Peroxidoxin gene expression in Leishmania

Leishmania protozoans are the etiologic agents of the disease leishmaniasis. The parasite exists in two morphological forms: promastigote and amastigote. Promastigotes are found in the gut of the sandfly vector while amastigotes reside inside the vertebrate macrophage. Leishmania, an obligate intracellular parasite, resists toxic reactive oxygen species (ROS) from both endogenous and exogenous sources. Like other protozoa, Leishmania lacks some of the antioxidant defence enzymes such as catalase and glutathione peroxidase (Gpx) that are usually found in aerobic cells. Instead they possess the antioxidant thiol compound trypanothione, in association with specific trypanothione linked antioxidant enzymes such as peroxidoxins. The transformation from promastigote to amastigote is a crucial step for parasite infection and survival. The molecular basis for this transformation is not clearly understood. Recently it was shown that the peroxidoxin gene is present in multiple copies in Leishmania. In the present study we examined the potential of antisense RNA and double stranded RNA (dsRNA) to perform functional knockout of the peroxidoxin gene. Towards that end antisense RNA and dsRNA expressing plasmids, targeting the peroxidoxin gene, were constructed. Leishmania promastigotes were subsequently transfected with these plasmids and the levels of peroxidoxin gene expression were studied. The results from this study suggest that there is no apparent reduction in either the levels of peroxidoxin mRNA or the protein in the transfected promastigotes as compared to the non-transfected cells