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

摘要: The parasitic protozoa belonging to Leishmania (L.) donovani complex possess abundant, developmentally regulated cathepsin L-like cysteine proteases. Previously, we have reported the isolation of protease gene, Ldccys2 from chagasi. Here, further characterized this gene and demonstrated its role during infection survival chagasi within U937 macrophage cells. amastigote specific genes L. identical organization, as determined by southern blots. In vivo expression analyses Northern blots showed that is specific. Western blot using anti-Ldccys2 antibody confirmed protein expression. Recombinant Ldccys2, a 30 kDA protein, was functionally active in gelatin assay. Results heterozygous knockout mutants intra-macrophage parasites. Since attempts generate null failed, used antisense RNA inhibition regulate Ldcccys2 Not surprisingly, results studies mutants, reiterating importance proteases pathogenesis. study shows expressed only infectious stages parasite. collective both mRNA helps amastigotes Finally, technique can be an alternate approach knockout, for silencing chagasi, especially cases such this, where mutant cannot achieved homologous recombination.