肠道病原菌III型分泌系统效应蛋白调控宿主细胞NF-κB和MAPK信号通路的研究进展

病原细菌感染对人类健康构成了严重的威胁,一类具有III型分泌系统(Type III secretion system,T3SS)的肠道致病细菌可以通过T3SS将效应蛋白“注射”到宿主细胞中,模拟和操纵宿主细胞的多种信号转导通路,包括细胞凋亡、细胞自噬和炎症反应等,从而有效地逃逸宿主的防御,增强感染性和致病性。本文综述了肠道病原菌T3SS效应蛋白在调控宿主炎症反应中NF-κB和MAPK通路的最新研究进展。     

沙门菌感染与Caspase­-8介导的宿主细胞调控性死亡研究进展

沙门菌主要通过食物传播,严重威胁了人类健康。肠道上皮细胞作为抵抗沙门菌入侵的重要屏障,可通过多种方式抵抗沙门菌的定植与入侵。同时,肠道固有层巨噬细胞可特异性识别正常菌群与沙门菌,激活炎性小体并分泌白细胞介素(interleukin,IL)-1β等炎症因子诱导炎症反应清除沙门菌。Caspase家族属于半胱氨酸蛋白酶,它们被激活后可执行各种细胞功能。Caspase-1是炎性小体的重要组成部分,可切割消皮素D(gasdermin D)诱导细胞焦亡,引发炎症反应。研究发现,Caspase-8同样参与炎性小体复合物的形成,但其功能尚不明确。新近研究发现,在沙门菌感染所诱导的细胞焦亡被抑制时,Caspase-8在炎性小体中被强烈激活,并在肠道上皮细胞和巨噬细胞中调控细胞死亡与炎症反应,以限制沙门菌感染。因此,Caspase-8在沙门菌感染期间也是调节宿主抗感染免疫的关键分子,研究其调控宿主细胞死亡以及炎症因子释放的机制对深入了解沙门菌感染与宿主抗感染免疫应答之间的关系具有重要意义。     

鼠伤寒沙门氏菌Ⅲ型分泌系统抗感染类抑制剂的筛选

【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(type Ⅲ secretion 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...     

肠道菌群代谢产物在鼠伤寒沙门菌感染中的作用研究进展

人和动物肠道内生存着多种多样的微生物群体,它们与宿主共同进化,对宿主的健康至关重要。肠道菌群可以发酵宿主难以消化的复杂碳水化合物,为宿主肠道细胞提供能量,同时其代谢产物对肠道病原菌沙门菌的感染产生着重要影响。正常情况下,肠道菌群代谢产物如丁酸与丙酸可以抑制沙门菌在肠道中的定植或者毒力基因的表达,而在肠道菌群受到扰乱时,其代谢的琥珀酸盐和1,2-丙二醇等物质却能促进沙门菌增殖。近年来,越来越多的研究揭示了肠道菌群代谢产物对沙门菌感染的影响。本综述通过总结近年来关于鼠伤寒沙门菌入侵时肠道菌群代谢产物改变的研究,综合阐述了肠道菌群代谢产物影响沙门菌感染的机制。     

沙门菌毒力效应蛋白对宿主NF-κB信号通路的调控

沙门菌(Salmonella)通过向宿主细胞分泌毒力效应蛋白(effector protein)来调控细胞内一系列的信号传导通路,从而有利于沙门菌的侵染和繁殖。NF-κB信号通路在宿主对病原菌的炎症反应及免疫应答中发挥着重要的作用,也是很多毒力效应蛋白调控的靶点。沙门菌致病岛(Salmonella pathogenicity island,SPI)-1上的毒力效应蛋白Sip A、Sop E、Sop E2和Sop B都能激活宿主细胞的NF-κB信号通路,而毒力效应蛋白Spt P、Avr A、Ssp H1以及SPI-2上的Sse L能有效地抑制NF-κB信号通路。研究这些毒力效应蛋白对NF-κB信号通路的时相调控和协同作用,将进一步揭示沙门菌的致病机制。     

沙门菌效应蛋白对宿主细胞的影响及分子机制

沙门菌(Salmonella spp.)作为胞内病原菌,通过侵入宿主细胞,导致人类和多种动物感染疾病。在与宿主细胞的长期斗争中,沙门菌进化出多种机制来逃避宿主的监视与防御,从而完成侵入并生存增殖的过程。尽管一些效应蛋白靶向的宿主因子已经被发现,但大多数效应蛋白的靶点尚且未知。本文综述了沙门菌效应蛋白对宿主细胞生理活动的影响,包括对细胞骨架的变化、炎症应答、胞膜修饰和滤泡的胞内移动现象及其分子机制进行阐述。     

SipC的C端能结合并促使纤维型肌动蛋白成束从而促进鼠伤寒沙门菌入侵

鼠伤寒沙门菌通过效应蛋白注入,利用宿主细胞的肌动蛋白骨架网络入侵非吞噬细胞。已知SipC能使肌动蛋白成核,使纤维型肌动蛋白成束,并转运鼠伤寒沙门菌Ⅲ型分泌系统的效应蛋白。但SipC如何使纤维型肌动蛋白成束的分子机制及发挥此活性功能域的作用仍不清楚。该研究利用一系列SipC删除/插入突     

鼠伤寒沙门菌rtsB基因缺失株的构建及其生物学特性

【背景】鼠伤寒沙门菌(Salmonella typhimurium)是一种重要的人畜共患病原菌,严重危害养殖业及人类健康。调控蛋白在病原菌的生存及感染过程中发挥重要作用。【目的】构建鼠伤寒沙门菌调控基因rtsB缺失株和互补株,分析调控蛋白RstB对鼠伤寒沙门菌生物学特性和致病性的影响。【方法】利用Red同源重组的方法构建鼠伤寒沙门菌SAT52的rtsB基因缺失株,并利用互补质粒构建互补株。然后比较分析野生株SAT52、缺失株?rtsB和互补株C?rtsB的生长特性、运动性、生物被膜形成能力、黏附入侵能力、胞内存活能力及致病性的差异。【结果】缺失rtsB基因不影响SAT52的生长速度,但导致运动能力增强,生物被膜形成能力减弱。细胞感染试验结果表明,rtsB基因有助于鼠伤寒沙门菌对Hela细胞的黏附入侵及RAW264.7细胞内的存活。动物试验结果表明rtsB基因缺失显著降低鼠伤寒沙门菌的致病力。【结论】rtsB基因在鼠伤寒沙门菌感染过程中发挥重要作用,可为阐释鼠伤寒沙门菌的致病机制提供参考。     

鼠伤寒沙门菌感染巨噬细胞铁稳态相关基因mRNA表达谱

用荧光定量PCR法检测鼠RAW264.7巨噬细胞感染与未感染鼠伤寒沙门菌后18种铁穗态相关基因的表达,评估宿主与病原体相互作用中铁稳态效应。研究显示,活的鼠伤寒沙门菌感染巨噬细胞1 h后可以诱导转铁蛋白受体表达,引起细胞内动态铁池相关基因的mRNA水平上长。基因表达分析显示,沙门菌通过诱导铁氧还原酶(Steap3)、铁膜转运蛋白(Dmt1)、铁调节因子Tfr2/Hfe以及铁调节蛋白(Irp1和Irp2)的表达主动吸收铁,而经铁转运蛋白(Fpn1)的铁外流并无明显改变。沙门菌在感染后1h积极地驱动了转铁蛋白介导的铁吸收程序。     

沙门菌致病岛2 Ⅲ型分泌系统研究进展

沙门菌(Salmonella)是革兰氏阴性的兼性胞内菌,可引起其广泛宿主的一系列疾病,严重时可导致全身性感染,威胁生命安全。沙门菌致病岛2(SPI2)是与沙门菌全身性感染密切相关的重要毒力基因簇,其编码的Ⅲ型分泌系统2(T3SS2)在沙门菌侵入宿主细胞后开始组装合成,经该装置分泌的多种效应蛋白对沙门菌在宿主细胞内的生存和增殖起着重要作用。近些年来,与沙门菌T3SS2相关的研究一直都是病原微生物领域关注的焦点之一。本文简要综述了SPI2的基因特征、SPI2基因表达的调控、T3SS2的结构和组成、T3SS2的效应蛋白及与T3SS2相关的疫苗研究等方面的主要研究进展。     

细菌三型分泌系统效应蛋白转运的研究进展

三型分泌系统(Type 3 secretion system,T3SS)作为存在于革兰氏阴性菌中的分泌系统之一,对革兰氏阴性菌的致病有重要作用。T3SS的致病作用体现在T3SS能直接将效应蛋白转运至宿主细胞,进而通过效应蛋白调控细胞的一系列通路,促进细菌定殖于细胞。而效应蛋白的转运受到两方面因素的调控,一方面是效应蛋白本身的信号序列,另一方面是T3SS相关蛋白的辅助。本文围绕近年来T3SS的构成、效应蛋白转运机制方面的最新进展进行概要综述。     

副溶血弧菌Ⅲ型分泌系统（T3SS）效应蛋白及其对宿主细胞的操控

副溶血弧菌是典型的食源性病原菌,也是全球范围内引起肠胃炎的主要病原菌。针筒状的Ⅲ型分泌系统(T3SS)为该菌主要的毒力因子,细菌感染时可将其效应蛋白直接注射至宿主细胞中,通过效应蛋白操纵宿主细胞,介导毒力的发挥。多数临床分离的副溶血弧菌含有2套T3SSs,其中T3SS1分泌的效应蛋白主要通过诱导细胞自噬、变圆和裂解等过程来发挥其细胞毒性,而T3SS2分泌的效应蛋白则主要通过破坏细胞骨架和操控细胞信号传导来发挥肠毒性。本文主要对副溶血弧菌T3SSs的组成和目前已发现的效应蛋白及其对宿主细胞的操控进行介绍。该研究不仅对深入了解该菌的致病机制有重要意义,而且也为宿主细胞信号转导机制研究提供新视角。     

Bringing down the host: enteropathogenic and enterohaemorrhagic Escherichia coli effector‐mediated subversion of host innate immune pathways

Enteric bacterial pathogens commonly use a type III secretion system (T3SS) to successfully infect intestinal epithelial cells and survive and proliferate in the host. Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC; EHEC) colonize the human intestinal mucosa, form characteristic histological lesions on the infected epithelium and require the T3SS for full virulence. T3SS effectors injected into host cells subvert cellular pathways to execute a variety of functions within infected host cells. The EPEC and EHEC effectors that subvert innate immune pathways – specifically those involved in phagocytosis, host cell survival, apoptotic cell death and inflammatory signalling – are all required to cause disease. These processes are reviewed within, with a focus on recent work that has provided insights into the functions and host cell targets of these effectors.     

Activation of a RhoA/myosin II-dependent but Arp2/3 complex-independent pathway facilitates Salmonella invasion

Salmonella stimulates host cell invasion using virulence effectors translocated by the pathogen's type-three secretion system (T3SS). These factors manipulate host signaling pathways, primarily driven by Rho family GTPases, which culminates in Arp2/3 complex-dependent activation of host actin nucleation to mediate the uptake of Salmonella into host cells. However, recent data argue for the existence of additional mechanisms that cooperate in T3SS-dependent Salmonella invasion. We identify a myosin II-mediated mechanism, operating independent of but complementary to the Arp2/3-dependent pathway, as contributing to Salmonella invasion into nonphagocytic cells. We also establish that the T3SS effector SopB constitutes an important regulator of this Rho/Rho kinase and myosin II-dependent invasion pathway. Thus, Salmonella enters nonphagocytic cells by manipulating the two core machineries of actin-based motility in the host: Arp2/3 complex-driven actin polymerization and actomyosin-mediated contractility.     

肠出血性大肠杆菌Ⅲ型分泌系统效应因子与宿主细胞相互作用研究进展

肠出血性大肠杆菌(Enterohemorrhagic Escherichia coli,EHEC)通过其Ⅲ型分泌系统将效应因子注入到宿主细胞内,破坏宿主细胞内的多种信号通路从而有利于细菌的感染及定植。近年来对于EHEC Ⅲ型分泌系统效应因子与宿主细胞相互作用研究成为EHEC致病机制研究新的热点,研究表明,除了经典的效应因子外,一些新发现的效应因子在细菌的致病过程中也发挥着重要作用,有些效应因子能够抑制宿主细胞内正常的信号通路,有些效应因子还具有抑制细胞凋亡,干扰炎症信号通路和抑制吞噬的作用。这些发现揭示了EHEC效应因子具有多种功能,它们通过与宿主细胞间的相互作用,在细菌的感染过程中发挥着重要作用。     

The cell death response to enteropathogenic Escherichia coli infection

Given the critical roles of inflammation and programmed cell death in fighting infection, it is not surprising that many bacterial pathogens have evolved strategies to inactivate these defences. The causative agent of infant diarrhoea, enteropathogenic Escherichia coli (EPEC), is an extracellular, intestinal pathogen that blocks both inflammation and programmed cell death. EPEC attaches to enterocytes, remains in the gut lumen and utilizes a type III secretion system (T3SS) to inject multiple virulence effector proteins directly into the infected cell, many of which subvert host antimicrobial processes through the disruption of signalling pathways. Recently, T3SS effector proteins from EPEC have been identified that inhibit death receptor‐induced apoptosis. Here we review the mechanisms used by EPEC T3SS effectors to manipulate apoptosis and promote host cell survival and discuss the role of these activities during infection.     

Diverse toxic effectors are harbored by vgrG islands for interbacterial antagonism in type VI secretion system

The type VI secretion system (T6SS) is considered as one of the key competition strategies by injecting toxic effectors for intestinal pathogens to acquire optimal colonization in host gut, a microenviroment with high-density polymicrobial community where bacteria compete for niches and resources. Enterotoxigenic Escherichia coli (ETEC), a major cause of infectious diarrhea in human and animals, widely encode T6SS clusters in their genomes. In this report, we first identified VT1, a novel amidase effector in ETEC, significantly hydrolyzed D-lactyl-L-Ala crosslinks between N-acetylmuramoyl and L-Ala in peptidoglycan. Further study showed that the VT1/VTI1 effector/immunity pair is encoded within a typical vgrG island, and plays a critical role for the successful establishment of ETEC in host gut. Numerous putative effectors with diverse toxin domains were found by retrieving vgrG islands in pathogenic E. coli, and designated as VT modules. Therein, VT5, a lysozyme-like effector widely encoded in ETEC, was confirmed to effectively kill adjacent cells, suggesting that VT toxin modules may be critical for pathogenic E. coli to seize a significantly competitive advantage for optimal intestinal colonization. To expand our analyses for large-scale search of VT antibacterial effectors based on vgrG island, >200 predicted effectors from 20 bacterial species were found and classified into 11 predicted toxins. This work reports a new retrieval strategy for screening T6SS effectors, and provides an example how pathogenic bacteria antagonize and displace commensal microbiome to successfully colonize in the host niches through a T6SS-dependent manner.     

A Comprehensive Proteomic Analysis of the Type III Secretome of Citrobacter rodentium

Enteropathogenic Escherichia coli, enterohemorrhagic E. coli, and Citrobacter rodentium belong to the family of attaching and effacing (A/E) bacterial pathogens. They intimately attach to host intestinal epithelial cells, trigger the effacement of intestinal microvilli, and cause diarrheal disease. Central to their pathogenesis is a type III secretion system (T3SS) encoded by a pathogenicity island called the locus of enterocyte effacement (LEE). The T3SS is used to inject both LEE- and non-LEE-encoded effector proteins into the host cell, where these effectors modulate host signaling pathways and immune responses. Identifying the effectors and elucidating their functions are central to understanding the molecular pathogenesis of these pathogens. Here we analyzed the type III secretome of C. rodentium using the highly sensitive and quantitative SILAC (stable isotope labeling with amino acids in cell culture)-based mass spectrometry. This approach not only confirmed nearly all known secreted proteins and effectors previously identified by conventional biochemical and proteomic techniques, but also identified several new secreted proteins. The T3SS-dependent secretion of these new proteins was validated, and five of them were translocated into cultured cells, representing new or additional effectors. Deletion mutants for genes encoding these effectors were generated in C. rodentium and tested in a murine infection model. This study comprehensively characterizes the type III secretome of C. rodentium, expands the repertoire of type III secreted proteins and effectors for the A/E pathogens, and demonstrates the simplicity and sensitivity of using SILAC-based quantitative proteomics as a tool for identifying substrates for protein secretion systems.     

Advances on Vibrio parahaemolyticus research in the postgenomic era

Vibrio parahaemolyticus is a leading cause of seafood-borne bacterial gastroenteritis in humans. Since its discovery in 1950, this bacterium has been isolated in widespread outbreaks and in sporadic cases of gastroenteritis worldwide. Although the exotoxin, thermostable direct hemolysin, had been the focus of extensive research on the pathogenicity of V. parahaemolyticus, the whole-genome sequencing of a clinical isolate, RIMD2210633 strain, was a breakthrough in this field. The possession of two sets of gene clusters for type III secretion systems (T3SS1 and T3SS2) was unveiled by that genome project. T3SS is a protein export apparatus that delivers bacterial proteins, called effectors, directly into the host's cytosol, to disrupt host cell function. The subsequent studies have established that T3SS2, which is encoded in an 80 kb pathogenicity island called V. parahaemolyticus pathogenicity island (Vp-PAI), is closely related to enteropathogenicity. Recent functional analyses of Vp-PAI-encoded genes revealed the sophisticated mechanisms in V. parahaemolyticus for sensing the intestinal environment and host cell contact, and a dozen T3SS2-exported proteins encoded in Vp-PAI. In this review, we summarize recent advances in V. parahaemolyticus research regarding the control of the expression of Vp-PAI-encoded genes, structural components and the secretory regulation of T3SS2, and the biological activities of T3SS2-exported effectors. Thus, Vp-PAI-encoded T3SS2 becomes an important key in the postgenomic era to shed light on the enteropathogenic mechanism of V. parahaemolyticus.