Analysis of gene function during Plasmodium development in the mosquito

Ookinete development and midgut invasion represent major population bottlenecks iri the malaria life cycle that can be successfully targeted by intervention strategies such as transmission blocking vaccines, provided essential parasite targets are known. From a recent proteomic analysis a large number of proteins expressed by the ookinete have been identified, yet the function of most of these molecules is unknown. This study aimed to characterise the function of ookinete proteins putatively involved in the interactions between the parasite and its environment by utilising gene targeting . approaches in Plasmodium' berghei. Twenty-six proteins, including thirteen hypothetical proteins, were selected based on their expression pattern 2lhd the possession of a signal peptide. While clonal knockout lines were successfully generated for half of the candidates, seven genes could not be disrupted, suggesting they might encode proteins essential for asexual blood stage development. To prioritise candidates for detailed phenotypic analysis, a basic phenotypic screen was first carried out to identify candidates that showed a significant reduction in parasite numbers at either oocyst or salivary gland sporozoite stage. This screen identified important roles at distinct points during parasite development for eight proteins, i.e. in more than half of the knockout lines. Seven of these could not be transmitted through mosquitoes due to a block before midgut invasion (two candidates),sporozoite formation (four candidates) or sporozoite invasion of the salivary gland (one candidate). Genetic crosses with parasite lines deficient in the production of either male or female fertile gametes demonstrated that the abnormal oocyst development observed. for the four sporulation-deficient mutants results from a lack of expression of the maternally-inherited gene copy. These findings suggest that sortie 'late' mutant oocyst phenotypes are caused by defects during the first few hours of parasite development in the mosquito, and that, like in mammals, early development may be maternally controlled.EThOS - Electronic Theses Online ServiceGBUnited Kingdo