans-splicing of mRNAs and novel control mechanisms involving differential processing of 3' -untranslated regions (3'-UTRs) of mRNAs. Therefore, gene transfection represents a useful tool for the identification and functional analysis of genes of interest as well as the mechanisms that direct their regulation. The development of genetic manipulation systems has provided opportunities for the study of genes involved in virulence, intracellular survival and drug resistance of Leishmania, as well as for the functional validation of specific parasite proteins as new ch...

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Molecular characterization and expression of a novel kinesin which localizes with the kinetoplast in the human pathogen, Leishmania donovani

Gerald

Coppens

Dwyer

2008

Cell Motil. Cytoskeleton

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Using a variety of molecular and cell biological approaches, we identified, characterized and expressed a novel kinesin, LdK39B in the protozoan pathogen Leishmania donovani. Results of RT-PCR revealed two distinct LdK39B products with different splice leader mini-exon sites, indicative of two potentially mature mRNA transcripts. Analyses indicated that LdK39B had a calculated molecular mass of >261, 327 Da and contained multiple amino acid repeat units. Several GFP-LdK39B fusion constructs were generated and used for episomal-expression in these parasites. Results of confocal and immunoelectron microscopy indicated that the GFP-LdK39B-fusion proteins localized to a region adjacent to the flagellar pocket and the kinetoplast i.e. the mitochondrial-DNA containing organelle that is physically tethered to the flagellar basal bodies. Sub-cellular fractionation results showed that GFP-LdK39B proteins were insoluble in nature and remained tightly associated with purified flagella/kinetoplasts following their extraction with detergent and high salts. Our cumulative results suggest that the LdK39B may play a scaffold-like role in facilitating and maintaining the unique spatial/structural association between the flagellum-basal body-kinetoplast complex in these parasites.

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Expression of a Mutant Form of Leishmania donovani Centrin Reduces the Growth of the Parasite

Cited by 74 publications

References 47 publications

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

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

Manipulación genética y el estudio del parásito protozoario Leishmania.

Molecular characterization and expression of a novel kinesin which localizes with the kinetoplast in the human pathogen, Leishmania donovani

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