Translational Regulation of Mosquito mRNAs during Plasmodium Infection

描述(申请人提供)：疟疾是世界上最重要的热带寄生虫病。它造成了全球2%的死亡和非洲9%的死亡。超过30亿人生活在疟疾的威胁下。蚊子传播疟疾寄生虫的能力在不同的蚊子物种之间有所不同，甚至在同一物种的不同地理株之间也是如此。我们的长期目标是确定在赋予蚊子对疟疾寄生虫疟疾的易感性或耐受性方面发挥核心作用的蚊子基因，并利用这一知识帮助未来控制疟疾传播的努力。了解寄生虫-媒介相互作用的分子细节在很大程度上依赖于对蚊子感染疟原虫期间新生蛋白质合成的准确测量。目前的表达谱在很大程度上监测了稳定状态下mRNA水平的变化。然而，由于各种转录后和翻译后调控机制，转录组中每个给定mRNA的丰度并不一定反映蛋白质组中编码蛋白的表达。一些蚊子因子可能没有被识别出来，因为在蚊子摄入了受疟原虫感染的血餐后，它们是在翻译上而不是在转录上受到调控。这项研究计划将信使核糖核酸图谱与全基因组的信使核糖核酸翻译状态分析相结合，以同时监测蚊子转录组和新生蛋白质合成的变化。我们将使用蔗糖密度梯度离心法将与多核糖体相关的mRNA从翻译上不活跃的mRNAs中分离出来。这两个组分中单个蚊子基因的mRNA水平将通过微阵列杂交来确定。这种方法不仅将提供关于蛋白质合成速度的更准确的信息，还将导致发现应对疟原虫感染的一般性和基因特异性的翻译调控。该项目的具体目标是：1)发现蚊子基因在中肠对疟原虫感染的翻译调节。2)研究恶性疟原虫感染过程中敏感蚊虫和耐药蚊虫的中肠基因转录调控是否存在差异。公共卫生相关性：项目叙述疟疾寄生虫必须在蚊子媒介内经历一系列发育变化，才能感染脊椎动物宿主。我们将使用一种新的全基因组方法来研究蚊子在与血餐一起摄入寄生虫后的蛋白质合成。这项研究的结果将增进我们对蚊子和疟疾寄生虫相互作用的了解，并揭示决定蚊子传播寄生虫能力的遗传因素。这些新的见解反过来可能导致疟疾控制的新战略。

DESCRIPTION (provided by applicant): Malaria is the world's most important tropical parasitic disease. It is responsible for 2 percent of all deaths worldwide and 9 percent of deaths in Africa. More than 3 billion people live under the threat of malaria. The ability of mosquitoes to transmit malaria parasites varies among different mosquito species, even between different geographical strains of the same species. Our long term goal is to identify mosquito genes that play central roles in imparting susceptibility or refractoriness of mosquitoes to the malaria parasite Plasmodium, and to use this knowledge to aid future efforts to control malaria transmission. Understanding molecular details of the parasite-vector interactions relies heavily on precise measurement of nascent protein synthesis in the mosquito during Plasmodium infection. Current expression profiling largely monitors alterations in steady-state levels of mRNA. However, due to various post-transcriptional and translational regulatory mechanisms, the abundance of each given mRNA in the transcriptome does not necessarily mirror the expression of the encoded proteins within the proteome. Some mosquito factors might have gone unrecognized because they are translationally rather than transcriptionally regulated after mosquitoes ingest a Plasmodium-infected blood meal. This research plan combines mRNA profiling with genome-wide analysis of mRNA translation states to simultaneously monitor changes in the transcriptome and nascent protein synthesis in the mosquito. We will use sucrose density gradient centrifugation to separate mRNAs associated with polyribosomes from those that are translationally inactive. The mRNA levels of individual mosquito genes in each of these two fractions will be determined using microarray hybridization. This approach will not only provide more accurate information regarding the rate of protein synthesis, it also will lead to the discovery of general and gene-specific translational regulation in response to Plasmodium infection. The specific aims of this project are: 1) To discover translational regulations of mosquito genes in the midgut in response to Plasmodium infection. 2) To determine whether the translational regulations of midgut mRNA differ between the Plasmodium falciparum-susceptible and -refractory mosquitoes during Plasmodium infection. PUBLIC HEALTH RELEVANCE: Project Narrative The malaria parasites must undergo a series of developmental transformations inside the mosquito vector to become infectious to vertebrate hosts. We will use a new genome-wide approach to study protein synthesis in mosquitoes after ingestion of parasites with a blood meal. The results from this study will advance our knowledge of mosquito-malaria parasite interaction and reveal genetic factors that determine the ability of the mosquitoes to transmit the parasites. The new insights in turn may lead to new strategies for malaria control.