家蚕BmDUOX蛋白在抵抗家蚕微孢子侵染中的功能研究

Insect intestinal epithelia are in direct contact with microbes, which range from beneficial symbionts to pathogens. Accordingly, hosts must have a conflicting strategy to combat pathogens efficiently while tolerating symbionts. It is considered to be an important line of insects to resist microbial infection. As the first identified microsporidia, Nosema bombycis (Nb) is the pathogen of silkworm pébrine which initially infect silkworm larvae from midgut cells, and it has been pronounced as the only legal quarantine in sericultural industry. In our previous study, we found that high concentration of H2O2 can disturb the spore germination in vitro, and then we indentified the gene members involved in DUOX-ROS signal pathway from the silkworm genome database. Recent progress has revealed that DUOX-ROS signal pathway is involved in mucosal immunity and dual oxidase (DUOX) plays a key role in mucosal immunity in organisms that range from flies to humans. Our results indicated that the transcriptional level of BmDUOX can be up-regulated by the infection of Nb, thus, we speculated that BmDUOX played important roles in resisting the infection of microsporidia in silkworm midgut through generating reactive oxygen species (ROS). Our proposal focus on the BmDUOX function study on three aspects as followings: the induction of ROS in silkworm BmE cells by Nb, the Cellular localization of BmDUOX，the analysis of catalytic activity and functional mechanism of BmDUOX. We try to elucidate the role of BmDUOX in resisting the infection of Nb in silkworm midgut immunity. Our project based on the idea to find a new clue to control the infection of microsporidia in sericulture. To carry out such a project is very helpful to uncover the silkworm intestinal immunomechanism and the mechanism of silkworm to resist the infection of microsporidia.

家蚕微孢子虫（Nb）是家蚕微粒子病的病原，对蚕业生产破坏性极大。本团队前期研究发现高浓度过氧化物能抑制孢子体外发芽；随后在家蚕基因组数据中检索到与活性氧（过氧化物、超氧化物等）产生相关的DUOX-ROS通路成员，据报道该通路参与了果蝇等物种的肠道上皮免疫。Nb对家蚕的最初侵染起始于中肠，是否中肠上皮细胞能通过DUOX-ROS通路产生活性氧来抵抗微孢子虫的侵染成为本团队研究的重点，至今该方面的研究尚无报道。进一步的研究表明Nb能诱导家蚕中肠BmDUOX表达量上调，暗示肠道上皮细胞可能通过调控通路关键蛋白BmDUOX来发挥其免疫功能。本研究拟从Nb对家蚕细胞活性氧诱导情况、BmDUOX细胞定位特征和BmDUOX催化活性与作用机制分析三方面深入研究该蛋白在家蚕抵抗微孢子虫侵染中的功能。本项目旨在从宿主角度寻找防控微孢子虫侵染的新方法，对揭示家蚕肠道免疫方式和对微孢子虫侵染的防御机制具有重要意义。

作为首个被鉴定的微孢子虫，家蚕微孢子虫（Nosema bombycis，Nb）是引起家蚕微粒子病的病原体，已被列为养蚕业的唯一法定检疫对象。对家蚕微孢子虫引起的宿主应答进行分析并对相关免疫通路与免疫分子进行鉴定，可以为了解家蚕微孢子虫与宿主互作关系、宿主的自我保护与防御机制提供依据。我们在家蚕基因组数据中检索到与活性氧（过氧化物、超氧化物等）产生相关的DUOX-ROS通路成员，据报道该通路参与了果蝇等物种的肠道上皮免疫。Nb对家蚕的最初侵染起始于中肠，且Nb能诱导家蚕中肠BmDUOX表达量上调，是否中肠上皮细胞能通过DUOX-ROS通路产生活性氧来抵抗微孢子虫的侵染成为研究的重点。首先，利用原核表达系统获得 BmDUOX 蛋白 N 端 peroxidase 区域的原核表达蛋白BmDUOX_POD和BmDUOX_OM，以其作为抗原免疫昆明鼠制备了鼠多克隆抗血清；间接免疫荧光分析（IFA）和免疫组化实验表明BmDUOX定位于家蚕BmE细胞和中肠细胞的细胞膜。随后，在细胞水平上分析了家蚕微孢子虫对细胞活性氧的诱导情况：其中，家蚕微孢子虫（Nb）和家蚕微孢子虫可溶性提取物（NbSME）均能够诱导 BmE 细胞内过氧化物的水平提高；同时 Nb 和 NbSME 也能够诱导 BmE 细胞内的超氧化物的水平显著提高，其总活性氧物质（ROS）的含量被诱导提高。另外，家蚕BmDUOX蛋白在抵抗微孢子虫侵染过程中发挥的功能与作用机制：在细胞水平上，通过抗体封闭实验表明 BmDUOX 参与了孢子对BmE细胞的粘附与侵染；RNA干涉实验显示BmDUOX表达量的降低利于微孢子虫的侵染，表明其可能参与了宿主对微孢子虫入侵的抵抗与清除过程。在个体水平上，通过喂食peroxidase结构域特异性抑制剂，表明BmDUOX可能参与家蚕中肠抵抗微孢子虫的入侵过程。本课题的研究成果有助于从宿主角度寻找防控微孢子虫侵染的新方法，对揭示BmDUOX在家蚕中的生物学功能、家蚕肠道免疫方式和对微孢子虫侵染的防御机制具有重要意义。