Properties of Plasmodium falciparum with a deleted apicoplast DNA gyrase

Malaria parasites have three genomes: a nuclear genome, a mitochondrial genome, and an apicoplast genome. Since the apicoplast is a plastid organelle of prokaryotic origin and has no counterpart in the human host, it can be a source of novel targets for antimalarials. Plasmodium falciparum DNA gyrase ( Pf Gyr) A and B subunits both have apicoplast-targeting signals. First, to test the predicted localization of this enzyme in the apicoplast and the breadth of its function at the subcellular level, nuclear encoded Pf GyrA was disrupted using CRISPR/Cas9 gene-editing. Isopentenyl pyrophosphate (IPP) is known to rescue parasites from apicoplast inhibitors. Indeed, successful growth and characterization of Pf ΔGyrA was possible in the presence of IPP. Pf GyrA disruption was accompanied by loss of plastid acyl-carrier protein (ACP) immunofluorescence and the plastid genome. Second, ciprofloxacin, an antibacterial gyrase inhibitor, has been used for malaria prophylaxis but there is a need for a more detailed description of the mode-of-action of ciprofloxacin in malaria parasites. As predicted Pf ΔGyrA clone supplemented with IPP was less sensitive to ciprofloxacin, but not the nuclear topoisomerase inhibitor etoposide. At high concentrations, however, ciprofloxacin continued to inhibit IPP-rescued Pf ΔGyrA possibly suggesting that ciprofloxacin may have an additional non-apicoplast target in P. falciparum . Overall, we confirm that Pf GyrA is an apicoplast enzyme in the malaria parasite, essential for blood-stage parasites, and a possible target of ciprofloxacin but perhaps not the only target.