马链球菌兽疫亚种荚膜与CD44相互作用调控TLR信号通路的机制

Streptococcus zooepidemicus (SEZ) is an important pathogen associated with opportunistic infections of a wide range of species, including horses, cows, and mice. In addition, SEZ is a major cause of swine streptococcal diseases in China. Although recent studies revealed a role for CD44 during host defense, little was known about its mechanism during infections with pathogenic microorganisms. The present study sought to investigate the role of CD44 in the host response, including the activity of SEZ clearance, inflammatory cells recruitment and inflammatory cytokine production. The interaction of CD44 with capsule was identified by flow cytometry. Then using siRNA, over expression and purified recombinant CD44, determine whether CD44 could mediate phagocytosis in macrophages. Besides, CD44-deficient and wide-type control mice were intranasally inoculated SEZ to induce pneumonia models. Following the treatment, mice inflammatory responses were characterized by inflammatory cell recruitment and chemokine expression in bronchoalveolar lavage fluid; several regulators of TLR signaling such as SOCS-3, A20, and IRAK-M were examined to reveal the role of CD44 in the regulation of capsue-TLR signaling. To confirm the regulation of CD44 in inflammation in vivo, the bacteria outgrowth and dissemination to distant organs were examined during SEZ infection. The results would not only elucidate the mechanism of CD44 in regulate macrophage phagocytic SEZ, but also suggest that targeting CD44 may be an effective strategy for treating and/or preventing SEZ infection.

马链球菌兽疫亚种（SEZ）不仅引起马、牛、小鼠等多种动物的感染，而且是我国猪链球菌病的主要病原之一。近年来有研究表明，细胞表面跨膜蛋白CD44能影响巨噬细胞吞噬微生物，但其作用机制尚不清楚。本项目拟在前期基础上，采用流式细胞术、超表达、RNA干扰等手段，研究CD44与SEZ荚膜的互作能力，分析CD44表达水平对巨噬细胞吞噬SEZ能力的影响。建立CD44基因敲除小鼠感染模型，并通过炎症细胞募集与炎症因子分泌两方面探索CD44对炎症的影响。在此基础上，分析TLR信号通路中SOCS-3、A20等调控因子的变化规律，揭示SEZ荚膜与CD44调控炎症的信号通路。最后通过两种菌株在CD44 KO和野生型小鼠体内增殖与扩散的能力验证SEZ荚膜与CD44互作对巨噬细胞吞噬功能的影响。研究结果不仅从理论上阐明SEZ荚膜与CD44作用调控TLR信号通路的机制，还为研发治疗链球菌病的新型生物阻断制剂提供思路。

马链球菌兽疫亚种（SEZ）不仅引起马、牛、小鼠等多种动物的感染，而且是我国猪链球菌病的主要病原之一。近年来有研究表明，细胞表面跨膜蛋白CD44能影响巨噬细胞吞噬微生物，但其作用机制尚不明确。本研究构建了SEZ感染小鼠模型，发现感染SEZ后小鼠巨噬细胞CD44的表达水平显著提高。此外，CD44缺失型小鼠腹腔巨噬吞噬SEZ的能力显著低于WT小鼠，而CD44缺失促进SEZ在小鼠体内的增殖与扩散并加速小鼠的死亡，表现为CD44 KO小鼠腹腔渗出液以及实质组织中细菌载量显著高于WT小鼠。另外一方面，CD44缺失也影响小鼠感染SEZ的炎症应答过程。CD44 缺失导致感染早期小鼠白细胞向腹腔中的募集增强。CD44 KO小鼠炎症细胞IL-1β、IL-6、KC、MIP-2以及MIP-1β等炎症因子表达水平高于WT小鼠。进一步研究发现CD44 KO小鼠TLR通路中的负调控因子A20的表达水平显著性降低。. 进一步深入探讨CD44在宿主感染SEZ的保护作用发现，CD44基因敲除小鼠感染SEZ表现在支气管肺泡聚集的巨噬细胞减少且细菌的生长和传播能力增强，从而导致小鼠的存活率降低。此外，CD44蛋白可以直接与SEZ结合，且缺失CD44的巨噬细胞吞噬SEZ的能力减弱。. 本研究发现SEZ的新型免疫原性蛋白SeseC_01411，通过免疫荧光与流式细胞术分析表明SeseC_01411分布于SEZ菌体的表面，通过免疫重组表达的SeseC_01411蛋白，可以诱导小鼠体产生强烈的体液免疫反应，并具有较强的免疫保护作用。此外，SeseC_01411蛋白的免疫血清在吞噬试验中能有效杀灭SEZ，免疫原蛋白SeseC_01411是SEZ的一种新型表面保护抗原。本研究阐明SEZ荚膜与CD44作用调控TLR的信号通路，发现了新型保护抗原SeseC_01411，为我国猪链球菌的防控提供理论依据。