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

描述（由申请人提供）：昆虫必须识别病原体才能产生免疫反应。昆虫中病原体的识别由模式识别受体（PRR）如C型凝集素介导。在烟草天蛾（Manduca sexta）中，C型凝集素已显示出作为刺激性PRR以增强免疫应答的功能。在蚊子冈比亚按蚊中，疟疾的载体和两种C型凝集素，CTL 4和CTLMA 2，以某种方式阻止伯氏疟原虫黑化。而LRIM-1和TEP-1可促进伯氏疟原虫黑化。然而，在A.由于免疫逃避，冈比亚对恶性疟原虫（人类寄生虫）的发育没有影响。恶性疟原虫是如何逃避A. gambiae，而P. berghei不能？我们推测A.冈比亚血浆或细胞外基质是疟原虫表面寄生的关键。本文旨在研究A.冈比亚C型凝集素作为推定的PRR调节针对疟原虫的免疫应答。具体目标是：1.继续研究昆虫原型C-型凝集素的详细分子相互作用，Manduca immulectin-2，作为理解潜在的凝集素-碳水化合物相互作用和凝集素-蛋白质相互作用的指导，影响模式识别和先天免疫应答的结果。我们将构建immulectin-2的羧基末端碳水化合物识别结构域中的缺失和点突变，预测其影响与微生物多糖或与酚氧化酶原活化复合物的蛋白质组分的相互作用。2.研究9个A.冈比亚按蚊C型凝集素（包括CTL 4和CTLM 2）和两种嵌合凝集素以及TEP-1（含硫酯蛋白-1）和LRIM-1（富含亮氨酸重复免疫基因-1），研究这些蛋白与伯氏疟原虫动合子和动合子表面蛋白的相互作用，并鉴定这些冈比亚按蚊能识别的伯氏疟原虫动合子表面蛋白。冈比亚蛋白。3.研究了9种A. gambiae凝集素、两种嵌合凝集素TEP-1和LRIM-1在蚊虫体内的包封、黑化、吞噬作用和酚氧化酶激活等方面的作用，并研究了这些蛋白在A.冈比亚。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。公共卫生相关性昆虫必须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，以刺激对细菌或真菌感染的免疫反应。然而，人们对昆虫免疫系统如何识别非真菌真核寄生虫知之甚少：这些生物体上的病原体相关分子模式（PAMP）是什么以及PRR与它们结合的是什么？本研究旨在探讨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.