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

疟疾是一种由疟原虫寄生虫引起的急性疾病，仅由按蚊传播。据估计，每年有2.19亿疟疾病例，造成约584 000人死亡，其中大多数是5岁以下的儿童。该疾病还对流行国家造成毁灭性的社会经济影响。疟原虫通过按蚊的叮咬在人与人之间传播。通过蚊子传播疟疾是维持疾病周期的链条中最薄弱的环节：寄生虫数量在蚊子的发育阶段达到最低点，其中雄性和雌性配子相互识别并受精，从而完成寄生虫生命周期。这个瓶颈代表了一个合理的干预点，在这一点上杀死寄生虫，产生传播阻断效应。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，除了推进我们对疟原虫和其他顶复体受精过程的基本理解之外。尽管这个有吸引力的（和逻辑的）命题，目前对Plamarin的受精知之甚少，目前只确定了少数在这个过程中发挥作用的分子。目前，我们对雌雄配子如何相互作用以使其受精的了解甚少。此外，疟原虫，特别是伯氏疟原虫（一种啮齿动物的寄生虫，对人类没有致病性），由于了解疟疾的分子基础的重要性，已经成为研究寄生虫/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。最近在我们实验室鉴定了伯氏疟原虫配子中的蛋白质。然后使用增强的基于计算的工具检查这些蛋白质，以确定可能位于雄配子表面（膜）上的47个分子。为了增加我们目前对疟原虫受精的知识，并测试我们基于计算机的预测，我们建议详细检查其中41种蛋白质，首先确认它们在细胞中的位置。其次，如果蛋白质被证实位于雄配子的表面，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，然后将它们用作“诱饵”，通过从制备的寄生虫材料中提取新的相互作用的蛋白质来识别它们在雌配子中的功能性相互作用伴侣。一旦确定，我们将通过产生另一系列的转基因伯氏疟原虫来检测这些雌性配子蛋白在受精和性发育中的潜在作用，以准确地检测雌性配子上的基因功能（通过进行基因敲除）和蛋白定位（转基因标签）。作为一个补充的方法，我们将评估配子表面分子在受精中的重要性，通过提高抗体对蛋白质确认存在于配子表面。我们将在一系列试验中使用这些抗体来检查它们阻断疟疾传播的能力，为我们提供有关抑制受精和有性生殖的关键分子的新信息，可能使未来开发阻断传播的疫苗成为可能。通过结合两种互补的方法（转基因技术和抗体研究），我们将确定参与疟原虫受精的关键分子，增加我们对配子表面的了解，并确定男性和女性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.