基于CRISPR技术筛选鉴定微孢子虫感染导致宿主细胞死亡的关键基因

Microsporidia are a group of fungi-related unicellular parasites that lead an obligate intracellular parasitic life. The infestation of microsporidia caused enormous financial losses in economical animals, even poses a threat to human health. However, the mechanism of how host response to microsporidia is remain unclear. In this study, we performed a genome-scale CRISPR/Cas9 knockout screen in BmE cells with the first identified microsporidia, Nosema bombycis (N.b). Several genes involved in STING, autophagy, Apoptosis and ECM receptor interaction pathways were highly enriched post-N.b selection. Then we found the survival rate of BmSTING knockout silkworm were increased post N.b infection, suggest that the resistance of silkworm to microsporidia were enhanced via knockout of BmSTING. We also observed that BmSTING overexpression effectively inhibits N.b replication in BmE cells, the cell proliferation promoted by N.b were also inhibited. Combined with references, we hypothesized that STING activates autophagy and apoptosis to response to microsporidia. Based on the results of pre-experimental methods, we will constructed the knockout pool based on STING mechanism and further study the relative molecular mechanisms that how host response to microsporidia, which provides new molecular targets for protecting host from microsporidia.Based on the results of pre-experimental methods, we will further study the relative molecular mechanisms that how host response to microsporidia, which provides new molecular targets for protecting host from microsporidia.

微孢子虫是一种单细胞真核专性寄生微生物，其感染对经济动物饲养甚至人类健康都具有重大威胁。目前宿主应答微孢子虫感染增殖的关键机制尚未阐明。我们利用微孢子虫对基于CRISPR-cas9技术构建的全基因组敲除细胞库筛选，发现STING，autophagy, 泛素蛋白酶体等信号机制在微孢子虫诱导的宿主细胞死亡中发挥着关键作用。在家蚕中敲除BmSTING，其微孢子虫感染后的死亡率显著下降；而在细胞中过表达BmSTING，细胞内的微孢子虫减少，同时由微孢子虫感染促进的细胞增殖受到显著抑制。结合文献报道，我们推测，STING可能通过调控细胞的自噬和凋亡来控制微孢子虫的感染增殖。在预实验基础上，本项目拟通过构建基于STING相关机制的细胞敲除文库进一步筛选影响微孢子感染的关键基因，并在家蚕和果蝇中利用细胞自噬、细胞凋亡，免疫沉淀等实验方法，深入探讨宿主应答微孢子虫的详细分子机制，为微孢子虫病防治寻找靶标。

微孢子虫是一种单细胞真核专性寄生微生物，其感染对经济动物饲养甚至人类健康都具有 重大威胁。目前宿主应答微孢子虫感染增殖的关键机制尚未阐明。我们利用微孢子虫对基于CR ISPR-cas9技术构建的全基因组敲除细胞库筛选，发现在全基因组编辑细胞库中存在不感染微孢子虫和微孢子虫感染后增殖较少的细胞；通过流式筛选活细胞测序，在单轮和多轮富集的gRNA中分别筛选到779和728个对应基因;富集通路分析发现TGF-beta、phototransduction、pentose and glucuronate interconversions、Glycerophospholipid metabolism、ECM-receptor interaction和STING等信号机制在微孢子虫感染导致的宿主死亡中发挥着关键作用；进一步筛选和验证发现Src42a、Roc1a、Snx6、BmSTING和 AKR2E4等关键基因在微孢子虫感染中的作用；在细胞水平敲除STING后，微孢子虫感染后死亡率降低，而在个体水平敲除后，在微孢子虫感染早期，敲除STING的家蚕可以较好存活，而在感染后期则更快死亡；进一步对STING激活机制进行检测发现，随着微孢子虫感染时间的延长，STING和自噬相关基因显著诱导上调；当STING敲除后，自噬信号分子LC3的激活下降，微孢子虫的增殖增多，从而促进个体更易死亡。此外，细胞中微孢子虫增殖较多时，能够促进下游Relish分子的活化入核。这些研究结果不仅可以为深入探讨宿主应答微孢子虫的详细分子机制提供依据，也将为微孢子虫病抗性素材创制提供靶标。

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