Efficient site-specific integration in Plasmodium falciparum chromosomes mediated by mycobacteriophage Bxb1 integrase

Abstract

Here we report an efficient, site-specific system of genetic integration into Plasmodium falciparum malaria parasite chromosomes. This is mediated by mycobacteriophage Bxb1 integrase, which catalyzes recombination between an incoming attP and a chromosomal attB site. We developed P. falciparum lines with the attB site integrated into the glutaredoxin-like cg6 gene. Transfection of these attB + lines with a dual-plasmid system, expressing a transgene on an attPcontaining plasmid together with a drug resistance gene and the integrase on a separate plasmid, produced recombinant parasites within 2 to 4 weeks that were genetically uniform for single-copy plasmid integration. Integrase-mediated recombination resulted in proper targeting of parasite proteins to intra-erythrocytic compartments, including the apicoplast, a plastid-like organelle. Recombinant attB × attP parasites were genetically stable in the absence of drug and were phenotypically homogeneous. This system can be exploited for rapid genetic integration and complementation analyses at any stage of the P. falciparum life cycle, and it illustrates the utility of Bxb1-based integrative recombination for genetic studies of intracellular eukaryotic organisms.In response to the worsening burden of Plasmodium falciparum malaria, intense research efforts have led to the development of various tools for the genetic manipulation of this organism. The application of techniques such as gene disruption and allelic replacement has afforded important insights into parasite mechanisms of drug resistance, pathogenesis, host cell invasion and transmission 1 . Yet genomic integration studies are plagued by low transfection and recombination efficiencies. In addition, transgene expression assays have been COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests. restricted to episomally replicating plasmids that parse unequally during mitotic segregation and hence cannot be selected in sexual stages and propagated through the life cycle 1 . NIH Public AccessIntegrative recombination offers a powerful approach to target foreign DNA into specific genomic locations. Bacteriophage serine integrase-based systems are particularly suitable because of their intrinsically high efficiency of recombination between host attB and phage attP sites, with integration and excision having distinct reaction requirements that preclude spontaneous reversion events 2 . These recombinases, unlike their tyrosine counterparts such as lambda integrase (used to develop the Invitrogen GATEWAY site-specific recombination system), are sufficient to catalyze recombination and do not require bacterial host factors. Among the serine integrases, the most extensively used has been Streptomyces sp. phage phiC31, which efficiently integrates plasmid vectors into prokaryotic or eukaryotic hosts containing att or pseudo-att sites 2,3 .The serine integrase of the mycobacteriophage Bxb1, which is a temperate phage whose genome recombines into an attB site located wi...

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