| 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.
In a parallel alternative approach, we expressed in a stage specific
manner (under ookinete and sporozoite specific promoters), serine protease inhibitors (serpins) of ovalbumin and Kazal families. The derived transgenic parasites exhibited normal development in the vertebrate. Moreover, in vitro and in vivo gametocyte to ookinete transition was not affected. However, while the LGI-expressing parasites (under CTRP promoter) showed a mild reduction in
oocyst formation, the NcPI-S-expressing parasites showed a severe (97-98%) inability to form oocysts.
Detailed characterization of NcPI-S-expressing ookinetes, involving
biochemical studies using in vitro ookinete cultures, as well as cell biology studies implicating gametocyte and ookinete feeding experiments, revealed an inability of the transgenic ookinetes, despite their normal motility, to associate tightly with mosquito midgut epithelium, a pre-requisite step for invasion. At the
molecular level although expression of two surface associated proteins (P25, CTRP), critical for the mosquito midgut invasion, was found unaffected in NcPI-S expressing ookinetes, protein levels of the secreted essential micronemal proteins WARP and SOAP, were severely reduced.
In a remarkable contrast to the NcPI-S expression in the ookinete, NcPI-S expression in sporozoites under CS promoter did not affect their development or their ability to invade mosquito salivary glands, and subsequently to establish a successful infection in the vertebrate host with similar pre-patent periods to the wt control.
In summary, this study has established P. berghei ookinetes as a test tube system to screen in vivo macromolecular protease inhibitors. Such molecules could be subsequently used to generate attenuated Plasmodium parasites as part of the efforts towards live attenuated vaccine. Alternatively, MPIs can be used for the development of innovative transmission blocking interventions.
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Validation of reverse genetic approaches aiming to reveal critical proteolytic steps in early sporogony of Plasmodium parasites
