Dissecting the molecular basis for gamete recognition in the malaria parasite, and its targeting to block transmission

This proposal sets to examine 41 proteomically-identified proteins, bioinformatically predicted to be present on the surface of the male gamete of P. berghei for localisation, and their interacting partners on the female gamete. Briefly, the project will produce GFP- or c-myc tagged parasites for each proteins of interest. Transgenic parasites expressing tagged proteins will be cloned following drug selection, and analysed by UV microscopy and IFA. Using these methods (with tubulin co-staining) it will help identify which putative fertilisation-related proteins are located on the surface of the male gamete, and are potentially involved in interaction with female gametes. Only proteins located on the gamete surface will be used in co-IP. Proteins will be co-IP’d from P. berghei female gamete preparations, identified by MS/MS, and analysed by the production of further transgenics to characterise function (by gene KO) and localisation (protein tagging). Each KO parasite will be analysed for the ability to; produce gametocytes, exflagellate, form ookinetes/oocysts. The project will also assess if male and female gametes bind; if male gametes fuse to females, and if nuclear fusion occurs. Tagged parasites will be analysed to assess when they are expressed, and if they co-localise with other fertilisation implicated molecules. It will additionally assess the importance of individual molecules in fertilisation by raising mAbs against proteins confirmed as present on the male gamete surface. It will then use these in exflagellation, ookinete, and SMFA assays to assess their ability to block transmission, identifying molecules that mediate reproduction while informing the development of novel transmission blocking vaccines. The use of these assays allows us to elucidate the point of action in the lifecycle for each protein examined. In this manner, we will identify molecules involved in plasmodial fertilisation, increase our knowledge of the gamete surface, and establish how male and female gametes interact.

UKRI Project details

Basic Science

Jan 2016 — Apr 2019

$741,958

Burkina Faso
United Kingdom