Malaria parasites including the most virulent species, Plasmodium falciparum, switch between a mammalian host and a mosquito vector, passing through multiple developmental stages and host tissues in the process. If malaria elimination is to be achieved, interrupting transmission will be essential yet it is the least understood part of the malaria cycle. Transmission forms, or gametocytes, upon reaching maturity after development in red blood cells, are the only stages that can be transmitted to the mosquito vector via an infected blood meal. Stage conversion into gametocytes occurs in a small subpopulation of late red blood cell stage parasites, whereby one parasite either produces only asexually or sexually committed invasive daughter cells. After invasion of sexual parasites into red blood cells, additional factors determine developmental progression during development toward transmission competent forms. The proportion of gametocytes derived from one replication cycle (i.e., conversion rate) and the ratio of female and male gametocytes (i.e., sex ratio) vary between strains, both in vivo and in in vitro models. Determinants of stage conversion remain elusive except that several chromosomal deletions have been linked to the loss of sexual development in vitro. We therefore hypothesize that Plasmodium stage conversion has a strong genetic component. No parasite (and host) factors involved in Plasmodium stage conversion have been identified so far, mostly due to lack of methodology to study this process systematically. Specifically, there has not been an efficient and reproducible system for studying conversion and genetics are inherently difficult because of the low transfection efficiency in P. falciparum. We have recently established a reproducible and high throughput compatible protocol for stage conversion and gametocyte development, which we have developed into a drug assay. More recently, we have also successfully applied this assay as a phenotypic readout in the first forward genetic screen in P. falciparum, using the piggyBac transposon. Here we propose systematic characterization of the pathways involved in stage conversion using a second-generation piggyBac screen that is optimized for high throughput mutant generation and phenotyping. This study combines several highly innovative approaches by linking an efficient conversion system and a forward genetic screen. In combination with our ongoing efforts to investigate pathways involved in stage conversion using chemical probes and transcriptional profiling, this study provides the unique opportunity for a comprehensive analysis of an essential yet so far elusive process in the malaria life cycle.
Malaria remains a major public health issue, and resistance to most current treatments is widespread. We propose a genetic screen to identify determinants that are essential for the formation of malaria transmission stages. We anticipate that the proposed experiments will significantly improve our understanding about a critical phase of the malaria life cycle and as such form the basis for novel transmission blocking intervention strategies.
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