Untangling the complex molecular interplay in vector-pathogen interactions using bioinformatics, gene silencing, and gene editing.

利用生物信息学、基因沉默和基因编辑来阐明载体-病原体相互作用中复杂的分子相互作用。

• 批准号: RGPIN-2019-04259
• 负责人: Lowenberger, Carl
• 金额: $ 4.23万
• 依托单位: Simon Fraser University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=744783
• 关键词: Untangling; complex; molecular; interplay; vector

Insect vectors transmit parasites and pathogens to humans, yet the vast majority of parasites that enter vectors are killed by components of the insects' innate immune system. This response is germ-line encoded and is neither learned nor acquired, unlike the characteristics of vertebrate immunity. Several mechanisms are used by the insects to recognize parasites and pathogens as "non-self". Recognition activates immune pathways (Toll, IMD, RNAi and Jak-STAT) to encapsulate, melanixe, or express potent antimicrobial peptides (AMPs) all of which can kill parasites. Despite the power of innate immunity, many insects still transmit parasites. The relationship between vector and parasite is very specific. Each vector species transmits one, or a very limited number of, parasites or pathogens. If the immune system functions why do vectors not kill all parasites? In Cali Colombia, approximately 30% of field collected Ae. aegypti, the major vector of Dengue and Zika viruses, actually kill the viruses while 70% continue to transmit the viruses to humans. If being refractory to viruses is a benefit, why do all female Ae. aegypti not kill the viruses? Within Ae. aegypti, Dengue viruses exploit existing mosquito proteins to establish, enter cells, and replicate, but in the refractory strains of Ae. aegypti these genes are not expressed. We will determine the role and importance of these genes by knocking down their expression and exposing these mosquitoes to Dengue virus to confirm the role of these genes. We then will use gene editing techniques to render susceptible phenotype-determining genes non-functional to determine their absence on virus development and on general mosquito fitness parameters. The Immune deficiency pathway (IMD) is a highly conserved immune pathway in insects that eliminates infections with Gram-negative bacteria. In the kissing bug, Rhodnius prolixus, however, the IMD pathway was reported to be absent, non-functional, or significantly reduced. The IMD pathway was reported to be absent in highly related insects. Despite these claims, the antimicrobial peptides normally (AMPs) regulated by the IMD pathway are expressed in these insects. We will use a series of bioinformatics and wet bench approaches to determine if the pathway is intact, but hidden in the genome, whether new proteins have replaced "missing: IMD components, or whether these AMPs are controlled by other pathways. Preliminary data indicate that the IMD pathway is present and functional R. prolixus. All of these experiments and approaches are designed to identify the regulation of innate immune responses in insects, how insects respond to parasites and pathogens, what factors determine vector-parasite specificity, and the mechanisms they use to restrict or eliminate parasite development. Ultimately, we may identify essential molecules or pathways that we might exploit to induce incompatibility between vectors and pathogens, and therefore eliminate transmission.

昆虫媒介将寄生虫和病原体传播给人类，但绝大多数进入媒介的寄生虫都被昆虫先天免疫系统的成分杀死。与脊椎动物免疫的特征不同，这种反应是种系编码的，既不是习得的也不是获得的。昆虫使用几种机制来识别寄生虫和病原体为“非自我”。识别激活免疫途径（Toll、IMD、RNAi和Jak-STAT），以封装、黑化或表达有效的抗菌肽（AMP），所有这些都可以杀死寄生虫。尽管有先天免疫的力量，许多昆虫仍然传播寄生虫。病媒和寄生虫之间的关系非常特殊。每种病媒传播一种或数量非常有限的寄生虫或病原体。如果免疫系统起作用，为什么病媒不能杀死所有的寄生虫？在哥伦比亚的卡利，约30%的领域收集了Ae。埃及人是登革热和寨卡病毒的主要载体，实际上可以杀死病毒，而70%的人继续将病毒传播给人类。如果难治性病毒是一个好处，为什么所有女性Ae。埃及人不能杀死病毒吗？在Ae。在埃及伊蚊中，登革病毒利用现有的蚊子蛋白来建立、进入细胞和复制，但在Ae.埃及人不表达这些基因。我们将通过敲低这些基因的表达并将这些蚊子暴露于登革热病毒来确定这些基因的作用和重要性。然后，我们将使用基因编辑技术使易感表型决定基因失去功能，以确定它们在病毒发育和一般蚊子适应性参数中的缺失。 免疫缺陷途径（IMD）是昆虫中高度保守的免疫途径，可消除革兰氏阴性菌的感染。然而，在接吻虫Rhodnius prolixus中，IMD通路被报道为不存在、无功能或显著减少。据报道，IMD途径在高度相关的昆虫中不存在。尽管有这些声明，但通常由IMD途径调节的抗菌肽（AMP）在这些昆虫中表达。我们将使用一系列生物信息学和湿台方法来确定该途径是否完整，但隐藏在基因组中，是否有新的蛋白质取代了“缺失的：IMD组件，或者这些AMP是否由其他途径控制。初步数据表明，IMD途径是存在的和功能性R。冗长所有这些实验和方法都旨在确定昆虫先天免疫反应的调节，昆虫如何对寄生虫和病原体作出反应，哪些因素决定了媒介-寄生虫特异性，以及它们用于限制或消除寄生虫发育的机制。最终，我们可以确定我们可能用来诱导载体和病原体之间不相容的必要分子或途径，从而消除传播。