Molecular Mechanisms to Attenuate Leishmania Parasite

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Leishmania parasite causes human disease with clinical symptoms ranging from-self healing cutaneous lesions to a fatal visceral infection. Additionally, in endemic areas, people infected with HIV are especially prone to Leishmania infection and latent infections can reactivate upon acquisition of HIV. The lack of understanding of cell biology and pathogenic mechanisms of this parasite makes the task of controlling this grave, worldwide health risk difficult. Closer to home, it is particularly of concern to U.S. military personnel, their families and other travelers visiting or living in the endemic areas. To find novel methods for control of this pathogen, we have initiated study to understand the mechanism of parasite differentiation from the avirulent (promastigote) to virulent (amastigote) form. Membrane or secretory proteins of Leishmania have been implicated for its defense mechanism against killing by the host. Very little is known about the secretory pathway of trypanosomatids parasites such as Leishmania in general with particular importance to glycosyation/folding and intracellular transport of membrane and secretory proteins. The ER resident chaperone proteins are known to play an essential role in these processes. We argued that alteration of the secretion process via modulating the expression of chaperone proteins might result in attenuation of virulence in Leishmania. We have cloned several homologues of such chaperone proteins such as calreticulin and protein disulfide isomerase (PDI) from Leishmania donovani. Overexpression of the P-domain of Leishmania calreticulin resulted in a significant reduction in the secretion of the parasite secretory acid phosphatases, a putative virulent factor. This effect is associated with an intracellular accumulation of active enzyme inside the parasite. Such parasites where shown to have decreased survival inside human macrophages. Mutations in the active site and expression of another essential ER chaperone PDI, which is important for maintaining reduced environment inside the ER and for the formation of disulphide bonds in addition to chaperone activity also affected the secretion of s-acid phosphatase (sAcP). These studies suggest that alteration in the chaperone protein expression can modulate the secretion of Leishmanial putative virulent factors, which can result in the attenuation of its infectivity. Further, the Leishmania PDI proved to be significantly smaller in size than a typical mammalian PDI (e.g. human host), therefore, we have initiated studies to elucidate the 3D structure of the parasite enzyme. To that end, milligram quantities of E.coli expressed Leishmania PDI have been prepared under native conditions and are currently being purified to homogeneity. Such preparation will be subjected to protein crystallization and X-ray diffraction analyses in order to determine structure of the Leishmania PDI. Comparison of the parasite PDI with the human PDI may results in significant differences that could be exploited for the development of novel anti-leishmanial drugs.