Expression of a mutant form of Leishmania donovani centrin reduces the growth of the parasite.
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 Ca(2+) 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 Ca(2+) binding site suggest that Ca(2+) 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 G(2)/M stage than in control cells. These studies indicate that centrin may have a functional role in Leishmania growth.
Selvapandiyan
A
A
Laboratory of Bacterial, Parasitic, and Unconventional Agents, Division of Emerging and Transfusion Transmitted Disease, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892, USA.
Duncan
R
R
Debrabant
A
A
Bertholet
S
S
Sreenivas
G
G
Negi
N S
NS
Salotra
P
P
Nakhasi
H L
HL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D002453:Cell Cycle; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry*; D003001:Cloning, Molecular; D003599:Cytoskeleton / Q000378:metabolism; D004533:Egtazic Acid / Q000494:pharmacology; D005434:Flow Cytometry; D017353:Gene Deletion; D015151:Immunoblotting; D007893:Leishmania donovani / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010802:Phylogeny; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013997:Time Factors; D014162:Transfection
D002135:Calcium-Binding Proteins; D002868:Chromosomal Proteins, Non-Histone; D012333:RNA, Messenger; C056608:caltractin; D004533:Egtazic Acid; D012313:RNA; D002118:Calcium