致病细菌QseC和TRAP群体感受系统拮抗策略抗菌作用机理的研究

The wide spread of antibiotic resistant bacteria in the world has become a nightmare to human beings and also is a leading cause of death in severe infection patients. The main problem of antibiotics used in clinical infection treatment is inevitably induce bacterial resistance, resulting in effective antibiotics is dwindling. Therefore, the discovery and development of new antibiotic drugs, which do not induce antibiotic resistance among bacteria, is a new trend for new antibiotic agent research. Inhibition of bacterial quorum sensing system (QSS) can inhibit their pathogenicities, but does not cause selective growth pressure on bacteria and does not induce antibitoic resistance. Currently, the development of new type of antibiotic agents targeting QSS has attracted more attention in antibiotic drug research field. However, the antibacterial effect of the currently known QSS inhibitors had no any inhibitory effects on bacteria in vitro. QSS inhibitors only show antibiotic effects in vivo. So far the mechanism of this phenomenon is almost entirely unclear. In this project, we plan to explore the antibiotic mechanisms of QSS inhibitor based on our hypothesis - - - - QSS inhibitors inhibit bacterial virulence and immune evasion, and thereafter activate innate immune system and clean the bacteria in body respectively. The research includes evaluation of the antibacterial effect of the QSS inhibitor of bacteria, detect virulence and innate immune evasion factors after QSS blocking, and explore the molecular mechanisms of innate immune system activation including inflammasome formation and activation , as well as IL-1?, IL-18 secretion form immune cells. This study will reveal antibacterial mechanism of QSS inhibitors, provide a new idea and strategy on antibiotic drug research, as well as explore new avenues for drug-resistant infection treatment.

耐药细菌感染是临床抗感染治疗中极为棘手的问题，也是造成重症感染死亡的首要原因，已成为危害人类健康的严重灾难。抗生素治疗感染最主要的问题是诱导细菌耐药，使抗生素临床应用面临严峻挑战。研制不诱导细菌耐药的新型抗菌药物成为未来的研究方向。阻断细菌的群体感受系统(QSS)可以抑制其致病性，但不会给细菌造成选择生长压力，具有不诱导细菌耐药的独特优点。因此以QSS为靶点的不诱导耐药抗菌药物的研究倍受重视。然而目前已知所有QSS抑制剂在体外均无抑菌作用，仅在体内表现抗菌功效，目前这一现象的机制几乎完全不清楚。本课题从抑制细菌QSS，进而抑制细菌免疫逃逸，激活机体免疫抗菌应答效应思路入手，分别开展QSS抑制剂体内抗菌效果评价，调控细菌毒力因子和免疫逃逸分子表达的规律，以及激活机体免疫抗菌应答机理等方面研究。旨在揭示QSS抑制剂抗菌作用机理，为抑制QSS抗菌策略研究提供新思路，为耐药菌感染治疗探索新途径。

抗生素治疗感染最主要的问题是不可避免地诱导细菌耐药，使其临床应用面临严峻挑战。因此，研制不诱导耐药的抗菌药物是未来的新方向和热点。细菌群体感受器抑制剂可以显著抑制细菌致病性，但不会给细菌造成选择生长压力，具有不易诱导耐药的独特优点。然而，目前已知所有群体感受器抑制剂在体外均无抑菌作用，仅在体内发挥抗菌作用，其机制完全不清楚。本研究通过应用细菌群体感受器抑制剂LED209和RIP阻断细菌QseC和TRAP群体感受系统，评价和确认阻断细菌QseC和TRAP系统后的体内抗菌效果，探索细菌毒力致病因子和免疫逃逸相关分子释放的规律，评价阻断QseC、TRAP群体感受系统对细菌致病毒力和机体抗菌免疫应答效应的影响，明确抑制细菌免疫逃逸、激活机体抗菌免疫应答效应的效应分子，从而揭示细菌QseC和TRAP系统抑制剂体内抗菌作用的机理。结果表明，QseC抑制剂LED209体外确实不影响细菌生长，但能显著降低感染小鼠的死亡率和脏器荷菌量。阻断QseC抑制鼠伤寒沙门氏菌重要毒力基因的表达，抑制鼠伤寒沙门氏菌鞭毛动力、上皮细胞侵袭及胞内增殖。通过氯膦酸脂质体清除小鼠体内巨噬细胞，确证巨噬细胞是参与QseC抑制剂体内抗感染保护效应的主要靶细胞。通过体外细菌与巨噬细胞共培养模型，证实阻断QseC抑制鼠伤寒沙门氏菌感染诱导的巨噬细胞炎性复合体激活和巨噬细胞焦亡，促进巨噬细胞清除胞内细菌。阻断肠出血性大肠杆菌QseC促进感染巨噬细胞中炎性复合体活化，增加MMP12的表达，进而促进巨噬细胞清除胞内细菌，发挥胞内抗菌作用。TRAP抑制剂RIP1183体外不影响细菌生长，但能显著降低脓毒症和肺炎模型小鼠的死亡率和脏器荷菌量。荧光定量PCR结果表明，RIP1183能有效抑制MRSA RNAIII、α-溶血素、PSM等重要毒力因子的表达。通过腹腔注射环磷酰胺清除小鼠体内中性粒细胞，证实RIP1183发挥体内抗菌作用的靶细胞主要为中性粒细胞。通过体外细菌与中性粒细胞共培养模型，明确了MRSA 的毒力因子PSM可诱导中性粒细胞坏死性凋亡。上述结果将为抑制群体感受系统抗菌策略及抗细菌免疫逃逸和毒力新型抗菌药物研究提供新思路，为耐药菌感染治疗探索新途径。