Comprehensive genetic dissection of the hemocyte immune response in the malaria mosquito Anopheles gambiae.
Comprehensive genetic dissection of the hemocyte immune response in the malaria mosquito Anopheles gambiae.
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DOI：
10.1371/journal.ppat.1003145
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发表时间：
2013-01
期刊：
影响因子：
6.7
通讯作者：
Christophides GK
中科院分区：
文献类型：
--
作者：
Lombardo F;Ghani Y;Kafatos FC;Christophides GK
近年来，通过 RNAi 介导的基因沉默对冈比亚按蚊进行反向遗传学研究，使人们对蚊子针对细菌和疟疾寄生虫感染的免疫反应有了深入的了解。我们在 An 中开发了 RNAi 筛选。使用针对蚊子血细胞中高表达或特异性表达的 109 个基因的双链 RNA 文库，对冈比亚血细胞样细胞进行研究，以识别血细胞免疫反应的新调节因子。检测包括细菌生物颗粒的吞噬作用、抗菌肽 CEC1 的表达以及蚊子补体因子 LRIM1 的基础表达和诱导表达。还进行了细胞活力筛选，以评估 dsRNA 细胞毒性并鉴定参与细胞生长和存活的基因。我们的结果鉴定了 22 种新型免疫调节因子，包括推测参与吞噬体组装和成熟的蛋白质（Ca2+ 通道、v-ATP 酶和细胞周期蛋白依赖性蛋白激酶）、模式识别（纤维蛋白原域凝集素和 Nimrod）、免疫调节（肽酶和丝氨酸蛋白酶同源物）、免疫信号传导（Eiger 和 LPS 诱导因子）、细胞粘附和通讯 （层粘连蛋白 B1 和 Ninjurin）和免疫稳态（脂蛋白受体）。强大的基于细胞的功能检测的发展为全基因组功能筛选铺平了道路，以研究蚊子对人类病原体感染的免疫反应。蚊子的免疫系统依靠先天的体液和细胞反应来对抗感染，包括疟原虫感染，这些感染必须通过蚊子才能感染人类。因此，对这些反应的详细分子了解可以帮助设计控制疟疾和其他蚊媒疾病传播的新方法。在这里，我们使用一种技术来沉默蚊子培养细胞中在蚊子血细胞（相当于人类白细胞）中高度和/或特异性表达的基因，作为研究它们在蚊子免疫系统反应中的功能的手段。我们的研究确定了免疫反应的几种新型调节剂，包括吞噬作用、血细胞对细菌和其他病原体的吞噬和随后的破坏、抗菌肽的产生（直接杀死或抑制微生物的增殖）以及重要补体调节剂的基础和诱导产生。补体是蚊子通过吞噬、裂解或黑色化（将病原体封闭在黑色素胶囊中）对疟疾寄生虫和细菌的强烈反应。我们还揭示了这些反应之间有趣的分子联系，例如吞噬作用和补体调节。我们的研究为蚊子免疫系统及其对感染的反应提供了新的见解。
Reverse genetics in the mosquito Anopheles gambiae by RNAi mediated gene silencing has led in recent years to an advanced understanding of the mosquito immune response against infections with bacteria and malaria parasites. We developed RNAi screens in An. gambiae hemocyte-like cells using a library of double-stranded RNAs targeting 109 genes expressed highly or specifically in mosquito hemocytes to identify novel regulators of the hemocyte immune response. Assays included phagocytosis of bacterial bioparticles, expression of the antimicrobial peptide CEC1, and basal and induced expression of the mosquito complement factor LRIM1. A cell viability screen was also carried out to assess dsRNA cytotoxicity and to identify genes involved in cell growth and survival. Our results identify 22 novel immune regulators, including proteins putatively involved in phagosome assembly and maturation (Ca2+ channel, v-ATPase and cyclin-dependent protein kinase), pattern recognition (fibrinogen-domain lectins and Nimrod), immune modulation (peptidase and serine protease homolog), immune signaling (Eiger and LPS-induced factor), cell adhesion and communication (Laminin B1 and Ninjurin) and immune homeostasis (Lipophorin receptor). The development of robust functional cell-based assays paves the way for genome-wide functional screens to study the mosquito immune response to infections with human pathogens. The mosquito immune system relies on innate humoral and cellular reactions to fight infections, including those by malaria parasites that must pass through mosquitoes before they can infect humans. Therefore, a detailed molecular understanding of these reactions could assist the design of new ways to control the spread of malaria and other mosquito-borne diseases. Here we use a technique to silence in mosquito cultured cells genes that are highly and/or specifically expressed in mosquito hemocytes, the equivalent of human white blood cells, as a means to investigate their function in reactions of the mosquito immune system. Our study identifies several novel regulators of immune reactions including phagocytosis, the engulfment and subsequent destruction of bacteria and other pathogens by hemocytes, the production of antimicrobial peptides, which directly kill or inhibit the proliferation of microbes, and the basal and induced production of an important complement regulator. Complement is a robust reaction of mosquitoes against malaria parasites and bacteria through phagocytosis, lysis or melanization (the enclosure of pathogens in a melanin capsule). We also reveal intriguing molecular connections between these reactions such as phagocytosis and regulation of complement. Our study provides novel insights into mosquito immune system and its reactions against infections.
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