Table_1_The salmonella effector Hcp modulates infection response, and affects salmonella adhesion and egg contamination incidences in ducks.docx

Salmonella Entertidis (SE) often causes persistent infections and egg contamination in laying ducks. Hcp, the core structural and effector proteins of the Type VI Secretion System (T6SS) in SE, contributes to bacterial invasion, adhesion and virulence. However, little is known about the effect of Hcp on the host’s infection responses and egg contamination incidences in duck. Herein, we generated an hcp deletion mutant SE MY1△hcp and detected its ability to invade duck granulosa cells (dGCs) and contaminate eggs. In comparison with MY1-infected group, the SE adhesion decreased by 15.96% in MY1△hcp-infected dGCs, and the apoptosis in MY1△hcp-infected dGCs decreased by 26.58% and 30.99% at 3 and 6 hours postinfection, respectively. However, the expression levels of immunogenic genes TLR4, NOD1, TNFα, IL-1β and proinflammatory cytokines IL-6, IL-1β, TNF-α release were markedly lower in the dGCs inoculated with MY1△hcp than that of the wild type. Besides, the laying ducks were challenged with MY1 or MY1△hcp in vivo, respectively. The lower egg production and higher egg contamination were observed in MY1-infected ducks in comparison with MY1△hcp-infected birds. Furthermore, the host’s infection response of differentially abundant proteins (DAPs) to Salmonella effector Hcp was identified using quantitative proteomics. A total of 164 DAPs were identified between the MY1- and MY1△hcp-infected cells, which were mainly engaged in the immune, hormone synthesis, cell proliferation and cell apoptotic process. Among them, STAT3, AKT1, MAPK9, MAPK14, and CREBBP were the center of the regulatory network, which might serve as key host response regulators to bacterial Hcp. In conclusion, we demonstrated that effector Hcp contributed to not only SE invasion, induction of dGCs apoptosis, and trigger of immune responses, but also enhanced contamination incidences. Also, the STAT3, AKT1, MAPK9, MAPK14, and CREBBP were identified as host’s infection response regulators of bacterial Hcp in duck. Overall, these results not only offered a novel evidence of SE ovarian transmission but also identified some promising candidate regulators during SE infection.

肠炎沙门氏菌（Salmonella Entertidis，SE）常可引起产蛋鸭持续性感染与蛋品污染。SE的VI型分泌系统（Type VI Secretion System，T6SS）核心结构蛋白与效应蛋白Hcp，可参与细菌侵袭、黏附及致病过程。然而，目前对于Hcp对鸭宿主感染应答及蛋品污染发生率的影响尚不清楚。本研究构建了hcp基因缺失突变株SE MY1△hcp，并检测其侵袭鸭颗粒细胞（duck granulosa cells，dGCs）及污染蛋品的能力。与MY1感染组相比，MY1△hcp感染的dGCs中SE黏附能力下降15.96%；感染后3小时和6小时，MY1△hcp感染组dGCs的细胞凋亡率分别降低26.58%和30.99%。此外，与野生型菌株感染组相比，MY1△hcp感染的dGCs中免疫相关基因TLR4、NOD1、TNFα、IL-1β的表达水平，以及促炎细胞因子IL-6、IL-1β、TNF-α的释放量均显著下调。体内实验中，分别用MY1或MY1△hcp攻毒产蛋鸭。相较于MY1△hcp感染组，MY1感染组鸭的产蛋量更低，蛋品污染率更高。进一步通过定量蛋白质组学，鉴定宿主细胞对沙门氏菌效应蛋白Hcp的差异丰度蛋白（differentially abundant proteins，DAPs）应答谱。在MY1与MY1△hcp感染的细胞中共鉴定到164个差异丰度蛋白，这些蛋白主要参与免疫应答、激素合成、细胞增殖及细胞凋亡过程。其中，STAT3、AKT1、MAPK9、MAPK14及CREBBP构成调控网络的核心节点，可能是鸭宿主对细菌Hcp的关键应答调控因子。综上，本研究证实效应蛋白Hcp不仅可促进SE侵袭、诱导dGCs凋亡及触发免疫应答，还可增强蛋品污染发生率。同时，本研究鉴定出STAT3、AKT1、MAPK9、MAPK14及CREBBP作为鸭宿主对细菌Hcp的感染应答调控因子。上述研究结果不仅为SE的卵巢传播途径提供了新的实验证据，还为SE感染过程中的潜在候选调控靶点提供了新的研究方向。