马链球菌兽疫亚种SzM蛋白激活巨噬细胞NLRP3炎症小体的机制

Streptococcal disease is one of the most important bacterial diseases in the pig industry. Streptococcus equi subsp. zooepidemicus (SEZ) is one of the important pathogens of streptococcal infection in China. Recent studies revealed NLRP3 inflammasome activation in macrophages play an important role in host defense, but little was known about its mechanism during infections with pathogenic microorganisms. Our recent results have confirmed that SEZ capsular influence the interaction between SEZ cell wall components with host cell so as to inhibit NLRP3 expression. In the present study, cells were treated with well-characterized inhibitors to determine macrophage uptake of SzM through a specific endocytic pathways. To visualize SzM protein untakes into macrophages, SzM-GFP co-lacalized with early endosome markers. Recombinant SzM protein and SzM-deficient mutant were used to examine that SzM protein provided a signal specifically triggers IL-1β signalling in macrophages. Then the caspase-1 knockout mice were used to reveal SzM protein activated NLRP3 inflammasome and triggered IL-1β release in a caspase-1 dependent manner. To confirm the regulation of NLRP3 inflammasome in vivo, the different strains outgrowth and dissemination to distant organs were examined during SEZ infection. Besides, the effects of wild type and caspase-1 knockout mice infected ΔCPS or ΔCPSΔSzM strain on NLRP3 activation, IL-1β and IL-18 expression were analyzed by ELISA and Western Blot. Understanding the roles and regulatory mechanisms of the NLRP3 inflammasome is essential for developing potential treatment approaches against pathogenic infections.

猪链球菌病是当今危害养猪业最重要的细菌性疾病之一，马链球菌兽疫亚种（SEZ）是我国猪链球菌病的重要病原。近期研究表明，巨噬细胞NLRP3炎症小体在抵御SEZ感染过程发挥重要作用，项目组前期发现荚膜影响SEZ表面蛋白与宿主细胞的相互作用从而抑制NLRP3的表达，但其作用机制尚不清楚。本项目拟在前期基础上，采用共定位配合吞噬途径抑制剂探索巨噬细胞吞噬SEZ表面蛋白SzM的作用途径；同时，用SzM蛋白及SzM缺失菌株分析SzM诱导巨噬细胞分泌IL-1β的信号通路；然后通过caspase-1基因敲除小鼠揭示SzM蛋白激活NLRP3炎症小体的机制；最后建立两种小鼠感染模型，分析ΔCPS、ΔCPSΔSzM菌株感染对NLRP3炎症小体活化及IL-1β分泌的影响，验证SzM调控NLRP3炎症小体的机制。研究结果不仅阐明SzM蛋白调控NLRP3炎症小体活化的机制，还为研发治疗链球菌病的新型阻断制剂提供思路。

马链球菌兽疫亚种（Streptococcus equi subsp. zooepidemicus，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。近期研究表明，巨噬细胞NLRP3炎症小体在抵御SEZ感染过程中发挥重要作用，项目组前期发现荚膜影响SEZ表面蛋白与宿主细胞的相互作用从而抑制NLRP3的表达，但其作用机制尚不清楚。本项目用不同比例的FAMSE染色的SEZ与巨噬细胞孵育，通过流式细胞术分析巨噬细胞的吞噬SEZ的能力，结果显示其阳性率、平均荧光强度随着感染复数的增加而上升，并与细菌计数呈正相关。.本项目通过比较不同日龄的小鼠和不同细菌接种量的致病力，成功建立WT与ΔCPS鼻腔攻毒感染小鼠肺炎模型。在此基础上，通过组织病理学观察发现ΔCPS感染小鼠后，其肺部小叶间质显著增宽、细支气管周围出现炎性细胞浸润，揭示SEZ的SzM蛋白参与调控SEZ炎症的过程。遂表达纯化SzM蛋白，通过免疫攻毒保护试验发现，重组SzM蛋白可诱导高水平的IgG抗体，并能保护小鼠免受致死量SEZ的攻击。.试验通过CCK8实验测定SzM蛋白的细胞毒性，通过LDH检测、PI染色实验证实SzM蛋白可以诱发巨噬细胞死亡。进一步研究发现，SzM蛋白可促进巨噬细胞分泌IL-1β和IL-18，且能促进caspase-1的表达与活化。遂使用caspase-1抑制剂Ac-YVAD-cmk、SzM蛋白处理巨噬细胞，发现SzM蛋白可抑制caspase-1的活化，且可使细胞焦亡标志蛋白GSDMD-N的表达显著下降；caspase-1抑制剂Ac-YVAD-cmk可抑制细胞上清IL-18和IL-1β的分泌。结果表明，SzM蛋白通过caspase-1途径激活IL-1β和IL-18炎症因子释放。研究结果不仅阐明SzM蛋白调控NLRP3炎症小体活化的机制，还为研发治疗链球菌病的新型阻断制剂提供思路。