基于蚊媒唾液腺探讨wolbachia感染蚊虫抗登革病毒的分子机制

Wolbachia are maternally transmitted symbiotic bacteria that can spread within insect populations because of their unique ability to manipulate host reproduction. When introduced to non-native mosquito hosts, Wolbachia induce resistance to a number of human pathogens, including dengue virus (DENV), Plasmodium, and filarial nematodes, but the molecular mechanism involved is unclear. To determine the contribution of salivary gland barrier to the anti-dengue resistance for the wolbachia infected mosquito, the present study is conducted to decipher the role of salivary gland in wolbachia induced anti-dengue resistance. Two gene expression profiles will be constructed on wolbachia-induced and dengue virus-induced mosquitoes by a whole genomic microarray assay and real-time RT-PCR. We expect to get many different expression genes so that we can priliminary elucidate the difference of salivary gland microenvironment between wolbachia-induced and dengue virus-induced mosquitoes. Then we will select several target genes to discuss the gene function on replication and distribution of wolbachia and dengue virus in mosquito salivary gland by overexpression and RNAi in vivo and in vitro. At last, a role of mosquito salivary gland and molecular mechanism will be discussed on wolbachia-induced anti-dengue resistance. Understanding the mechanism will improve the antiviral efficiency and facilitate development of novel method to control dengue transmission.

wolbachia是胞质遗传的共生菌，因其操控宿主的生殖系统能力特别强，因此可在宿主种群迅速传播。将wolbachia导入非天然蚊虫宿主，可诱导蚊虫对人类病原产生抗性，如疟原虫，登革病毒等，但其抗性分子机制并不清楚。为了探讨蚊虫唾液腺屏障在wolbachia调适的抗登革病毒传播中作用，本研究以蚊虫唾液腺为研究对象，采用全基因组芯片，构建 wolbachia 感染蚊虫唾液腺表达谱，Dengue感染蚊虫唾液腺表达谱，获得差异表达基因并验证芯片结果；通过对差异表达基因生物信息学分析，探讨wolbachia感染对蚊虫唾液腺微环境影响以及登革病毒感染对蚊虫唾液腺微环境影响的差异；最后通过对差异功能基因进行过表达和RNAi，探讨蚊媒唾液腺在wolbachia感染蚊虫抗登革病毒传播的可能作用及其分子机制。

wolbachia 是胞质遗传的共生菌，因其操控宿主的生殖系统能力特别强，可在宿主种群迅速传播。将wolbachia导入非天然蚊虫宿主，可诱导蚊虫对人类病原产生抗性，如疟原虫、登革病毒等，但其抗性分子机制尚不十分清楚。唾液腺是蚊虫传播病原的重要器官，为了探讨蚊虫唾液腺在wolbachia调适的抗登革病毒中作用，本研究开展了如下研究工作。首先采用全基因组芯片，构建wolbachia感染蚊虫唾液腺表达谱，发现598个基因有表达差异，获得上调基因324个，其中以免疫相关基因、redox/stress/mitochondrion相关基因上调为主；构建Dengue感染蚊虫唾液腺表达谱，发现412个基因表达差异，上调基因180个，下调基因232个，其中以免疫相关基因、redox/stress/mitochondrion相关基因下调为主；发现wolbahica感染蚊虫唾液腺与脂肪代谢相关基因上调（Malic enzyme，Annexin X，Fatty acyl-CoA reductase，glycerol-3-phosphate acyltransferase，neutral Sphingomyelinase，steroid dehydrogenase）；同时还发现几个唾液组分蛋白（yellow-c，serpin21）可同时被wolbachia，Dengue病毒异向调控。进而采用RNAi技术knockdown yellow-c，serpin21对登革病毒复制有一定影响，但无统计学差异; 最后我们克隆并表达3个唾液组分蛋白，其中有2个重组蛋白（yellow-c，34-2kDa）可以促进dengue病毒在人角质形成细胞中复制。基于以上研究结果可以初步得出如下3个结论。1. wolbachia调适的免疫触发也是其在唾液腺水平抗登革病毒机制之一；2. wolbachia 调适的差异唾液组分蛋白通过干扰哺乳动物细胞免疫微环境来控制病毒的传播；3. wolbachia调适蚊虫唾液腺上皮细胞内的脂代谢变化可能是wolbachia抗病毒机制之一，但尚需进一步的研究证实。