Ⅵ型分泌系统研究进展

Ⅵ型分泌系统(Type Ⅵ secretion system,T6SS)是存在于约25%革兰阴性细菌中的一种分泌系统。研究表明T6SS不仅具有对抗其他细菌和杀伤真核宿主细胞的能力,还与细菌的生存及致病能力等方面有着密切的联系。T6SS在不同菌种中表达与功能差异较大,多种致病菌T6SS得到了广泛的研究及讨论。本文主要对T6SS的结构、表达及其功能进行综述,为后续相关研究提供思路。     

肺炎克雷伯菌Ⅵ型分泌系统的研究进展

肺炎克雷伯菌可引起多系统感染, 是临床常见的病原菌。Ⅵ型分泌系统(T6SS)是广泛存在于革兰阴性菌膜内的分泌装置, 通过接触依赖的方式将毒性效应物质传递给靶细胞, T6SS与细菌运动能力、黏附能力、定植能力、细菌间竞争能力、生物膜形成、细菌耐药相关的基因水平转移等相关。对T6SS的结构及其在肺炎克雷伯菌中作用的研究进展(包括肺炎克雷伯菌T6SS的调节、效应因子和临床研究)进行阐述, 以期为肺炎克雷伯菌T6SS的进一步研究提供参考依据。     

空肠弯曲菌VI型分泌系统结构特征和菌株中分布

目的 明确中国空肠弯曲菌VI型分泌系统基因簇的结构特征及其在菌株中的分布。方法 对中国不同来源空肠弯曲菌菌株进行全基因组测序,并与数据库不同来源空肠弯曲菌菌株基因组序列和VI型分泌系统基因簇做比较分析。结果 52株空肠弯曲菌经基因组系统发育分析被分为了6个进化分支,对应不同来源的菌株。空肠弯曲菌的VI型分泌系统基因簇大小约14 kb,包含14个基因,其中hcp等9个主要基因与其他细菌T6SS相同基因的同源性较高,但仍具有空肠弯曲菌独特的基因簇结构。有36.5%(19/52)空肠弯曲菌菌株含有VI型分泌系统基因簇,主要为鸡来源分支和混合来源分支中的鸡来源菌株;有83.3%(10/12)中国的鸡来源菌株携带VI型分泌系统基因簇。结论 空肠弯曲菌的VI型分泌系统与鸡来源菌株密切相关。     

志贺菌Ⅲ型分泌系统研究进展

Ⅲ型分泌系统（T3SS）是革兰阴性致病菌重要的分泌系统,细菌通过T3SS将毒力蛋白注入宿主细胞。志贺菌在与宿主肠道上皮细胞接触后,激活T3SS并将效应子蛋白注入真核宿主细胞内,引起细菌性痢疾。本文综述了志贺菌T3SS的结构与功能,从分子水平揭示了志贺菌的致病机理。     

细菌III型分泌系统及其效应蛋白致病性研究进展

细菌III型分泌系统(T3SS)是细菌分泌毒力因子的重要分泌系统之一,也是抗菌药物抑制细菌繁殖生长的有效靶标。细菌III型分泌系统是革兰阴性菌不断进化而来的毒力决定簇之一,是细菌在长期进化中逐渐形成的入侵宿主细胞的特异性机制。近几年对T3SS结构、装配和致病作用的深入研究取得重大的突破。细菌III型分泌系统的致病机制和蛋白细胞结构装置对疾病的预防和控制有着重要的意义。     

空肠弯曲菌Ⅵ型分泌系统结构特征和菌株中分布

目的明确中国空肠弯曲菌Ⅵ型分泌系统基因簇的结构特征及其在菌株中的分布。方法对中国不同来源空肠弯曲菌菌株进行全基因组测序,并与数据库不同来源空肠弯曲菌菌株基因组序列和VI型分泌系统基因簇做比较分析。结果 52株空肠弯曲菌经基因组系统发育分析被分为了6个进化分支,对应不同来源的菌株。空肠弯曲菌的Ⅵ型分泌系统基因簇大小约14 kb,包含14个基因,其中hcp等9个主要基因与其他细菌T6SS相同基因的同源性较高,但仍具有空肠弯曲菌独特的基因簇结构。有36.5%(19/52)空肠弯曲菌菌株含有Ⅵ型分泌系统基因簇,主要为鸡来源分支和混合来源分支中的鸡来源菌株;有83.3%(10/12)中国的鸡来源菌株携带Ⅵ型分泌系统基因簇。结论空肠弯曲菌的Ⅵ型分泌系统与鸡来源菌株密切相关。     

衣原体T3SS效应蛋白的研究进展

衣原体是一种专性细胞内寄生的革兰氏阴性菌,可在广泛的宿主中引起疾病。衣原体Ⅲ型分泌系统效应蛋白是指衣原体通过Ⅲ型分泌系统(TypeⅢsecretion system,T3SS)分泌的,与其存活及毒力相关的蛋白质。衣原体T3SS效应蛋白对于调控宿主细胞功能具有重要作用,如诱导肌动蛋白细胞骨架的重组,改变宿主细胞信号传导机制和抑制宿主细胞凋亡等。因此,研究衣原体T3SS效应蛋白对研究衣原体的致病机制以及研发衣原体疫苗具有重要意义。     

沙门氏菌毒力岛及Ⅲ型分泌系统研究进展

沙门氏菌(Salmonella)是寄生于人和动物肠道内的革兰氏阴性杆菌,能引起人和动物多种不同临床症状表现,为人类食物中毒主要病原菌之一。沙门氏菌的侵袭力与毒力岛(pathogenicity island,PI)及其编码的Ⅲ型分泌系统(Type Ⅲ secretion system,T3SS)直接相关。本文对沙门氏菌毒力岛、T3SS组成、T3SS分泌及调控机制、T3SS与沙门氏菌致病性、应用研究等进行综述,以期为深入研究提供参考。     

沙门菌Ⅲ型分泌系统组成与装配研究进展

沙门菌(Salmonella)是引发人和动物食物中毒、胃肠炎的主要食源性病原菌,该菌III型分泌系统(T3SS)对其入侵宿主细胞发挥着重要作用,近年来,有关沙门菌T3SS的组成、装配以及相关致病机理的研究取得了一定进展。本文对沙门菌T3SS的组成与装配等研究作一综述,为深入研究沙门菌的致病机制以及预防、治疗该菌引发的疾病提供新的策略和手段。     

分枝杆菌Ⅶ型分泌系统的结构、底物与功能

细菌分泌系统对于细菌的发育、种内和种间竞争、营养获取、毒力、生理、遗传、生态都非常重要。细菌利用分泌系统将蛋白质分泌到细胞外,也是细菌适应生境所必需的。分枝杆菌Ⅶ型分泌系统及其底物与结核分枝杆菌致病密切相关,笔者将对Ⅶ型分泌系统的组成、结构、底物和功能进行介绍,尤其是与宿主相互作用中的功能,以期为研发结核病新的控制措施提供基础。     

Virulence blockers as alternatives to antibiotics: type III secretion inhibitors against Gram-negative bacteria

In recent years mounting problems related to antibiotic-resistant bacteria have resulted in the prediction that we are entering the preantibiotic era. A way of preventing such a development would be to introduce novel antibacterial medicines with modes of action distinct from conventional antibiotics. Recent studies of bacterial virulence factors and toxins have resulted in increased understanding of the way in which pathogenic bacteria manipulate host cellular processes. This knowledge may now be used to develop novel antibacterial medicines that disarm pathogenic bacteria. The type III secretion system (T3SS) is known to be a potent virulence mechanism shared by a broad spectrum of pathogenic Gram-negative bacteria that interact with human, animal and plant hosts by injecting effector proteins into the cytosol of host cells. Diseases, such as bubonic plague, shigellosis, salmonellosis, typhoid fever, pulmonary infections, sexually transmitted chlamydia and diarrhoea largely depend on the bacterial proteins injected by the T3SS machinery. Recently a number of T3SS inhibitors have been identified using screening-based approaches. One class of inhibitors, the salicylidene acylhydrazides, has been subjected to chemical optimization and evaluation in several in vitro and ex vivo assays in multiple bacterial species including Yersinia spp., Chlamydia spp., Salmonella spp. and Pseudotuberculosis aeruginosa. Reports published up to date indicate that T3SS inhibitors have the potential to be developed into novel antibacterial therapeutics.     

Comparative genomics reveal the mechanism of the parallel evolution of O157 and non-O157 enterohemorrhagic Escherichia coli

Among the various pathogenic Escherichia coli strains, enterohemorrhagic E. coli (EHEC) is the most devastating. Although serotype O157:H7 strains are the most prevalent, strains of different serotypes also possess similar pathogenic potential. Here, we present the results of a genomic comparison between EHECs of serotype O157, O26, O111, and O103, as well as 21 other, fully sequenced E. coli/Shigella strains. All EHECs have much larger genomes (5.5–5.9 Mb) than the other strains and contain surprisingly large numbers of prophages and integrative elements (IEs). The gene contents of the 4 EHECs do not follow the phylogenetic relationships of the strains, and they share virulence genes for Shiga toxins and many other factors. We found many lambdoid phages, IEs, and virulence plasmids that carry the same or similar virulence genes but have distinct evolutionary histories, indicating that independent acquisition of these mobile genetic elements has driven the evolution of each EHEC. Particularly interesting is the evolution of the type III secretion system (T3SS). We found that the T3SS of EHECs is composed of genes that were introduced by 3 different types of genetic elements: an IE referred to as the locus of enterocyte effacement, which encodes a central part of the T3SS; SpLE3-like IEs; and lambdoid phages carrying numerous T3SS effector genes and other T3SS-related genes. Our data demonstrate how E. coli strains of different phylogenies can independently evolve into EHECs, providing unique insights into the mechanisms underlying the parallel evolution of complex virulence systems in bacteria.     

In vivo actin cross-linking induced by Vibrio cholerae type VI secretion system is associated with intestinal inflammation

Type VI secretion systems (T6SSs) have recently been recognized as potential virulence determinants of many Gram-negative bacterial pathogens. Although mechanistic studies are lacking, T6SS-dependent phenotypes can be observed in various animal models of infection. Presumably translocation of T6SS effectors into target cells is involved in virulence, but few such effectors have been identified. A hallmark of T6SS function is the in vitro secretion of Hcp and VgrG proteins, which are thought to form part of an extracellular secretion apparatus. One well-characterized effector domain is the C-terminal actin cross-linking domain (ACD) of the VgrG-1 protein, constitutively secreted by the T6SS of Vibrio cholerae strain V52. Previous work indicated that translocation of VgrG-1 occurred only after endocytic uptake of bacteria into host cells. VgrG-1-induced actin cross-linking impaired phagocytic activity of host cells, eventually causing cell death. To determine whether V. cholerae T6SS is functional during animal infection, derivatives of V52 were used to infect infant mice. In this infection model a diarrheal response occurred, and actin cross-linking could be detected. These host responses were dependent on a functional T6SS and on the ACD of VgrG-1. Gene expression and histologic studies showed innate immune activation and immune cell infiltration in the intestinal lumen. The T6SS-dependent inflammatory response was also associated with increased recovery of V. cholerae from the intestine. We conclude that the T6SS of V52 induces an inflammatory diarrhea that facilitates replication of V. cholerae within the intestine.     

The Vibrio cholerae type VI secretion system displays antimicrobial properties

The acute diarrheal disease cholera is caused by the marine bacterium Vibrio cholerae. A type VI secretion system (T6SS), which is structurally similar to the bacteriophage cell-puncturing device, has been recently identified in V. cholerae and is used by this organism to confer virulence toward phagocytic eukaryotes, such as J774 murine macrophages and Dictyostelium discoideum. We tested the interbacterial virulence of V. cholerae strain V52, an O37 serogroup with a constitutively active T6SS. V52 was found to be highly virulent toward multiple Gram-negative bacteria, including Escherichia coli and Salmonella Typhimurium, and caused up to a 100,000-fold reduction in E. coli survival. Because the T6SS-deficient mutants V52ΔvasK and V52ΔvasH showed toxicity defects that could be complemented, virulence displayed by V. cholerae depends on a functional T6SS. V. cholerae V52 and strains of the O1 serogroup were resistant to V52, suggesting that V. cholerae has acquired immunity independently of its serogroup. We hypothesize that the T6SS, in addition to targeting eukaryotic host cells, confers toxicity toward other bacteria, providing a means of interspecies competition to enhance environmental survival. Thus, the V. cholerae T6SS may enhance the survival of V. cholerae in its aquatic ecosystem during the transmission of cholera and between epidemics.     

Killing of Gram-negative and Gram-positive bacteria by a bifunctional cell wall-targeting T6SS effector

The type VI secretion system (T6SS) is a powerful tool deployed by Gram-negative bacteria to antagonize neighboring organisms. Here, we report that Acinetobacter baumannii ATCC 17978 (Ab17978) secretes D-lysine (D-Lys), increasing the extracellular pH and enhancing the peptidoglycanase activity of the T6SS effector Tse4. This synergistic effect of D-Lys on Tse4 activity enables Ab17978 to outcompete Gram-negative bacterial competitors, demonstrating that bacteria can modify their microenvironment to increase their fitness during bacterial warfare. Remarkably, this lethal combination also results in T6SS-mediated killing of Gram-positive bacteria. Further characterization revealed that Tse4 is a bifunctional enzyme consisting of both lytic transglycosylase and endopeptidase activities, thus representing a family of modularly organized T6SS peptidoglycan-degrading effectors with an unprecedented impact in antagonistic bacterial interactions.

Bacteria live in dense communities and are often in constant competition with other bacterial species to secure nutrients and space. Bacterial warfare is mediated by the production of diffusible antimicrobial compounds and by sophisticated molecular weapons, such as the type VI secretion system (T6SS). The T6SS is a dynamic nanomachine that delivers toxic effector proteins from an attacking cell (predator) to nearby competitors (prey) in a contact-dependent manner. Although bacteria possess a diverse arsenal of toxic effector proteins to kill Gram-negative bacteria, current evidence suggests that Gram-positive bacteria are immune to T6SS attacks. Immunity to T6SS-dependent killing between Gram-negative kin cells is accomplished by the expression of immunity proteins, which specifically bind and inactivate their cognate effector. Broad-spectrum mechanisms of protection against nonkin T6SS attacks in Gram-negative bacteria have only recently been uncovered (1, 2).Due to its essentiality, the bacterial cell wall peptidoglycan (PG), also known as murein, is targeted by various T6SS effectors. PG is composed of glycan chains of alternating N-acetylglucosamine and N-acetylmuramic acid (MurNAc) that are crosslinked through MurNAc-attached peptides. PG-degrading (PGase) effectors induce bacterial cell lysis, which facilitates the complete clearance of bacterial competitors by preventing dead cells from shielding susceptible prey from T6SS attacks, a phenomenon known as the “corpse barrier” effect (3). The PGase effectors characterized to date contain one lytic enzymatic activity and can be categorized as muramidases, which cleave the β-(1,4)-glycosidic bond (4, 5), N-acetylmuramyl-L-alanine amidases, which unlink the peptides chains from MurNAc (6) and LD (4, 7), or DD-endopeptidases (7), which cleave PG crosslinks.T6SS-dependent bacterial warfare generates an arms race in which both predator and prey evolve tools to prevail. We recently showed that some Acinetobacter baumannii strains, such as strain ATCC 17978 (Ab17978), use the periplasmic racemase RacK to produce the noncanonical D-amino acid (NCDAA) D-lysine (D-Lys) from L-Lys. Incorporation of D-Lys into the PG of Ab17978 underlies a defensive strategy against the PGase activity of T6SS effectors from competing bacteria (2). Beyond being incorporated into the PG, most of the D-Lys that is produced is secreted into the extracellular milieu (2). It has been recently suggested that NCDAAs carry out diverse biological roles in bacterial ecosystems (8). In this work, we investigate the role of extracellular D-Lys in bacterial warfare.     

A multidrug resistance plasmid contains the molecular switch for type VI secretion in Acinetobacter baumannii

Infections with Acinetobacter baumannii, one of the most troublesome and least studied multidrug-resistant superbugs, are increasing at alarming rates. A. baumannii encodes a type VI secretion system (T6SS), an antibacterial apparatus of Gram-negative bacteria used to kill competitors. Expression of the T6SS varies among different strains of A. baumannii, for which the regulatory mechanisms are unknown. Here, we show that several multidrug-resistant strains of A. baumannii harbor a large, self-transmissible resistance plasmid that carries the negative regulators for T6SS. T6SS activity is silenced in plasmid-containing, antibiotic-resistant cells, while part of the population undergoes frequent plasmid loss and activation of the T6SS. This activation results in T6SS-mediated killing of competing bacteria but renders A. baumannii susceptible to antibiotics. Our data show that a plasmid that has evolved to harbor antibiotic resistance genes plays a role in the differentiation of cells specialized in the elimination of competing bacteria.Antibiotic-resistant bacteria that cause hospital-acquired infections are a mounting concern for health care systems globally (1). Multidrug-resistant (MDR) Acinetobacter baumannii is emerging as a frequent cause of difficult-to-treat nosocomial infections, and some isolates are resistant to all clinically relevant antibiotics (2, 3). A. baumannii is often isolated from polymicrobial infections and therefore spends at least a part of its time competing with other bacteria (4). Antagonistic interactions between bacteria manifest in a variety of different ways (5), and the type VI secretion system (T6SS) is a potent weapon used by many Gram-negative bacteria to kill competitors (6–8). The multicomponent T6SS apparatus facilitates a dynamic contact-dependent injection of toxic effector proteins into prey cells (9, 10), and expression of cognate immunity proteins prevents self-inflicted intoxication (9, 11). The T6SS is composed of several conserved proteins involved in the formation of the secretory apparatus (12, 13). One of these components, hemolysin-coregulated protein (Hcp), forms hexameric tubule structures that are robustly secreted to the culture supernatants in bacteria with an active T6SS, allowing it to be used as a molecular marker for T6SS activity (6, 14).T6SS is a dynamic apparatus (15). Its biogenesis follows energetically costly cycles of assembly/disassembly, and therefore, in most bacteria, T6SS appears to be exquisitely regulated. T6SS is silenced in most strains and only activated under specific conditions, such as an attack from another bacterium or in environments leading to membrane perturbations (16–19). Many Acinetobacter spp. encode the genes for a T6SS, including Acinetobacter noscomialis and Acinetobacter baylyi, which possess a constitutively active antibacterial T6SS (20–24). A. baumannii strains have been shown by us and others to secrete Hcp (21, 25), but to our knowledge a T6SS-dependent phenotype has not been ascribed to this species. Furthermore, our previous results showed that Hcp secretion is highly variable between A. baumannii strains, with some isolates carrying an inactive system (21). The precise regulatory mechanism(s) underlying T6SS suppression in some A. baumannii is unknown.Here, we show that a large resistance plasmid of A. baumannii functions to repress the T6SS by encoding negative regulators of its activity. Analysis of colonies from a clinical isolate showed that the plasmid is readily lost in a subset of the population. This leads to the activation of the T6SS, which imparts the ability to kill other bacteria, with the simultaneous loss of antibiotic resistance. We propose that the differentiation into T6SS+ MDR– and T6SS– MDR+ phenotypes may constitute a novel survival strategy of this organism.     

What's the point of the type III secretion system needle?

Recent work by several groups has significantly expanded our knowledge of the structure, regulation of assembly, and function of components of the extracellular portion of the type III secretion system (T3SS) of Gram-negative bacteria. This perspective presents a structure-informed analysis of functional data and discusses three nonmutually exclusive models of how a key aspect of T3SS biology, the sensing of host cells, may be performed.