Expression of a mutant form of Leishmania donovani centrin reduces the growth of the parasite

Leishmania donovani, a protozoan parasite, causes visceral disease in human s. To identify genes that control growth, we have isolated for the first ti me in the order Kinetoplastida a gene encoding for centrin from L. donovani . Centrin is a calcium-binding cytoskeletal protein essential for centrosom e duplication or segregation. Protein sequence similarity and immunoreactiv ity confirmed that Leishmania centrin is a homolog of human centrin 2. Immu nofluorescence analysis localized the protein in the basal body. Calcium bi nding analysis revealed that its C-terminal Ca2+ binding domain binds 16-fo ld more calcium than the N-terminal domain. Electrophoretic mobility shift of centrin treated with EGTA and abrogation of the shift in its mutants lac king a Ca2+ binding site suggest that Ca2+ binding to these regions may hav e a role in the protein conformation. The levels of centrin mRNA and protei n were high during the exponential growth of the parasite in culture and de clined to a low level in the stationary phase. Expression of N-terminal-del eted 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 th an in control cells. These studies indicate that centrin may have a functio nal role in Leishmania growth.