EarlyGam - Exploring early sexual differentiation in the malaria parasite Plasmodium falciparum
Project Abstract
Plasmodium falciparum, an obligate intracellular parasite of the phylum Apicomplexa, is the causative agent of the most severe form of malaria in humans. Despite the massive implementation of control interventions, malaria still causes over 200 million clinical cases and half a million deaths annually. The widespread prevalence of malaria in endemic regions is maintained by a complex yet highly efficient transmission cycle between the human host and mosquito vector. For transmission to occur, mosquitoes must ingest mature female and male gametocytes, which are constantly produced during blood infection at low frequency from the pool of asexual parasites that undergo repeated rounds of replication inside erythrocytes. Gametocytes are non-replicating cells that need twelve days to differentiate into the transmission-competent mature quiescent forms. Unlike the asexual parasites, gametocytes are not efficiently killed by front-line antimalarial drugs. A detailed understanding of gametocyte biology will be essential to support the development of urgently needed new medicines for malaria elimination.
Seminal work in the past ten years (including our own) demonstrated that asexual parasites commit to sexual development via an epigenetic switch that activates expression of AP2-G, the master transcriptional regulator of sexual conversion. These discoveries transformed our possibilities to study gametocyte biology at the molecular and functional level. We propose to capitalize on these advancements to investigate the earliest phase of sexual differentiation and the mechanisms underlying sex determination in P. falciparum. We developed an experimental approach that will allow us for the first time to study early sex-specific gene expression. We will also ask whether facultative heterochromatin contributes to the non-genetic sex determination mechanism of malaria parasites. Lastly, we will perform functional studies on a number of uncharacterized early gametocyte genes. Carefully engineered fluorescent reporter strains able to discriminate between asexual parasites and early female and male gametocytes will be used throughout this project.
To achieve our objectives, we will mainly employ CRISPR/Cas9-based gene editing to modify target genes, DiCre-dependent conditional mutagenesis for loss-of-function studies, RNA-seq and CUT&Tag to profile transcriptome and heterochromatin landscapes, respectively, a newly designed in vivo proximity-dependent biotinylation approach to identify protein interactomes, and ultrastructure expansion microscopy for high-resolution protein localization data. This comprehensive and innovative set of approaches will provide unprecedented functional insight into P. falciparum gametocytogenesis and will generate new tools and datasets as important resources for the wider malaria research community.
