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

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Malaria is an acute disease caused by Plasmodium parasites, which are transmitted exclusively by Anopheles mosquitoes. There are an estimated 219 million malaria cases annually, causing ~584,000 deaths, the majority of whom are children under the age of five. The disease additionally inflicts a devastating socio-economic impact on endemic countries. Plasmodium is transmitted from person to person by the bite of an anopheline mosquito. Transmission of malaria through the mosquito is the weakest link in the chain that maintains the disease cycle: parasite numbers reach their lowest ebb during their developmental stages in the mosquito, where male and female gametes recognise and fertilise each other, allowing the completion of the parasitic lifecycle. This bottleneck represents a logical point for interventions that kill the parasite at this point, creating a transmission-blocking effect. As such, research on malarial fertilisation may open novel avenues for future malaria control measures, in addition to advancing our basic understanding of the process of fertilisation in Plasmodium, and other apicomplexa. Despite this attractive (and logical) proposition, very little is currently known about fertilisation in Plamodium, and only a handful of molecules that play a role in the process are currently identified. We currently have very little understanding of how male and female gametes interact with each other to allow fertilisation.Additionally, Plasmodium, and notably Plasmodium berghei (a parasite of rodents that is not pathogenic to man) has become a model organism for the study of parasite/host interactions because of the importance of understanding the molecular basis of malaria. The male gamete is a particularly attractive model molecule to study fertilisation, as it is very simple, with only 4 cellular compartments. The proteins in the gamete of P. berghei have recently been identified in our laboratory. These proteins were then examined using enhanced computing-based tools to identify 47 molecules that are potentially located on the surface (membrane) of the male gamete. In order to increase our currently sparse knowledge of fertilisation in Plasmodium, and to test our computer-based predictions, we propose to examine 41 of these proteins in detail, by firstly confirming their location in the cell. Secondly, if proteins are confirmed as located on the surface of the male gamete, we will make transgenic parasites where the proteins of interest are tagged, and then use them as "bait" to identify their functional interacting partners in female gametes, by pulling novel, interacting proteins out of prepared parasite material. Once identified, we will examine the potential roles of these female gamete proteins in fertilisation and sexual development by the generating a further series of transgenic P. berghei parasites to accurately characterise gene function on the female gamete (by making gene knockouts), and protein localisation (transgenic tagging). As a complimentary approach, we will assess the importance of gamete surface molecules in fertilisation by raising antibodies against proteins confirmed as present on the surface of the gamete. We will use the antibodies in a range of assays to examine their ability to block malarial transmission, giving us new information about key molecules that inhibit fertilisation and sexual reproduction, potentially enabling the future development of a transmission-blocking vaccine. By combining two complimentary approaches (transgenic technology and antibody studies), we will identify key molecules involved in fertilisation of Plasmodium, increase our knowledge of the surface of the gametes, and establish how male and female gametes interact to allow malarial transmission.