Metabolic Regulation of Gametocytogenesis in Malaria

Abstract

Plasmodium sp. is an obligate intracellular protozoan parasite causing malaria and is responsible for more than 500,000 human deaths annually. P. falciparum and P. vivax are the two major species causing malaria in humans. In the human host, the parasite exists in two forms; the asexual stages (merozoite, ring, trophozoite and schizont) and the sexual stage called gametocytes. The asexual intraerythrocytic cycle is responsible for the symptoms of malaria in the diseased host while gametocytes are essential for onward transmission of the parasite from a diseased host to a healthy host via the female Anopheles mosquito. A very small fraction of parasites participating in the asexual cycle exit the cycle to commit to form gametocytes. Since this is the only transmissible form of the parasite and important for development of transmission blocking interventions, it is critical to understand the factors responsible for commitment and development of mature gametocytes. In the current study we have used a combination of cell biology and bioinformatics along with global and targeted metabolomics to understand the cues and mechanisms of sexual stage induction in the malaria parasite. Plasmodium being an intracellular pathogen closely shares its metabolic circuitry with host RBCs. In our study, we employed global and targeted metabolomics to investigate P. falciparum-induced host redox perturbation. We observed diminished GSH/GSSG ratio in parasitized RBC; indicative of redox imbalance in host cell. Further, we demonstrated that the parasite has a truncated TS pathway which leads to accumulation of redox active HCy in host RBCs and culture supernatant. This observation provides the mechanism behind the long-known occurrence of hyperhomocysteinemia in malaria patients. Additionally, we show that physiological concentration of HCy present in malaria patients is sufficient to induce sexual stage transition in the parasite to form gametocytes. Thus, we provide the functional significance of a disrupted metabolic pathway in the parasite. Using immunoblot and mass spectrometry we further observed a novel PTM called homocysteinylation in parasite histones which we hypothesize plays a role in epigenetic control of gametocytogenesis. Further, we have studied the effect of redox imbalance in host RBCs on gametocytogenesis by examining (1) naturally occurring hemoglobinopathies like sickle cell anaemia and thalassemia. (2) G6PD-deficient RBCs; and (3) by creating redox stress using chemical intervention via 2-AAPA, an inhibitor of glutathione reductase. Our results show that in all the above host RBCs, redox imbalance triggers gametocytogenesis in parasites. Further, we found that plasma from haemoglobinopathic patients have elevated homocysteine levels and show concomitant increase in histone homocysteinylation of parasites growing in these RBCs, emphasising its potential role in triggering gametocytogenesis. Therefore, our observations indicate that hemoglobinopathic patients are protected from severe malaria but may work as silent reservoir of gametocytes facilitating transmission of malaria. Altogether our results implicate the potential of redox imbalance in triggering commitment and suggest the role of homocysteine and concomitant parasite histone homocysteinylation as a potential mechanism for autocrine/paracrine induction of gametocytogenesis. Apart from redox metabolites, our global metabolomics study revealed pipecolic acid to be uniquely present in parasite infected in vitro and in vivo samples. Thus, pipecolic acid may serve as a metabolite marker for malaria infections.