C-type lectins in innate immune responses of Anopheles gambiae and Manduca sexta

描述(申请人提供)：昆虫必须先识别病原体，然后才能产生免疫反应。昆虫对病原体的识别是由模式识别受体(PRRs)介导的，如C型凝集素。在烟草天牛Manduca sexta中，C型凝集素已被证明具有刺激PRR的功能，以增强免疫反应。在冈比亚按蚊体内，疟疾的媒介，以及两种C型凝集素CTL4和CTLMA2，以某种方式防止伯氏疟原虫黑化。但LRIM-1和TEP-1能促进伯氏假单胞菌的黑化。然而，由于免疫逃避，在冈比亚疟原虫中沉默CTL4、CTLMA2和LRIM-1对恶性疟原虫的发育没有影响。恶性疟原虫如何逃避冈比亚按蚊的免疫系统，而伯氏疟原虫却不能？我们推测冈比亚按蚊血浆或细胞外基质中的蛋白质与疟原虫表面的相互作用是关键。本建议旨在研究冈比亚按蚊的C型凝集素作为可能的PRRs来调节对疟原虫的免疫反应。具体目标是：1.继续研究昆虫C型凝集素原型Manduca imulectin-2的详细分子相互作用，以此作为了解潜在的凝集素-碳水化合物相互作用和凝集素-蛋白质相互作用的指南，这些相互作用影响模式识别和先天免疫反应的结果。我们将在免疫凝集素-2的羧基末端碳水化合物识别结构域上构建缺失和点突变，预测会影响与微生物多糖或与酚氧化酶原激活复合体的蛋白质成分的相互作用。2.研究9种冈比亚按蚊C型凝集素(包括CTL4和CTLMA2)和两种嵌合凝集素以及TEP-1(含硫酯的蛋白-1)和LRIM-1(富含亮氨酸的重复免疫基因-1)的配基结合特异性和亲和力，研究这些蛋白与伯氏疟原虫和动子表面蛋白的相互作用，并鉴定可被这些冈比亚按蚊识别的动植物表面蛋白。3.研究9种冈比亚按蚊凝集素和2种嵌合凝集素TEP-1和LRIM-1在蚊体包膜、黑化、吞噬和酚氧化酶激活中的作用，并探讨这些蛋白在冈比亚按蚊免疫应答过程中的相互作用。我们的长期目标是了解寄生虫识别和寄生虫-模式识别受体在媒介昆虫中的相互作用。与公共卫生相关的昆虫必须先识别病原体和寄生虫，然后才能产生免疫反应。昆虫对非自身病原体的识别是由模式识别受体(PRRs)介导的。我们对昆虫中作为PRR的蛋白质有一些了解，这些蛋白质可以刺激对细菌或真菌感染的免疫反应。然而，关于昆虫免疫系统如何识别非真菌真核生物寄生虫，人们知之甚少：这些生物上的病原体相关分子模式(PAMP)是什么，PRR与它们结合在一起？本课题旨在探讨C型凝集素在冈比亚按蚊抗疟原虫天然免疫应答中的潜在作用。

DESCRIPTION (provided by applicant): Insects must recognize pathogens before they can mount an immune response. Recognition of pathogens in insects is mediated by pattern recognition receptors (PRRs) such as C-type lectins. In the tobacco hornworm, Manduca sexta, C-type lectins have been shown to function as stimulatory PRRs to enhance immune responses. In the mosquito Anopheles gambiae, a vector for malaria, and two C-type lectins, CTL4 and CTLMA2, somehow prevent Plasmodium berghei from melanization. But LRIM-1 and TEP-1 can enhance P. berghei melanization. However, silencing of CTL4, CTLMA2 and LRIM-1 in A. gambiae has no effect on development of P. falciparum, the human parasite, due to immune evasion. How can P. falciparum evade the immune system of A. gambiae, while P. berghei can not? We hypothesize that interaction of proteins from A. gambiae plasma or extracellular matrix with the surface of Plasmodium parasites are the keys. This proposal aims to investigate A. gambiae C-type lectins as putative PRRs to modulate immune responses against Plasmodium. The specific aims are: 1. Continue to investigate detailed molecular interactions of an insect prototype C-type lectin, Manduca immulectin-2, as a guide to understanding potential lectin-carbohydrate interactions and lectin-protein interactions that influence the outcome of pattern recognition and innate immune responses. We will construct deletions and point mutations in the carboxyl-terminal carbohydrate recognition domain of immulectin-2 predicted to affect interactions with microbial polysaccharides or with protein components of the prophenoloxidase activation complex. 2. Investigate the ligand-binding specificity and affinity of nine A. gambiae C-type lectins (including CTL4 and CTLMA2) and two chimeric lectins, as well as TEP-1 (thioester-containing protein-1) and LRIM-1 (leucine-rich repeat immune gene-1), study interactions of these proteins with P. berghei ookinetes and ookinete surface proteins, and identify P. berghei ookinete surface proteins that can be recognize by these A. gambiae proteins. 3. Study functions of nine A. gambiae lectins, two chimeric lectins, TEP-1 and LRIM-1 in encapsulation, melanization, phagocytosis and phenoloxidase activation in mosquitoes, and investigate interactions of these proteins during immune responses in A. gambiae. Our long term goal is to understand parasite recognition and parasite-pattern recognition receptor interaction in vector insects. PUBLIC HEALTH RELEVANCE Insects must recognize pathogens and parasites before they can mount an immune response. Recognition of nonself pathogens in insects is mediated by pattern recognition receptors (PRRs). We have some understanding of the proteins that serve as PRRs in insects to stimulate immune responses to bacterial or fungal infection. However, very little is known about how insect immune systems recognize non-fungal eukaryotic parasites: what are the pathogen-associated molecular patterns (PAMPs) on such organisms and what PRRs bind to them? This project is to investigate C-type lectins as potential PRRs in innate immune responses of Anopheles gambiae against Plasmodium parasites.