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Originally published In Press as doi:10.1074/jbc.M106806200 on September 5, 2001

J. Biol. Chem., Vol. 276, Issue 46, 43253-43261, November 16, 2001
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Expression of a Mutant Form of Leishmania donovani Centrin Reduces the Growth of the Parasite*

Angamuthu SelvapandiyanDagger , Robert DuncanDagger , Alain DebrabantDagger , Sylvie Bertholet§, Gannavaram Sreenivas, Narender S. Negi||, Poonam Salotra, and Hira L. NakhasiDagger **

From the Dagger  Laboratory of Bacterial, Parasitic, and Unconventional Agents, Division of Emerging and Transfusion Transmitted Disease, and the § Laboratory of Parasitic Biology and Biochemistry, Division of Bacterial, Parasitic and Allergenic Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892, and the  Institute of Pathology (Indian Council of Medical Research) and || Safdarjung Hospital, New Delhi, 110 029 India

Leishmania donovani, a protozoan parasite, causes visceral disease in humans. To identify genes that control growth, we have isolated for the first time in the order Kinetoplastida a gene encoding for centrin from L. donovani. Centrin is a calcium-binding cytoskeletal protein essential for centrosome duplication or segregation. Protein sequence similarity and immunoreactivity confirmed that Leishmania centrin is a homolog of human centrin 2. Immunofluorescence analysis localized the protein in the basal body. Calcium binding analysis revealed that its C-terminal Ca2+ binding domain binds 16-fold more calcium than the N-terminal domain. Electrophoretic mobility shift of centrin treated with EGTA and abrogation of the shift in its mutants lacking a Ca2+ binding site suggest that Ca2+ binding to these regions may have a role in the protein conformation. The levels of centrin mRNA and protein were high during the exponential growth of the parasite in culture and declined to a low level in the stationary phase. Expression of N-terminal-deleted centrin in the parasite significantly reduces its growth rate, and it was found that significantly more cells are arrested in the G2/M stage than in control cells. These studies indicate that centrin may have a functional role in Leishmania growth.

* This work was supported by INDO-US Vaccine Action Program Y3-AI-9319-01 through interagency agreement between NIAID, National Institutes of Health and CBER/Food and Drug Administration.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF406767 and to GenPept Data Bank with accession number(s) AAL01153.

** To whom correspondence should be addressed. Tel.: 301-496-2205; Fax: 301-480-7928; E-mail: nakhasi@cber.fda.gov.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.


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