Regulation of guanylyl cyclase by intracellular Ca²⁺ in relation to the infectivity of the protozoan parasite, Leishmania donovani

Abstract

A neuronal type Ca²⁺ stimulated nitric oxide synthase was earlier reported by us to be present in the protozoan parasite Leishmania donovani. As part of nitric oxide-cyclic GMP transduction signaling operative in higher eukaryotes and involved in the long-term potentiation, a soluble guanylyl cyclase has also been detected in this lower eukaryote. However, detailed biochemical characterization revealed the enzyme to be Ca²⁺ modulated and unstimulated by nitric oxide donors as opposed to higher eukaryotes. The possible role of intracellular Ca²⁺ level in the regulation of guanylyl cyclase activity as well as L. donovani infectivity was explored by measuring the intracellular survival of the parasites in mammalian macrophages after treatments, which decrease or elevate the intracellular Ca²⁺. Parasites loaded with intracellular Ca²⁺ chelators displayed significantly decreased infectivity and cyclic GMP level. In contrast, pretreatment with Ca²⁺ ionophores, which elevated Ca²⁺ levels in L. donovani, significantly enhanced the cyclic GMP level as well as the infectivity of the parasites. Moreover, treatment with selective inhibitors of soluble guanylyl cyclase also reduced infectivity, even in cases of calcium ionophore-treated parasites. The gene encoding the soluble guanylyl cyclase was cloned, sequenced and over expressed in bacterial system. The recombinant protein showed enzyme characteristics similar to that obtained in L. donovani promastigote cytosol. Together these results suggest a possible link between guanylyl cyclase, intracellular Ca²⁺ content and parasite infectivity.