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: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=737514
• 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，但进入媒介的绝大多数寄生虫都会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，也不是后天获得的。昆虫使用多种机制将寄生虫和病原体识别为“非自身”。识别会激活免疫途径（Toll、IMD、RNAi 和 Jak-STAT）来封装、黑色素化或表达有效的抗菌肽 (AMP)，所有这些都可以杀死寄生虫。尽管先天免疫能力很强，但许多昆虫仍然传播寄生虫。媒介和寄生虫之间的关系非常具体。每种媒介物种传播一种或非常有限数量的寄生虫或病原体。如果免疫系统起作用，为什么载体不能杀死所有寄生虫？在哥伦比亚卡利，大约 30% 的田地收集了白伊菌。埃及伊蚊是登革热和寨卡病毒的主要传播媒介，实际上可以杀死这些病毒，而 70% 的病毒继续将病毒传播给人类。如果对病毒具有抵抗力是一种好处，那为什么所有雌性伊蚊都如此呢？埃及伊蚊不能杀死病毒吗？在Ae内。埃及伊蚊、登革热病毒利用现有的蚊子蛋白来建立、进入细胞并复制，但在伊蚊的难治性菌株中。埃及伊蚊这些基因不表达。我们将通过降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，据报道，在接吻蝽（Rhodnius prolixus）中，IMD 通路不存在、无功能或显着减少。据报道，高度相关的昆虫中不存在 IMD 途径。尽管有这些说法，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，新的蛋白质是否取代了“缺失的：IMD成分，或者这些AMP是否受其他途径控制。初步数据表明IMD途径存在并且具有功能性。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，昆虫如何调节昆虫的先天免疫反应。” 对寄生虫和病原体作出反应，哪些因素决定媒介寄生虫的特异性，以及它们用来限制或消除寄生虫发育的机制。最终，我们可以识别重要的分子或途径，利用它们来诱导载体和病原体之间的不相容，从而消除传播。