虫菌体特异表面蛋白与球孢白僵菌逃避昆虫免疫识别的关系及作用机制

Fungal pathogens could evade or avoid hemolymph immune recognition of the host insect by means of modification of cell surface and cell wall structure. However, the mechanism of fungal pathogen modifying cell surface feature to avoid immune detection of the host insect is still unclear. The entomopathogenic fungus, Beauveria bassiana, elaborate free floating cells termed hyphal bodies once penetrating hyphae reaching the hemocoel. It is demonstrated that a highly ordered outermost brush-like structure is uniquely present in the cell surface of B. bassiana hyphal bodies, which might be involved in evading the insect hemolymph immune response by masking the pathogen-associated molecular patters (PAMPs). We firstly revealed that the brush-like structure is mainly composed of proteins. And then, seven cell surface proteins specially expressed in the hyphal bodies has been screened and identified using comparative proteomic and bioinformatic methods, and by tagging the gene with eGFP. These findings suggested a valuable cue explore the mechanism of the fungal pathogen that evades the host insect hemolymph immune response. In this project, we are going to investigate formation of the cell surface proteins and position relationship of the different proteins in the cell surface of the hyphal bodies using immunofluorescene method as well as reverse transcription PCR (RT-PCR). Gene disruption and overexpression strains will be constructed to investigate effect of the cell wall surface proteins on hyphal body differentiation, cell surface feature and construction, and to characterize roles of the B. bassiana hyphal body-specific cell surface proteins in evading immune recognition of the host insect. One of our goals of the project is to expect to make a breakthrough on understanding mechanism of interaction between the fungal pathogen and host insect. Another goal is to hope to provide a theoretical basis for improvement of fungal the fungal agent using genetic engineering.

病原真菌通过表面组成或结构特化，逃避宿主昆虫免疫识别或攻击。然而，有关病原真菌表面结构特化与逃避昆虫免疫识别的机制，尚缺乏明晰的认识。球孢白僵菌在昆虫体腔内分化形成虫菌体，在表面特化出一层规则的“刷状”突起，推测其与隐蔽病原相关分子模式而逃避宿主免疫识别相关。课题组研究发现，虫菌体表面的突起结构主要由蛋白质组成。利用比较蛋白组学、生物信息学以及eGFP标签法，初步筛选了7个虫菌体特异的表面蛋白。该发现为揭示病原真菌逃避昆虫免疫识别的机制提供了重要线索。本项目拟采用免疫荧光等方法分析表面蛋白在虫菌体分化过程中的表达次序及空间关系，结合基因破坏和过表达等技术，探究特异表面蛋白对虫菌体分化以及表面特性和组成的影响，解析表面蛋白与白僵菌逃避昆虫免疫识别的关系及作用机制。项目的开展，有望在揭示病原真菌与昆虫的互作机制方面取得新的突破，而且研究结果将可能为改良和提升真菌生防效果提供理论依据。

病原真菌通过细胞特化或表面修饰，逃避宿主昆虫免疫识别，但其机制尚不明晰。球孢白僵菌侵入虫体后分化的虫菌体表面特化出一层规则的“刷状”凸起，推测与逃避免疫反应相关。课题组研究发现，该凸起结构主要由蛋白组成，并通过蛋白组学鉴定了6个虫菌体特异表达的蛋白。本项目解析了其中3个蛋白与病原菌逃避昆虫免疫防御的关系及作用机制。表面蛋白Laccase 2 (BbLac2)受昆虫营养、氧化胁迫和昆虫酚类物质的诱导，不仅分布于细胞表面，还可分泌到胞外。基因敲除导致菌株对氧化胁迫敏感性增加，毒力削弱，激活昆虫免疫反应，而过表达菌株则对氧化胁迫耐受性增强，毒力增强并伴随“沉默”昆虫免疫反应。结合体外表达蛋白研究和细胞表面特性研究结果表明，病原菌特异性分泌BbLac2可清除宿主免疫反应产生的ROS和通过竞争PO底物酚类化合物而干扰PO介导的免疫反应。而且，BbLac2作为表面蛋白，“隐蔽”了病原相关分子模式，沉默Toll免疫途径，逃避昆虫免疫识别。表面蛋白Concanavalin A-like lectin/glucanase (CAL1)受昆虫营养诱导表达，且大量分泌到昆虫血腔。体外表达研究发现，CAL1具有水解β-1,3/1,4-糖苷键活性。超量表达CAL1显著提高了菌株结合葡聚糖活性，生长加快，细胞壁几丁质含量降低，葡聚糖含量显著增加，虫菌体细胞表面凸起明显，细胞表面病原相关分子模式(PAMPs)葡聚糖水平显著降低，易逃避昆虫免疫反应，毒力显著提高。而基因破坏导致虫菌体几丁质含量增加，且细胞表面葡聚糖等PAMPs水平显著增加，易激活昆虫免疫反应，毒力降低。由此表明，病原真菌特异的分泌CAL1降解昆虫营养，同时参与细胞壁重塑，促进病原菌增殖。另外，CAL1作为表面蛋白，“隐蔽”病原相关分子模式，帮助病原真菌逃避免疫识别。尽管分泌性的磷脂酶A2 (BbPLA2)分离于虫菌体细胞表面/分泌蛋白库，但研究发现其主要分布于虫菌体脂滴。在病原菌侵染过程中，BbPLA2分泌到胞外降解昆虫脂类(体壁或血腔)物质，供给菌体生长所需能量，促进病原菌侵染致病。项目研究拓宽了病原真菌逃避免疫防御反应的认识，并为菌株遗传改良提供了新的理论依据。