Validation of reverse genetic approaches aiming to reveal critical proteolytic steps in early sporogony of Plasmodium parasites

Abstract

With the rapid emergence of resistance of mosquitoes and Plasmodium parasites to the available insecticides and anti-malarial drugs respectively, and the lack of an effective vaccine, malaria remains one of the deadliest diseases claiming more than a million lives annually mostly from developing countries. Therefore, there is an urgent need to identify new targets for developing new antimalarial strategies. During Plasmodium development both in vertebrate host and mosquito vector, central roles in invasion of the host cells/tissues, parasite metabolism, growth and differentiation, as well as, in host immune evasion, invasion/egress of the parasite from the host cell are played by parasitic proteases. As a consequence, in addition to numerous candidates so far identified, Plasmodium proteases as potential candidates for malaria interventions have been recently on the rise. However, since the most promising protease targets are the ones that are essential during the parasite life cycle, and therefore resistant to the conventional Plasmodium gene knock-out strategies, alternative approaches leading to either conditional silencing, or functional inactivation should develop. In this study, we explore two such approaches. First we targeted specifically P. berghei SUB2 gene, which encodes for a subtilisin-like serine protease expressed both in asexual and mosquito stages. SUB2 is an essential sheddase in merozoites, which makes its disruption unachievable. In an attempt to overcome this obstacle, and identify its functional involvement, as well as, putative substrates in mosquito stages we generated transgenic P. berghei parasites in which a ~1kb genomic sequence upstream of the SUB2 open reading frame (ORF), was replaced with the merozoite specific promoter, PbAMA-1. This strategy aimed to restrict expression of SUB2 in merozoites and abolish it in the subsequent stages. Indeed the resultant transgenic parasites developed normally in the vertebrate host. However, contrary to our expectations, the levels of PbSUB-2 in ookinetes were unaffected indicating a more complex regulation of gene expression than initially thought.