Type VI secretion system effector proteins: Effective weapons for bacterial competitiveness

The Type VI secretion system (T6SS) is a protein translocation nanomachine widespread among Gram‐negative bacteria and used as a means to deliver effectors directly into target bacterial or eukaryotic cells. These effectors have a wide variety of functions within target cells that ultimately help the secreting cell gain a competitive fitness advantage. Here, we discuss the different ways in which these effectors can be delivered by the T6SS and the diverse mechanisms by which they exert their noxious action upon recipient cells. We also highlight the existence of roles for T6SS effectors beyond simply the killing of neighbouring cells.     

Extracellular Production of a Serratia marcescens Serine Protease in Escherichia coli

The Serratia marcescens serine protease (SSP) is one of the extracellular enzymes secreted from this Gram-negative bacterium. When the ssp gene, which encodes a SSP precursor (preproSSP) composed of a typical NH₂-terminal signal peptide, a mature enzyme domain, and a large COOH-terminal pro-region, is expressed in Escherichia coli, the mature protease is excreted through the outer membrane into the medium. The COOH-terminal pro-region, which is integrated into the outer membrane, provides the essential function for the export of the mature protein across the outer membrane. This is a very simple pathway, in contrast to the general secretory pathway exemplified by the secretion of a pullulanase from Klebsiella oxytoca, in which many separately encoded accessory proteins are required for the transport through the outer membrane. Moreover, the NH₂-terminal region of 71 amino acid residues of the COOH-terminal pro-sequence plays an essential role, as an “intramolecular chaperone,” in the folding of the mature enzyme in the medium. In addition to ssp, the S. marcescens strain contains two ssp homologues encoding proteins similar to SSP in amino acid sequence and size, but with no protease activity. Characterization of the homologue proteins and chimeric proteins between the homologues and SSP, all of which are produced in E. coli, has shown that they are membrane proteins that are localized in the outer membrane in the same manner as for SSP. By use of the COOH-terminal domain of SSP, pseudoazurin was exported to the cell surface of E. coli, which proves the usefulness of the SSP secretory system in the export of foreign proteins across the outer membrane.     

A major outer membrane protein of Serratia marcescens which was easily solubilized and had a capacity to bind to calcium

Easily solubilized major outer membrane protein was found in Serratia marcescens. The protein was originally obtained as a membrane-associated, insoluble protein in the outer membrane when the cells were slightly disrupted. However, the amount of this protein in the outer membrane gradually decreased with the time of sonication. The decrease was not due to decomposition of the protein but to solubilization into a soluble fraction after a long period of disruption. The molecular weight of this protein was 47 kDa and it bound calcium. Another 40 kDa calcium binding protein was also found in the outer membrane of S. marcescens.     

The effect of nuclease on transformation efficiency in Serratia marcescens

No differences in the efficiency of transformation were observed from both plasmid and chromosomal DNA in Serratia marcescens 2170 and an extracellular nuclease defective isogenic strain. The efficiency of transformation was the same for Escherichia coli 5K and E. coli containing a recombinant plasmid conferring the ability to synthesize a S. marcescens nuclease. From these results we conclude that the extracellular nuclease of S. marcescens 2170 is not the main cause of the low efficiency of transformation observed in this bacterium.     

鲍曼不动杆菌VI型分泌系统溶血素-联合调节蛋白研究进展

鲍曼不动杆菌是一种革兰氏阴性的非发酵致病菌,在医院环境中广泛存在,并且已经成为医院获得性感染的重要病原体之一。近年来,由于抗菌药物的广泛应用,导致多重耐药鲍曼不动杆菌引起的感染和暴发流行,给临床治疗带来了极大的挑战。有研究表明,细菌Ⅵ型分泌系统与细菌的致病性相关。本文综述了鲍曼不动杆菌Ⅵ型分泌系统及主要功能蛋白(溶血素-联合调节蛋白)的研究进展,以期为进一步研究鲍曼不动杆菌的致病机制提供基础。     

空肠弯曲菌Ⅵ型分泌系统研究进展

Ⅵ型分泌系统(Type Ⅵ Secretion System,T6SS)是一种倒置于细胞膜上的类噬菌体样结构,能够输送效应蛋白并在定殖和生态位建立中发挥作用.近年来,在空肠弯曲菌(Campylobacter jejuni)中发现了T6SS同源基因且能够表达组装成结构完整的T6SS,但T6SS对空肠弯曲菌的毒力影响尚不清...     

High-level production of Serratia proteamaculans metalloprotease using a recombinant ABC protein exporter-mediated secretion system in Pseudomonas fluorescens

Serratia proteamaculans metalloprotease (SPP) was successfully secreted by a heterologous ABC protein exporter, the Pseudomonas fluorescens TliDEF, in recombinant host strains. Escherichia coli and P. fluorescens cells containing the SPP-encoding gene showed the extracellular protease activity only when the TliDEF-encoding gene cluster was coexpressed. Recombinant P. fluorescens produced an approximately 34.8-fold higher amount of extracellular SPP than did E. coli. The use of a more nutrient-rich medium and controlled dissolved oxygen conditions was effective in increasing SPP secretion in P. fluorescens batch fermentation (an 8.7-fold increase from 41.8 U/mL to 365.2 U/mL). Therefore, SPP, which could not be secreted without an ABC protein exporter, was produced in large quantities by applying the heterologous TliDEF exporter in P. fluorescens. The results also suggest that the use of the ABC protein exporter in P. fluorescens could be an efficient production platform for an industrially promising type I secretion pathway-dependent enzyme.     

The type VI secretion toolkit

Bacterial secretion systems are macromolecular complexes that release virulence factors into the medium or translocate them into the target host cell. These systems are widespread in bacteria allowing them to infect eukaryotic cells and survive or replicate within them. A new secretion system, the type VI secretion system (T6SS), was recently described and characterized in several pathogens. Genomic data suggest that T6SS exist in most bacteria that come into close contact with eukaryotic cells, including plant and animal pathogens. Many research groups are now investigating the underlying mechanisms and the way in which the effector proteins translocated through this machine subvert host defences. This review provides an overview of our current knowledge about type VI secretion, focusing on gene regulation, components of the secretion machine, substrate secretion and the cellular consequences for the host cell.     

Cryo‐EM reconstruction of Type VI secretion system baseplate and sheath distal end

The bacterial Type VI secretion system (T6SS) assembles from three major parts: a membrane complex that spans inner and outer membranes, a baseplate, and a sheath–tube polymer. The baseplate assembles around a tip complex with associated effectors and connects to the membrane complex by TssK. The baseplate assembly initiates sheath–tube polymerization, which in some organisms requires TssA. Here, we analyzed both ends of isolated non‐contractile Vibrio cholerae sheaths by cryo‐electron microscopy. Our analysis suggests that the baseplate, solved to an average 8.0 Å resolution, is composed of six subunits of TssE/F₂/G and the baseplate periphery is decorated by six TssK trimers. The VgrG/PAAR tip complex in the center of the baseplate is surrounded by a cavity, which may accommodate up to ~450 kDa of effector proteins. The distal end of the sheath, resolved to an average 7.5 Å resolution, shows sixfold symmetry; however, its protein composition is unclear. Our structures provide an important step toward an atomic model of the complete T6SS assembly.     

Type VI secretion system sheath inter‐subunit interactions modulate its contraction

Secretion systems are essential for bacteria to survive and manipulate their environment. The bacterial type VI secretion system (T6SS) generates the force needed for protein translocation by the contraction of a long polymer called sheath. The sheath is a six‐start helical assembly of interconnected VipA/VipB subunits. The mechanism of T6SS sheath contraction is unknown. Here, we show that elongating the N‐terminal VipA linker or eliminating charge of a specific VipB residue abolishes sheath contraction and delivery of effectors into target cells. Mass spectrometry analysis identified the inner tube protein Hcp, spike protein VgrG, and other components of the T6SS baseplate significantly enriched in samples of the stable non‐contractile sheaths. The ability to lock the T6SS in the pre‐firing state opens new possibilities for understanding its mode of action.     

The Serratia marcescens hemolysin is secreted but not activated by stable protoplast-type L-forms of Proteus mirabilis

The outer-membrane protein ShlB of Serratia marcescens activates and secretes hemolytic ShlA into the culture medium. Without ShlB, inactive ShlA (termed ShlA*) remains in the periplasm. Since Proteus mirabilis L-form cells lack an outer membrane and a periplasm, it was of interest to determine in which compartment recombinant ShlA* and ShlB are localized and whether ShlB activates ShlA*. The cloned shlB and shlA genes were transcribed in P. mirabilis stable L-form cells by the temperature-inducible phage T7 RNA polymerase. Radiolabeling, Western blotting, and complementation with C-terminally truncated ShlA (ShlA255) identified inactive ShlA* in the culture supernatant. ShlB remained cell-bound and did not activate ShlA without integration in an outer membrane. Although hemolytic ShlA added to L-form cells had access to the cytoplasmic membrane, it did not affect L-form cells. Synthesis of the large ShlA protein (165 kDa) in P. mirabilis L-form cells under phage T7 promoter control demonstrates that L-form cells are suitable for the synthesis and secretion of large recombinant proteins. This property and the easy isolation of released proteins make L-form cells suitable for the biotechnological production of proteins. Received: 17 February 1998 / Accepted: 30 June 1998     

Type VI secretion and bacteriophage tail tubes share a common assembly pathway

The Type VI secretion system (T6SS) is a widespread macromolecular structure that delivers protein effectors to both eukaryotic and prokaryotic recipient cells. The current model describes the T6SS as an inverted phage tail composed of a sheath‐like structure wrapped around a tube assembled by stacked Hcp hexamers. Although recent progress has been made to understand T6SS sheath assembly and dynamics, there is no evidence that Hcp forms tubes in vivo. Here we show that Hcp interacts with TssB, a component of the T6SS sheath. Using a cysteine substitution approach, we demonstrate that Hcp hexamers assemble tubes in an ordered manner with a head‐to‐tail stacking that are used as a scaffold for polymerization of the TssB/C sheath‐like structure. Finally, we show that VgrG but not TssB/C controls the proper assembly of the Hcp tubular structure. These results highlight the conservation in the assembly mechanisms between the T6SS and the bacteriophage tail tube/sheath.     

细菌Ⅵ型分泌系统效应蛋白的装配及功能的研究进展

蛋白分泌作为细胞之间传递信号的途径之一,在微生物生存竞争中也扮演着重要的角色。革兰氏阴性菌可以通过Ⅵ型分泌系统(type Ⅵ secretion system, T6SS)将效应蛋白传递至胞外或原核和真核微生物中,从而介导微生物间的竞争或宿主-细菌的相互作用,最终建立竞争优势。本文主要总结了T6SS的结构与组成,并重点对效应蛋白的装配以及其与免疫蛋白的作用机制的研究进展进行阐述,为以后靶向T6SS抗菌药物的研制提供新思路。     

细菌VI型分泌系统调节因素的研究进展

细菌的VI型分泌系统(type VI secretion system,T6SS)是一种新发现的分泌系统,在病原菌对宿主黏附、侵入及杀伤等方面均发挥了重要作用。目前的研究主要集中在T6SS在细菌致病、细菌间竞争等作用方面。然而对于其调控因素的研究尚处于初级阶段。对于大多数细菌而言,T6SS的表达并不是恒定的。现已发现温度、渗透压、抗生素、离子等环境因素均可调节T6SS。此外,在分子层面,H-NS蛋白、RpoN转录因子、c-di-GMP等也可发挥对T6SS的调节作用。在这些调控因素的调节下,细菌可以适时地开启或关闭其T6SS的表达,从而更好地感知并适应环境。对T6SS调控因素的研究对于充分认识细菌致病性并进行有效控制至关重要。本文将对调节T6SS的环境因素与调节因子做一综述。     

铜绿假单胞菌Ⅵ型分泌系统的研究进展

铜绿假单胞菌是一种能引起多部位急、慢性感染且难以用抗生素控制的机会致病菌,近年来已成为院内感染的主要致病菌之一。大量研究表明,细菌将毒力因子精准输送至宿主细胞是其致病的关键,分泌系统在这一过程中扮演重要作用,其中近期发现的Ⅵ型分泌系统(type Ⅵ secretion system,T6SS)在铜绿假单胞菌与宿主间的相互作用和促进生物膜的形成等机制中发挥重要作用,已引起国内外学者高度关注。着重对铜绿假单胞菌T6SS的结构组成、效应功能和调节机制等相关研究进行简要综述,旨在为铜绿假单胞菌感染患者的治疗提供新策略。     

Domestication of a housekeeping transglycosylase for assembly of a Type VI secretion system

The type VI secretion system (T6SS) is an anti‐bacterial weapon comprising a contractile tail anchored to the cell envelope by a membrane complex. The TssJ, TssL, and TssM proteins assemble a 1.7‐MDa channel complex that spans the cell envelope, including the peptidoglycan layer. The electron microscopy structure of the TssJLM complex revealed that it has a diameter of ~18 nm in the periplasm, which is larger than the size of peptidoglycan pores (~2 nm), hence questioning how the T6SS membrane complex crosses the peptidoglycan layer. Here, we report that the MltE housekeeping lytic transglycosylase (LTG) is required for T6SS assembly in enteroaggregative Escherichia coli. Protein–protein interaction studies further demonstrated that MltE is recruited to the periplasmic domain of TssM. In addition, we show that TssM significantly stimulates MltE activity in vitro and that MltE is required for the late stages of T6SS membrane complex assembly. Collectively, our data provide the first example of domestication and activation of a LTG encoded within the core genome for the assembly of a secretion system.     

细菌Ⅵ型分泌系统的调控与功能研究进展

Ⅵ型分泌系统(Type Ⅵ Secretion System,T6SS)是近年来研究较多的一种细菌分泌系统,广泛存在于革兰氏阴性菌中,在细菌的毒力、定殖、扩散及竞争遗传中发挥着重要的作用.本文综述了细菌T6SS的结构、调控以及生物学功能的最新研究进展,以期为基于T6SS的抗菌药物研制及细菌感染的诊断与防控提供新思路.     

VI型分泌系统核心组分VgrG的致病功能

正VI型分泌系统(Type VI secretion system,T6SS)是一种接触依赖性分泌系统,能够将效应因子分泌至细菌胞外,具有多种不同功能,包括增强致病菌毒力、抗细菌毒力、增加机会致病菌的菌间竞争力。T6SS存在于超过四分之一的革兰阴性菌中[1-2]。禽致病性大肠杆菌(Avian Pathogenic Escherichia coli,APEC)可引起鸡、鸭及其他禽类的肠道外疾病,严重制约养禽业的健康发展,同时对食品安全构成威胁。APEC中存