Leishmania parasite causes human disease (Leishmaniasis) with clinical symptoms ranging from self healing cutaneous lesions to fatal visceral infection. The lack of understanding of the mechanism by which Leishmania parasite causes disease poses a serious public health risk worldwide and in particular for U. S. military personnel their families and tourists either living or travelling in endemic areas. As a first step towards understanding the molecular mechanism of Leishmania pathogenesis, we have began to analyze the processes that are involved in parasite life cycle in transformation from an avirulent to virulent form. We have identified several differentially regulated genes of Leishmania donovani, a viscerotropic parasite, using various molecular biology techniques such as differential hybridization and arbitarily primed polymerase chain reaction (AP-PCR). We have characterized one such gene that codes for a homologue of a mammalian chaperone protein calretciculin (CR) which is differentially regulated. We have also recently cloned a gene for protein disulfide isomerase (PDI) that is involved in formation of disulfide bond of proteins in the ER. CR along with other chaperones such as PDI have been shown to be important for glycosylation/folding and intracellular transport of membrane and secretory proteins in higher eukaroyotes. Very little is known about this process in parasites in general and Leishmania in particular. Further, membrane proteins or secretory proteins of Leishmania have been implicated for its defense mechanism against killing by the host. We attempted to alter the expression of CR gene in L. donovani so as to manipulate the biology of this protein and to create an attenauted parasite. Deletion of CR gene was lethal for the parasite. Next, we overexpressed the complete coding region or the putative domains of CR such as N-terminal(N), Proline rich (P), and C-terminal (C) regions. Mutant parasites expressing N-, P- and C-domains had a siginificnt decrease in one of the essential secretory proteins, s-acid phosphatase (sAcP). Reduction in the sAcP secretion was due to the accumulation of the protein in the ER. Further analysis showed that these mutants were less resitant to killing by the macrophages. Thus, these studies suggest that alteration in the secretion of leishmanial putative virulent factors can result in the attenuation of its infectivity.
