Harnessing single-cell transcriptomics for the determination of sexual dimorphism during Plasmodium falciparum gametocytogenesis

About This Grant

PROJECT SUMMARY/ABSTRACT Unlike higher eukaryotes, unicellular organisms like the malaria parasite Plasmodium falciparum rely on the precise regulation of molecular events that stabilize the development of one sex lineage and/or repress the alternative fate. The development of P. falciparum is closely tied to its complex life cycle where sexual differentiation within a subpopulation of host red blood cells is critical for transmission to the mosquito vector. This sexual lifecycle stage, known as gametocytogenesis is a lengthy process, taking approximately 12 days to transition from immature sexual stages (stage I) to mature, differentiated male and female stages (stage V) that can be taken up by the female anopheles mosquito. Despite extensive research efforts, the molecular mechanisms governing sexual development and dimorphism in P. falciparum remain poorly understood. Recent studies have begun to shed light on sexual development using single-cell RNA sequencing (scRNA-Seq) and have defined a Malaria Cell Atlas,. These studies suggest that sexual fate in malaria parasites is likely determined downstream of commitment, following a branch point during gametocytogenesis. However, significant knowledge gaps persist, particularly regarding the timing and regulation of sex dimorphism. To address these gaps, we propose a comprehensive study leveraging scRNA-Seq to uncover the molecular mechanisms of cell fate determination using a combination of parasite genotypes from different geographical locations and unique genetic mutants in key regulators of gametocytogenesis. Our approach is structured around two specific aims. The first aim will determine the temporal, single-cell transcriptional landscape of gametocytogenesis using the 10x Genomics chromium platform with short-read (Illumina) sequencing using P. falciparum strains from varying genetic backgrounds of Africa, South America, and Southeast Asia origin. We will also refine the classification of gametocyte clusters associated with distinct stages (I-V) of gametocytogenesis and better delineate the earliest lineage bifurcations along the sexual trajectory. To accomplish this, we will measure and compare temporal, scRNA-Seq data from genetically modified strains with defects in known regulators of sexual-stage development. The second aim will capture the evolutionary dynamics of sex-specific transcript isoform diversity. To assess the downstream impact of isoform regulators on gametocyte development, differentiation efficiency, morphology, and reproductive success, we will couple long-read (PacBio) sequencing to better elucidate masked transcriptional modules and differential exon usage during gametocytogenesis. Additionally, we will validate the role of candidate hub isoform regulator(s) using genetic manipulation techniques such as DiCre-inducible conditional knockout (cKO). The outcomes of this proposal include identifying key regulators of sexual dimorphism and lineage-specific transcript isoforms used by malaria parasites to prepare for mosquito transmission.