Role of Leishmania (Leishmania) chagasi amastigote cysteine protease in intracellular parasite survival: studies by gene disruption and antisense mRNA inhibition

The amastigote specific Ldccys2 genes of L. (L.) chagasi and L. (L.) donovani have identical gene organization, as determined by southern blots. In vivo expression analyses by Northern blots showed that Ldccys2 is amastigote specific. Western blot using anti-Ldccys2 antibody confirmed the amastigote specific protein expression. Recombinant expression of Ldccys2, a 30 kDA protein, was functionally active in a gelatin assay. Results from Ldccys2 heterozygous knockout mutants showed its role during macrophage infection and in intra-macrophage survival of the parasites. Since attempts to generate null mutants failed, we used antisense RNA inhibition to regulate Ldcccys2 gene expression. Not surprisingly, the results from antisense studies further confirmed the results from heterozygous knockout mutants, reiterating the importance of amastigote specific cysteine proteases in Leishmania infection and pathogenesis.The study shows that Ldccys2 is a developmentally regulated gene and that Ldccys2 is expressed only in infectious amastigote stages of the parasite. The collective results from both the heterozygous knockout mutants and antisense mRNA inhibition studies shows that Ldccys2 helps in infection and survival of L. (L.) chagasi amastigotes within the macrophage cells. Finally, antisense RNA technique can be used as an alternate approach to gene knockout, for silencing gene expression in L. (L.) chagasi, especially in cases such as this, where a null mutant cannot be achieved by homologous recombination.Leishmania are the etiological agents of a variety of disease manifestations, collectively termed as leishmaniasis. Visceral leishmaniasis caused by Leishmania (L.) donovani and Leishmania (L.) chagasi is a serious health problem in many tropical and subtropical countries [1-3]. During the digenetic life cycles of Leishmania, it alternates between gut of sand fly vector as an extra cellular promastigote and in the acidic phagolysosome of macrophage as an intracellular am