Apicoplast isoprenoid precursor synthesis and molecular basis of fosmidomycin resistance in Toxoplasma gondii

Abstract

Apicomplexan parasites are important pathogens responsible for economically important as well as life threatening infections in humans and animals. Poor efficacy and emerging resistance against available drugs is a matter of concern, as drug treatment still remains the chief way to combat these infections. The identification of a divergent plant plastid like organelle called apicoplast in these parasites has invited much attention for the development of new drugs. This unique organelle is essential as it is used by these parasites to meet its metabolic demands. Apicoplast supplies three important metabolites, fatty acids, isoprenoid subunits and heme. Out of these three metabolites, pathway to make isoprenoid subunits seems to be the most important one evidenced by its conservation among all the members of the phylum. The pathway is also the target of the antibiotic fosmidomycin, a drug that is in the phase II of clinical trials to be used against Plasmodium, the causative agent of malaria. Although effective against Plasmodium and Babesia, many other members of the Phylum including Toxoplasma gondii is naturally resistant to the antibiotic at concentrations 100 fold higher than against the sensitive parasites. I have used genetic tools to dissect this surprising observation using Toxoplasma as the model organism. Using the conditional knock out strategy in Toxoplasma, we showed that the target pathway is essential in organisms resistant to fosmidomycin. By complementing the target enzyme with a known fosmidomycin sensitive enzyme, we then realized that lack of drug access is the reason for resistance. In support of this hypothesis, we engineer denovo drug sensitive T.gondii parasites by the heterologous expression of the bacterial glycerol-3-po4 transporter capable of importing fosmidomycin. This sensitivity engineered by the transporter expression in Toxoplasma was comparable to what is seen in Plasmodium and Babesia. Further biochemical analyses showed that parasite plasma membrane is the chief barrier for drug entry as the transporter-expressing parasites accumulated the drug in the cytoplasm with the help of the transporter. Lethal infections with these parasites in mice could be treated with intraperitoneal injections of fosmidomycin validating the chemotherapeutic potential of the pathway