鼠伤寒沙门氏菌Ⅲ型分泌系统抗感染类抑制剂的筛选

Screening of anti-virulence compounds inhibiting typeⅢsecretion system of Salmonella Typhimurium

【背景】肠道沙门氏菌(Salmonella enterica)是一种常见的食源性肠道致病菌,可以感染人畜并引发食物中毒、伤寒等疾病。近年来因抗生素滥用导致肠道沙门氏菌耐药性问题日益严峻,迫切需要开发新型抗感染药物。肠道沙门氏菌致病的关键在于与宿主细胞接触后可以通过Ⅲ型分泌系统(typeⅢsecretion system,T3SS)向宿主细胞内注射效应蛋白,进而调控宿主细胞囊泡运输和免疫应答等生理活动,以方便其高效侵染宿主细胞。T3SS是一类由超过20种蛋白质组成、高度复杂的跨膜分子机器,是革兰氏阴性病原菌中普遍存在的一类蛋白质运输系统和毒力系统。在不同病原菌中,其结构与功能非常保守。位于T3SS核心跨膜区的SctV家族蛋白是T3SS中最保守的组分之一,参与T3SS能量供应和效应蛋白的分泌过程,SctV蛋白的关键氨基酸突变失活后会导致鼠伤寒沙门氏菌丧失对宿主的入侵能力。【目的】以沙门氏菌SctV家族蛋白为靶点,尝试通过虚拟筛选技术筛选与SctV胞内区相互作用的抗感染类T3SS抑制剂。【方法】结合体外相互作用分析、细菌生长曲线实验、细菌分泌实验和细胞侵染实验等对候选分子进行抑制效果的分析和验证。【结果】最终筛选出小分子抑制剂C4(纽莫康定B0)和C5(茜草素),二者均可以显著抑制T3SS分泌效应蛋白,并进一步抑制鼠伤寒沙门氏菌对NCM460细胞的侵染。进一步的研究发现,化合物C4和C5并不是通过结合到SctV胞内区而是通过其他不同机制来抑制细菌的毒性。【结论】化合物C4通过未知通路调控T3SS效应蛋白的分泌过程,而C5可能通过下调调控基因hilD的表达来抑制T3SS分泌效应蛋白,并进一步抑制沙门氏菌对NCM460细胞的侵染。本研究为针对T3SS开发新型抗感染药物提供了新的靶点和思路,并为后续T3SS抗感染类抑制剂的优化和改造提供了分子理论基础。

[Background]Salmonella enterica is a common foodborne enteric pathogen that infects humans and animals and causes food poisoning,typhoid fever and other diseases.In recent years,antibiotic abuse has aggravated the antibiotic resistance of S.enterica,and it is urgent to develop new anti-infectives.After the contact with the intestinal epithelium,S.enterica can deliver several effector proteins into the host cells through the typeⅢsecretion system(T3SS).The effector proteins can manipulate many common cellular processes,including host immune responses,cytoskeletal dynamics,vesicle transport,and signaling pathways,which facilitate the infection in the host cells.T3SS,composed of more than 20 proteins,is a protein transport nanomachine ubiquitous in most Gram-negative bacterial pathogens and symbionts.Its structure and function are highly conserved in diverse pathogens.The SctV family protein,one of the most conserved components of T3SS,is located at the core part of T3SS and involved in energy supply and effector secretion of T3SS.The mutation of key residues in SctV results in the failure of S.enterica invasion.[Objective]To obtain the anti-virulence T3SS inhibitors with the SctV family proteins of Salmonella as the targets by virtual screening.[Methods]The in vitro interaction assay,secretion assay,bacterial growth curve assay,and bacterial invasion assay were combined to analyze and validate the inhibitory effect of candidate molecules.[Results]The compounds C4(pneumocandin B0)and C5(purpurin)significantly inhibited the secretion of T3SS effectors and further prevented S.Typhimurium from invading NCM460 cells.Instead of binding to the intracellular part of SctV,C4 and C5 employed other mechanisms to inhibit the virulence of S.Typhimurium.[Conclusion]How C4 inhibits the secretion of T3SS effectors remains unclear.C5 may down-regulate the expression of the regulator gene hilD to inhibit the secretion of T3SS effectors and further hinder the invasion of S.Typhimurium into NCM460 cells.This work provides new targets and ideas for the development of novel anti-infectives targeting T3SS.Moreover,it provides a theoretical basis for the optimization and modification of subsequent T3SS anti-virulence inhibitors.