Identification and characterization of novel parasite proteins involved in growth and transmission of Plasmodium falciparum

Abstract

We demonstrated that PfPHB1 (PF3D7_0829200) and PfPHB2 (PF3D7_1014700) are expressed in the asexual and sexual blood stages of the parasite. Immunostaining indicated these proteins as mitochondrial residents as they were found to be localized as branched structures. We further validated PfPHBs as organellar proteins with no sex-specific expression in male and female gametocytes residing in Plasmodium mitochondria, where they interact with each other. Bio-lipid Sphingosine-1-phosphate was found to be interacting specifically with PfPHB2. Functional characterization was done in Saccharomyces cerevisiae orthologous model by expressing PfPHB1 and PfPHB2 in cells harbouring respective mutants. The PfPHBs functionally complemented the yeast PHB1 and PHB2 mutants, where the proteins were found to be involved in stabilizing the mitochondrial DNA, retaining mitochondrial integrity and rescuing yeast cell growth. Further, Rocaglamide (Roc-A), a known inhibitor of PHBs and anti-cancerous agent, was tested against PfPHBs and as an antimalarial. Roc-A treatment retarded the growth of PHB1, PHB2, and ethidium bromide petite yeast mutants. Moreover, Roc-A inhibited growth of yeast PHBs mutants that were functionally complemented with PfPHBs, validating P. falciparum PHBs as one of the molecular targets for Roc-A. Roc-A treatment led to growth inhibition of artemisinin-sensitive (3D7), artemisinin-resistant (R539T) and chloroquine-resistant (RKL-9) parasites in nanomolar ranges. The compound was able to retard gametocyte and oocyst growth with significant morphological aberrations. In addition, efforts were made to generate knockouts and inducible-knockdowns of PfPHB1 and PfPHB2 in P. falciparum. However, due to the difficult location for homologous recombination, the strains could not be generated. Based on our findings, we propose the presence of functional mitochondrial PfPHB1 and PfPHB2 in P. falciparum and their druggability to block parasite growth.