Structural and SAXS analysis of Tle5–Tli5 complex reveals a novel inhibition mechanism of H2‐T6SS in Pseudomonas aeruginosa

Widely spread in Gram‐negative bacteria, the type VI secretion system (T6SS) secretes many effector‐immunity protein pairs to help the bacteria compete against other prokaryotic rivals, and infect their eukaryotic hosts. Tle5 and Tle5B are two phospholipase effector protein secreted by T6SS of Pseudomonas aeruginosa. They can facilitate the bacterial internalization process into human epithelial cells by interacting with Akt protein of the PI3K‐Akt signal pathway. Tli5 and PA5086‐5088 are cognate immunity proteins of Tle5 and Tle5B, respectively. They can interact with their cognate effector proteins to suppress their virulence. Here, we report the crystal structure of Tli5 at 2.8Å resolution and successfully fit it into the Small angle X‐ray scattering (SAXS) model of the complete Tle5–Tli5 complex. We identified two important motifs in Tli5 through sequence and structural analysis. One is a conserved loop‐β‐hairpin motif that exists in the Tle5 immunity homologs, the other is a long and sharp α‐α motif that directly interacts with Tle5 according to SAXS data. We also distinguished the structural features of Tle5 and Tle5B family immunity proteins. Together, our work provided insights into a novel inhibition mechanism that may enhance our understanding of phospholipase D family proteins.     

Type VI secretion system MIX‐effectors carry both antibacterial and anti‐eukaryotic activities

Most type VI secretion systems (T6SSs) described to date are protein delivery apparatuses that mediate bactericidal activities. Several T6SSs were also reported to mediate virulence activities, although only few anti‐eukaryotic effectors have been described. Here, we identify three T6SSs in the marine bacterium Vibrio proteolyticus and show that T6SS1 mediates bactericidal activities under warm marine‐like conditions. Using comparative proteomics, we find nine potential T6SS1 effectors, five of which belong to the polymorphic MIX‐effector class. Remarkably, in addition to six predicted bactericidal effectors, the T6SS1 secretome includes three putative anti‐eukaryotic effectors. One of these is a MIX‐effector containing a cytotoxic necrotizing factor 1 domain. We demonstrate that T6SS1 can use this MIX‐effector to target phagocytic cells, resulting in morphological changes and actin cytoskeleton rearrangements. In conclusion, the V. proteolyticus T6SS1, a system homologous to one found in pathogenic vibrios, uses a suite of polymorphic effectors that target both bacteria and eukaryotic neighbors.     

A phospholipase A1 antibacterial Type VI secretion effector interacts directly with the C‐terminal domain of the VgrG spike protein for delivery

The Type VI secretion system (T6SS) is a multiprotein machine that delivers protein effectors in both prokaryotic and eukaryotic cells, allowing interbacterial competition and virulence. The mechanism of action of the T6SS requires the contraction of a sheath‐like structure that propels a needle towards target cells, allowing the delivery of protein effectors. Here, we provide evidence that the entero‐aggregative Escherichia coli Sci‐1 T6SS is required to eliminate competitor bacteria. We further identify Tle1, a toxin effector encoded by this cluster and showed that Tle1 possesses phospholipase A₁ and A₂ activities required for the interbacterial competition. Self‐protection of the attacker cell is secured by an outer membrane lipoprotein, Tli1, which binds Tle1 in a 1:1 stoichiometric ratio with nanomolar affinity, and inhibits its phospholipase activity. Tle1 is delivered into the periplasm of the prey cells using the VgrG1 needle spike protein as carrier. Further analyses demonstrate that the C‐terminal extension domain of VgrG1, including a transthyretin‐like domain, is responsible for the interaction with Tle1 and its subsequent delivery into target cells. Based on these results, we propose an additional mechanism of transport of T6SS effectors in which cognate effectors are selected by specific motifs located at the C‐terminus of VgrG proteins.     

Chimeric adaptor proteins translocate diverse type VI secretion system effectors in Vibrio cholerae

Vibrio cholerae is a diverse species of Gram-negative bacteria, commonly found in the aquatic environment and the causative agent of the potentially deadly disease cholera. These bacteria employ a type VI secretion system (T6SS) when they encounter prokaryotic and eukaryotic competitors. This contractile puncturing device translocates a set of effector proteins into neighboring cells. Translocated effectors are toxic unless the targeted cell produces immunity proteins that bind and deactivate incoming effectors. Comparison of multiple V. cholerae strains indicates that effectors are encoded in T6SS effector modules on mobile genetic elements. We identified a diverse group of chimeric T6SS adaptor proteins required for the translocation of diverse effectors encoded in modules. An example for a T6SS effector that requires T6SS adaptor protein 1 (Tap-1) is TseL found in pandemic V. cholerae O1 serogroup strains and other clinical isolates. We propose a model in which Tap-1 is required for loading TseL onto the secretion apparatus. After T6SS-mediated TseL export is completed, Tap-1 is retained in the bacterial cell to load other T6SS machines.     

Structural basis for recognition of the type VI spike protein VgrG3 by a cognate immunity protein

The bacterial type VI secretion system (T6SS) is used by donor cells to inject toxic effectors into receptor cells. The donor cells produce the corresponding immunity proteins to protect themselves against the effector proteins, thereby preventing their self-intoxication. Recently, the C-terminal domain of VgrG3 was identified as a T6SS effector. Information on the molecular mechanism of VgrG3 and its immunity protein TsaB has been lacking. Here, we determined the crystal structures of native TsaB and the VgrG3C–TsaB complex. VgrG3C adopts a canonical phage-T4-lysozyme-like fold. TsaB interacts with VgrG3C through molecular mimicry, and inserts into the VgrG3C pocket.     

New secreted toxins and immunity proteins encoded within the Type VI secretion system gene cluster of Serratia marcescens

Protein secretion systems are critical to bacterial virulence and interactions with other organisms. The Type VI secretion system (T6SS) is found in many bacterial species and is used to target either eukaryotic cells or competitor bacteria. However, T6SS‐secreted proteins have proven surprisingly elusive. Here, we identified two secreted substrates of the antibacterial T6SS from the opportunistic human pathogen, Serratia marcescens. Ssp1 and Ssp2, both encoded within the T6SS gene cluster, were confirmed as antibacterial toxins delivered by the T6SS. Four related proteins encoded around the Ssp proteins (‘Rap’ proteins) included two specifically conferring self‐resistance (‘immunity’) against T6SS‐dependent Ssp1 or Ssp2 toxicity. Biochemical characterization revealed specific, tight binding between cognate Ssp–Rap pairs, forming complexes of 2:2 stoichiometry. The atomic structures of two Rap proteins were solved, revealing a novel helical fold, dependent on a structural disulphide bond, a structural feature consistent with their functional localization. Homologues of the Serratia Ssp and Rap proteins are found encoded together within other T6SS gene clusters, thus they represent founder members of new families of T6SS‐secreted and cognate immunity proteins. We suggest that Ssp proteins are the original substrates of the S. marcescens T6SS, before horizontal acquisition of other T6SS‐secreted toxins. Molecular insight has been provided into how pathogens utilize antibacterial T6SSs to overcome competitors and succeed in polymicrobial niches.     

Structural Insights into the Pseudomonas aeruginosa Type VI Virulence Effector Tse1 Bacteriolysis and Self-protection Mechanisms

Recently, it was identified that Pseudomonas aeruginosa competes with rival cells to gain a growth advantage using a novel mechanism that includes two interrelated processes as follows: employing type VI secretion system (T6SS) virulence effectors to lyse other bacteria, and at the same time producing specialized immunity proteins to inactivate their cognate effectors for self-protection against mutual toxicity. To explore the structural basis of these processes in the context of functional performance, the crystal structures of the T6SS virulence effector Tse1 and its complex with the corresponding immunity protein Tsi1 were determined, which, in association with mutagenesis and Biacore analyses, provided a molecular platform to resolve the relevant structural questions. The results indicated that Tse1 features a papain-like structure and conserved catalytic site with distinct substrate-binding sites to hydrolyze its murein peptide substrate. The immunity protein Tsi1 interacts with Tse1 via a unique interactive recognition mode to shield Tse1 from its physiological substrate. These findings reveal both the structural mechanisms for bacteriolysis and the self-protection against the T6SS effector Tse1. These mechanisms are significant not only by contributing to a novel understanding of niche competition among bacteria but also in providing a structural basis for antibacterial agent design and the development of new strategies to fight P. aeruginosa.     

Genetically distinct pathways guide effector export through the type VI secretion system

Bacterial secretion systems often employ molecular chaperones to recognize and facilitate export of their substrates. Recent work demonstrated that a secreted component of the type VI secretion system (T6SS), haemolysin co‐regulated protein (Hcp), binds directly to effectors, enhancing their stability in the bacterial cytoplasm. Herein, we describe a quantitative cellular proteomics screen for T6S substrates that exploits this chaperone‐like quality of Hcp. Application of this approach to the Hcp secretion island I‐encoded T6SS (H1‐T6SS) of Pseudomonas aeruginosa led to the identification of a novel effector protein, termed Tse4 (t ype VI s ecretion e xported 4), subsequently shown to act as a potent intra‐specific H1‐T6SS‐delivered antibacterial toxin. Interestingly, our screen failed to identify two predicted H1‐T6SS effectors, Tse5 and Tse6, which differ from Hcp‐stabilized substrates by the presence of toxin‐associated PAAR‐repeat motifs and genetic linkage to members of the valine‐glycine repeat protein G (vgrG) genes. Genetic studies further distinguished these two groups of effectors: Hcp‐stabilized effectors were found to display redundancy in interbacterial competition with respect to the requirement for the two H1‐T6SS‐exported VgrG proteins, whereas Tse5 and Tse6 delivery strictly required a cognate VgrG. Together, we propose that interaction with either VgrG or Hcp defines distinct pathways for T6S effector export.     

Pathogenic Streptococcus strains employ novel escape strategy to inhibit bacteriostatic effect mediated by mammalian peptidoglycan recognition protein

Pathogenic streptococcal species are responsible for some of the most lethal and prevalent animal and human infections. Previous reports have identified a candidate pathogenicity island (PAI) in two highly virulent clinical isolates of Streptococcus suis type 2, a causative agent of high‐mortality streptococcal toxic shock syndrome. This PAI contains a type‐IVC secretion system C subgroup (type‐IVC secretion system) that is involved in the secretion of unknown pathogenic effectors that are responsible for streptococcal toxic shock syndrome caused by highly virulent strains of S. suis. Both virulence protein B4 and virulence protein D4 were demonstrated to be key components of this type‐IVC secretion system. In this study, we identify a new PAI family across 3 streptococcal species; Streptococcus genomic island contains type‐IV secretion system, which contains a genomic island type‐IVC secretion system and a novel PPIase molecule, SP1. SP1 is shown to interact with a component of innate immunity, peptidoglycan recognition protein (PGLYRP‐1) and to perturb the PGLYRP‐1‐mediated bacteriostatic effect by interacting with protein PGLYRP‐1. Our study elucidates a novel mechanism by which bacteria escape by components of the innate immune system by secretion of the SP1 protein in pathogenic Streptococci, which then interacts with PGLYRP‐1 from the host. Our results provide potential targets for the development of new antimicrobial drugs against bacteria with resistance to innate host immunity.     

The Toxoplasma gondii rhoptry protein ROP18 is an Irga6‐specific kinase and regulated by the dense granule protein GRA7

In mice, avirulent strains (e.g. types II and III) of the protozoan parasite Toxoplasma gondii are restricted by the immunity‐related GTPase (IRG) resistance system. Loading of IRG proteins onto the parasitophorous vacuolar membrane (PVM) is required for vacuolar rupture resulting in parasite clearance. In virulent strain (e.g. type I) infections, polymorphic effector proteins ROP5 and ROP18 cooperate to phosphorylate and thereby inactivate mouse IRG proteins to preserve PVM integrity. In this study, we confirmed the dense granule protein GRA7 as an additional component of the ROP5/ROP18 kinase complex and identified GRA7 association with the PVM by direct binding to ROP5. The absence of GRA7 results in reduced phosphorylation of Irga6 correlated with increased vacuolar IRG protein amounts and attenuated virulence. Earlier work identified additional IRG proteins as targets of T. gondii ROP18 kinase. We show that the only specific target of ROP18 among IRG proteins is in fact Irga6. Similarly, we demonstrate that GRA7 is strictly an Irga6‐specific virulence effector. This identifies T. gondii GRA7 as a regulator for ROP18‐specific inactivation of Irga6. The structural diversity of the IRG proteins implies that certain family members constitute additional specific targets for other yet unknown T. gondii virulence effectors.     

Regulation of Vibrio parahaemolyticus T3SS2 gene expression and function of T3SS2 effectors that modulate actin cytoskeleton

Vibrio parahaemolyticus is a leading causative agent of seafood‐borne gastroenteritis worldwide. Most clinical isolates from patients with diarrhoea possess two sets of genes for the type III secretion system (T3SS) on each chromosome (T3SS1 and T3SS2). T3SS is a protein secretion system that delivers effector proteins directly into eukaryotic cells. The injected effectors modify the normal cell functions by altering or disrupting the normal cell signalling pathways. Of the two sets of T3SS genes present in V. parahaemolyticus, T3SS2 is essential for enterotoxicity in several animal models. Recent studies have elucidated the biological activities of several T3SS2 effectors and their roles in virulence. This review focuses on the regulation of T3SS2 gene expression and T3SS2 effectors that specifically target the actin cytoskeleton.     

Pseudomonas fluorescens MFE01 delivers a putative type VI secretion amidase that confers biocontrol against the soft-rot pathogen Pectobacterium atrosepticum

The type VI secretion system (T6SS) is a contractile nanomachine widespread in Gram-negative bacteria. The T6SS injects effectors into target cells including eukaryotic hosts and competitor microbial cells and thus participates in pathogenesis and intermicrobial competition. Pseudomonas fluorescens MFE01 possesses a single T6SS gene cluster that confers biocontrol properties by protecting potato tubers against the phytopathogen Pectobacterium atrosepticum (Pca). Here, we demonstrate that a functional T6SS is essential to protect potato tuber by reducing the pectobacteria population. Fluorescence microscopy experiments showed that MFE01 displays an aggressive behaviour with an offensive T6SS characterized by continuous and intense T6SS firing activity. Interestingly, we observed that T6SS firing is correlated with rounding of Pectobacterium cells, suggesting delivery of a potent cell wall targeting effector. Mutagenesis coupled with functional assays then revealed that a putative T6SS secreted amidase, Tae3^Pf, is mainly responsible for MFE01 toxicity towards Pca. Further studies finally demonstrated that Tae3^Pf is toxic when produced in the periplasm, and that its toxicity is counteracted by the Tai3^Pf inner membrane immunity protein.     

Modulation of innate immune responses by Yersinia type III secretion system translocators and effectors

The innate immune system of mammals responds to microbial infection through detection of conserved molecular determinants called ‘pathogen‐associated molecular patterns’ (PAMPs). Pathogens use virulence factors to counteract PAMP‐directed responses. The innate immune system can in turn recognize signals generated by virulence factors, allowing for a heightened response to dangerous pathogens. Many Gram‐negative bacterial pathogens encode type III secretion systems (T3SSs) that translocate effector proteins, subvert PAMP‐directed responses and are critical for infection. A plasmid‐encoded T3SS in the human‐pathogenic Yersinia species translocates seven effectors into infected host cells. Delivery of effectors by the T3SS requires plasma membrane insertion of two translocators, which are thought to form a channel called a translocon. Studies of the Yersinia T3SS have provided key advances in our understanding of how innate immune responses are generated by perturbations in plasma membrane and other signals that result from translocon insertion. Additionally, studies in this system revealed that effectors function to inhibit innateimmune responses resulting from insertion of translocons into plasma membrane. Here, we review these advances with the goal of providing insight into how a T3SS can activate and inhibit innate immune responses, allowing a virulent pathogen to bypass host defences.     

Crystal Structure of Hcp from Acinetobacter baumannii: A Component of the Type VI Secretion System

The type VI secretion system (T6SS) is a bacterial macromolecular machine widely distributed in Gram-negative bacteria, which transports effector proteins into eukaryotic host cells or other bacteria. Membrane complexes and a central tubular structure, which resembles the tail of contractile bacteriophages, compose the T6SS. One of the proteins forming this tube is the hemolysin co-regulated protein (Hcp), which acts as virulence factor, as transporter of effectors and as a chaperone. In this study, we present the structure of Hcp from Acinetobacter baumannii, together with functional and oligomerization studies. The structure of this protein exhibits a tight β barrel formed by two β sheets and flanked at one side by a short α-helix. Six Hcp molecules associate to form a donut-shaped hexamer, as observed in both the crystal structure and solution. These results emphasize the importance of this oligomerization state in this family of proteins, despite the low similarity of sequence among them. The structure presented in this study is the first one for a protein forming part of a functional T6SS from A. baumannii. These results will help us to understand the mechanism and function of this secretion system in this opportunistic nosocomial pathogen.     

Identification of a regulatory cascade controlling Type III Secretion System 3 gene expression in Burkholderia pseudomallei

A major and critical virulence determinant of many Gram‐negative bacterial pathogens is the Type III Secretion Systems (T3SS). T3SS3 in Burkholderia pseudomallei is critical for bacterial virulence in mammalian infection models but its regulation is unknown. B. pseudomallei is the causative agent of melioidosis, a potentially fatal disease endemic in Southeast Asia and northern Australia. While screening for bacterial transposon mutants with a defective T3SS function, we discovered a TetR family regulator (bspR) responsible for the control of T3SS3 gene expression. The bspR mutant exhibited significant virulence attenuation in mice. BspR acts through BprP, a novel transmembrane regulator located adjacent to the currently delineated T3SS3 region. BprP in turn regulates the expression of structural and secretion components of T3SS3 and the AraC family regulator bsaN. BsaN and BicA likely form a complex to regulate the expression of T3SS3 effectors and other regulators which in turn affect the expression of Type VI Secretion Systems (T6SS). The complete delineation of the bspR initiated T3SS regulatory cascade not only contributes to the understanding of B. pseudomallei pathogenesis but also provides an important example of how bacterial pathogens could co‐opt and integrate various regulatory motifs to form a new regulatory network adapted for its own purposes.     

空肠弯曲菌Ⅵ型分泌系统研究进展

Ⅵ型分泌系统(TypeⅥSecretion 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楚。本文就空肠弯曲菌T6SS结构组成、分布特征及效应功能等方面的研究进展进行综述,以期为进一步解析空肠弯曲菌毒力因子的调控机制提供新思路。     

Crystal structure of the C‐terminal domain of the Salmonella type III secretion system export apparatus protein InvA

InvA is a prominent inner‐membrane component of the Salmonella type III secretion system (T3SS) apparatus, which is responsible for regulating virulence protein export in pathogenic bacteria. InvA is made up of an N‐terminal integral membrane domain and a C‐terminal cytoplasmic domain that is proposed to form part of a docking platform for the soluble export apparatus proteins notably the T3SS ATPase InvC. Here, we report the novel crystal structure of the C‐terminal domain of Salmonella InvA which shows a compact structure composed of four subdomains. The overall structure is unique although the first and second subdomains exhibit structural similarity to the peripheral stalk of the A/V‐type ATPase and a ring building motif found in other T3SS proteins respectively.     

The SciZ protein anchors the enteroaggregative Escherichia coli Type VI secretion system to the cell wall

Type VI secretion systems (T6SS) are multi‐component machines encoded within the genomes of most Gram‐negative bacteria that associate with plant, animal and/or human cells, and therefore are considered as potential virulence factors. We recently launched a study on the Sci‐1 T6SS of enteroaggregative Escherichia coli (EAEC). The Sci‐1 T6SS is composed of all or a subset of the 21 gene products encoded within the cluster, 13 of which are shared by all T6SS identified so far. In the present work, we focussed our attention on the SciZ protein. We first showed that SciZ is required for the release of the Hcp protein in the culture supernatant and for efficient biofilm formation, demonstrating that SciZ is necessary for EAEC T6SS function. Indeed, SciZ forms a complex with SciP, SciS and SciN, three core components of the transport apparatus. Fractionation and topology studies showed that SciZ is a polytopic inner membrane protein with three trans‐membrane segments. Computer analyses identified a motif shared by peptidoglycan binding proteins of the OmpA family in the SciZ periplasmic domain. Using in vivo and in vitro binding assays, we showed that this motif anchors the SciZ protein to the cell wall and is required for T6SS function.