An.gambiae immune signaling gene SNPs and natural P. falciparum infection

描述（由申请人提供）：重要数据表明Toll/imd、胰岛素和TGF-β信号通路在冈比亚按蚊中肠疟疾寄生虫发育的调节中起作用。然而，没有数据可以证实这些信号通路在自然界中调节寄生虫的发育。我们选择使用SNP关联作图方法来解决这个问题，该方法使用来自马里和喀麦隆的实地收集的蚊子，这些国家具有高疟疾传播率，其中包括遗传上最分歧的An的地理分布。冈比亚人口尚未描述。我们将通过两个具体目标来实现这一目标。第一个目标包括群体遗传分析。感染和未感染的恶性疟原虫。冈比亚将从马里和喀麦隆的研究中心收集。这些位点经过精心选择，以包括该地区已知存在的所有遗传多样性（三种分子形式和染色体倒位多态性）。蚊子样本将根据感染、分子形式和核型按地点分组。然后，每只蚊子将进行大约384个免疫信号基因SNP的基因分型，并通过关联映射确定与感染相关的SNP。该分析将鉴定参与恶性疟原虫感染易感性的SNP。在第二个目标中，将分析选定的感兴趣的SNP以确定它们对蚊子蛋白质功能和对恶性疟原虫感染的易感性的影响。具体来说，我们将利用永生安。gambiae细胞系和过表达、敲低和基因转化技术的组合，以确认从序列预测的功能，并优先考虑用于体内研究的SNP。对于体内研究，我们将使用两种遗传上不同的An菌株。冈比亚，其已经针对感兴趣的SNP进行了基因分型。我们将使用抑制剂和转染（敲低，O 'nyong-nyong感染性克隆介导的过表达）方案来模拟含SNP的等位基因对感染蚊子中恶性疟原虫发育的影响。我们的工作将从我们的实验室和我们同事的实验室中获取功能数据，以检查现场收集的蚊子中选定的免疫信号通路的重要性。在这种情况下，本文提出的研究将有助于选择合适的基因靶点的转基因策略，并提供关键的新见解，在AN的群体遗传学的免疫。据我们所知，冈比亚目前还没有这些资料。 公共卫生相关性：冈比亚按蚊在撒哈拉以南非洲传播人类疟疾寄生虫恶性疟原虫。许多实验室研究都集中在蚊子的免疫系统如何响应并摧毁这些寄生虫，但关于这些反应在自然界中是否重要的信息很少。我们的研究将确定的反应，是重要的自然种群的一个。冈比亚的长期目标是，这些信息可以促进新的疟疾控制方法。

DESCRIPTION (provided by applicant): Significant data implicate the Toll/imd, insulin, and TGF-¿ signaling pathways in the regulation of malaria parasite development in the Anopheles gambiae midgut. However, no data are available to confirm that these signaling pathways regulate parasite development in nature. We have chosen to address this issue using a SNP association mapping approach with field-collected mosquitoes from Mali and Cameroon, countries with high malaria transmission that include the geographic distribution of the most genetically diverged An. Gambiae populations yet described. We will pursue this goal through two specific aims. The first Aim includes population genetic analyses. Plasmodium falciparum infected and uninfected An. gambiae will be collected from sites in Mali and Cameroon. These sites were carefully selected to include all of the genetic diversity known to exist in the region (three molecular forms and chromosome inversion polymorphism). Mosquito samples will be grouped by site with respect to infection, molecular form and karyotype. Each mosquito will then be genotyped for roughly 384 immune signaling gene SNPs and those SNPs correlated with infection identified by association mapping. This analysis will identify SNPs putatively involved in susceptibility to P. falciparum infection. In the second Aim, selected SNPs of interest will be analyzed to determine their effects on mosquito protein function and on susceptibility to P. falciparum infection. Specifically, we will utilize immortalized An. gambiae cell lines and a combination of over expression, knock-down, and gene conversion techniques to confirm predictions of function from sequence and to prioritize SNPs for in vivo studies. For the in vivo studies, we will use two genetically distinct strains of An. gambiae, which have been genotyped for the SNPs of interest. We will use inhibitors and transfection (knockdown, O'nyong-nyong infectious clone mediated overexpression) protocols to mimic the effects of SNP-containing alleles on P. falciparum development in infected mosquitoes. Our work will take functional data from our lab and from the labs of our colleagues to examine the importance of the selected immune signaling pathways in field-collected mosquitoes. In this light, the studies proposed herein will facilitate selection of appropriate gene targets for transgenesis strategies and provide critical new insights into the population genetics of immunity in An. gambiae that to our knowledge are not currently available. PUBLIC HEALTH RELEVANCE: The mosquito Anopheles gambiae transmits the human malaria parasite Plasmodium falciparum in sub-Saharan Africa. Many laboratory studies have focused on how the mosquito immune system responds to and destroys these parasites, but there is little to no information on whether these responses are important in nature. Our studies will identify responses that are important in natural populations of An. gambiae with the long-term goal that this information can contribute to novel malaria control methods.