Selective impairment of protein kinase C isotypes in murine macrophage by Leismania donovani

Leishmania donovani, an obligate intracellular parasite resides and multipl ies within macrophage of the reticuloendothelial system. The intracellular signalling mechanism involved in the impaired oxidative response in leishma niasis has not yet been clearly established. Generation of superoxide anion (O-2(-)) is supposed to be the first line of host defence during microbial invasion. We found a substantial inhibition of superoxide anion generation in parasitized macrophages, which was just the reverse in case of macropha ges challenged with Lipophosphoglycan (LPG) deficient attenuated leishmania l parasite UR-6. The generation of O-2(-) essentially needs the prior activ ation of protein kinase C (PKC) mediated phosphorylation events. Our study proposed that phosphorylation of 67, 54, 47 and 36 kDa proteins was attenua ted during infection. This was supported by PKC activity study, where Ca-de pendent PKC activity was inhibited but, Ca-independent PKC activity was enh anced. This result was further confirmed by using isotype specific pseudosu bstrate inhibitors of Ca-dependent PKC beta and Ca-independent PKC zeta. Ap plication of beta -pseudosubstrate could not alter the Ca-dependent PKC act ivity but zeta -pseudosubstrate inhibited the Ca-independent PKC activity i n infected macrophages. Our immunoblot analysis with specific antibody agai nst PKC beta and PKC zeta isotypes showed down regulation of PKC beta -II e xpression with concomitant induction of PKC zeta. Such inhibition of Ca-dep endent PKC beta was reversed in macrophages treated with UR-6. Taken togeth er, our observations revealed that infection with L. donovani selectively a ttenuates both the expression and activity of Ca-dependent PKC beta.