Development and application of a rapid site -specific integration system in the malaria parasite Plasmodium falciparum
Nkrumah, Louis John
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The current transfection techniques for genomic integration and complementation studies in the malaria parasite, Plasmodium falciparum, are inefficient and labor-intensive. It takes 3-5 months to achieve genetic integration in the parasite, many of which result in multi-copy or random integration of the plasmid into undesired regions of the genome. We have developed an efficient, site-specific integration system in P. falciparum, which uses the Bxb1 mycobacteriophage integrase to rapidly catalyze recombination between an attP plasmid and a chromosomal attB site. Transfection of attB+ P. falciparum lines with the attP plasmid produced recombinant attB x attP parasites within 2-4 weeks. The integration was stable in the absence of drug pressure and homogeneous for single-copy plasmid integration. The system supported expression and targeting of transgenic proteins to subcellular compartments in the parasite.;We employed the integrase-mediated attB x attP integration system to test whether pfenr ( Plasmodium falciparum enoyl-ACP reductase) is the target of the antimalarial agent triclosan. We generated transgenic P. falciparum parasites expressing the A217V mutant of pfenr that was previously reported to cause a 7000-fold decrease in triclosan binding affinity. However, the A217V mutation failed to confer resistance to triclosan in the parasites. Further studies revealed that neither pfenr transcript nor protein was detectable in the asexual stages of the parasite. Targeted disruption of pfenr in P. falciparum produced viable parasites with no growth defects. Our results suggest that pfenr is not essential in the asexual stages of P. falciparum, where triclosan has been reported to kill the parasites, and hence may not be the target of triclosan.;We used allelic exchange technique to investigate the role of PfNHE ( P. falciparum sodium-proton exchanger) in quinine resistance. Replacing the endogenous 3' untranslated region (UTR) of pfnhe with a truncated 3'UTR resulted in under-expression of PfNHE and a subsequent 30% reduction in quinine resistance in recombinant P. falciparum clones. The decrease in quinine resistance was observed only in parasites that were already resistant to quinine, suggesting that PfNHE contributes to quinine resistance, but only in a genome context already conferring a basal level of quinine resistance.