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

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.

We propose 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, we 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) we will 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. We will 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. We will additionally assess the importance of individual molecules in fertilisation by raising mAbs against proteins confirmed as present on the male gamete surface. We 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.

Plasmodium is the causative agent of malaria and is thus directly responsible for the death of ~ 584,000 people every year, with a particular burden in sub-Saharan Africa. Malaria has been eradicated from Europe largely through vector control. However, the disease burden greatly affects economic growth, and inflicts a devastating socio-economic impact on endemic countries. In addition, more people are travelling in affected areas and an increasing number of cases are imported into the UK every year. Although the problem is multifaceted, it is recognised that interventions targeted at the vector stages will be central in the fight against the disease. Transmission of malaria through its mosquito vector is the weakest link in the chain that maintains the disease cycle: parasite numbers reach their lowest ebb during their early developmental stages in the mosquito. This bottleneck represents a likely point for intervention that provides a transmission blocking effect. Therefore data produced by the proposed research may be of future relevance to development of disease control interventions and could be exploited by public or private bodies, ultimately improving the quality of human and animal life. If applicable, the PI of this application will be primarily responsible for coordinating activities with Imperial Innovations towards patenting of concepts and exploitation of data generated through the proposed research. The applicant will additionally undertake the communication of the findings to the wider scientific community through participation at national and international meetings and conferences, social media and outreach activities, and initiate new collaborations aimed at advancing and broadening the scope of the proposed research.

The proposed research aims to study the sexual stages of Plasmodium, and specifically, to provide novel insights into the mechanism of fertilisation in Plasmodium, a process for which we currently have sparse knowledge. As discussed in the "Academic Beneficiaries" section, the proposed research potentially impacts several diverse fields including molecular parasitology, host/parasite interactions, vector biology, disease control and public health.

Our studies on this process aim to generate new and fundamental knowledge that could directly benefit researchers working not only on the sexual stages of Plasmodium, but also on other apicomplexan and trypanasomatid parasites. DNA constructs, P. berghei transgenic parasites and antibodies generated by this project will be preserved in the laboratory and readily made available upon request for secondary research. Research and development skills of staff supported within this proposal will also be developed significantly, with transferrable lab and management skills passed to the applicant and requested researcher. Discovery of interacting partners will potentially lead to the discovery of new potential vaccine targets for future study. If findings within this proposal are of potential clinical relevance, they can rapidly be translated to human malaria parasites at a later date. Given his links with specialist vaccinology groups (Jenner Institute/Kanazawa University/Fraunhofer Institute), field-based labs (IRSS, Burkina Faso/ MVRC, Thailand) and NGOs (PATH-MVI/BMGF), the applicant is well positioned to achieve this, and has previous experience of performing basic scientific research, and translating findings into clinically relevant experimental vaccines (e.g. HAP2/P25). Data arising from the generation of transgenic parasites, or subsequent proteomic analysis of interacting proteins, will be deposited in relevant public databases and released upon publication, or soon after curation if publication is not applicable.