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

疟疾是由疟原虫引起的一种急性疾病，疟原虫只通过按蚊传播。据估计，每年有2.19亿疟疾病例，造成约58.4万人死亡，其中大多数是五岁以下的儿童。此外，该疾病还对流行国家造成毁灭性的社会经济影响。疟原虫通过按蚊叮咬在人与人之间传播。通过蚊子传播疟疾是维持疾病循环的链条中最薄弱的一环：在蚊子的发育阶段，寄生虫数量达到最低点，雄配子和雌配子相互识别并受精，从而完成寄生虫的生命周期。这一瓶颈代表了在这一点上杀死寄生虫的干预措施的合乎逻辑的点，产生了传播阻断效应。因此，对疟疾受精的研究可能会为未来的疟疾控制措施开辟新的途径，此外还可以促进我们对疟原虫和其他尖端复合体受精过程的基本了解。尽管这一命题很有吸引力(而且合乎逻辑)，但目前人们对Plamodium中的受精知之甚少，而且目前只发现了少数在这一过程中发挥作用的分子。目前我们对雄配子和雌配子如何相互作用以实现受精知之甚少。此外，由于了解疟疾的分子基础的重要性，疟疾，特别是伯氏疟原虫(一种对人类不具致病性的啮齿动物寄生虫)已成为研究寄生虫/宿主相互作用的模式生物。雄配子是研究受精的一个特别有吸引力的模型分子，因为它非常简单，只有4个细胞室。伯氏假单胞菌配子中的蛋白质最近在我们实验室被鉴定出来。然后使用增强的基于计算的工具对这些蛋白质进行检查，以识别可能位于雄配子表面(膜)上的47个分子。为了增加我们目前关于疟原虫受精的稀少知识，并检验我们基于计算机的预测，我们建议通过首先确认它们在细胞中的位置来详细检查这些蛋白质中的41个。其次，如果蛋白质被确认位于雄配子的表面，我们将在感兴趣的蛋白质被标记的地方制造转基因寄生虫，然后通过从准备好的寄生虫材料中提取新的相互作用的蛋白质，将它们作为“诱饵”来识别它们在雌配子中的功能相互作用伙伴。一旦确定，我们将研究这些雌配子蛋白在受精和性发育中的潜在作用，方法是产生另一系列转基因伯氏原虫寄生虫，以准确地描述雌配子上的基因功能(通过进行基因敲除)和蛋白质定位(转基因标记)。作为一种补充方法，我们将通过提高针对配子表面存在的蛋白质的抗体来评估配子表面分子在受精中的重要性。我们将在一系列检测中使用抗体来检查它们阻止疟疾传播的能力，为我们提供关于抑制受精和有性繁殖的关键分子的新信息，可能使未来开发传播阻断疫苗成为可能。通过结合两种互补的方法(转基因技术和抗体研究)，我们将识别参与疟原虫受精的关键分子，增加我们对配子表面的了解，并建立雄配子和雌配子如何相互作用以允许疟疾传播。

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.