IcmF family protein TssM exhibits ATPase activity and energizes type VI secretion

The type VI secretion system (T6SS) with diversified functions is widely distributed in pathogenic Proteobacteria. The IcmF (intracellular multiplication protein F) family protein TssM is a conserved T6SS inner membrane protein. Despite the conservation of its Walker A nucleotide-binding motif, the NTPase activity of TssM and its role in T6SS remain obscure. In this study, we characterized TssM in the plant pathogen Agrobacterium tumefaciens and provided the first biochemical evidence for TssM exhibiting ATPase activity to power the secretion of the T6SS hallmark protein, hemolysin-coregulated protein (Hcp). Amino acid substitutions in the Walker A motif of TssM caused reduced ATP binding and hydrolysis activity. Importantly, we discovered the Walker B motif of TssM and demonstrated that it is critical for ATP hydrolysis activity. Protein-protein interaction studies and protease susceptibility assays indicated that TssM undergoes an ATP binding-induced conformational change and that subsequent ATP hydrolysis is crucial for recruiting Hcp to interact with the periplasmic domain of the TssM-interacting protein TssL (an IcmH/DotU family protein) into a ternary complex and mediating Hcp secretion. Our findings strongly argue that TssM functions as a T6SS energizer to recruit Hcp into the TssM-TssL inner membrane complex prior to Hcp secretion across the outer membrane.     

Type VI secretion modulates quorum sensing and stress response in Vibrio anguillarum

Type VI protein secretion systems (T6SS) are essential for virulence of several Gram‐negative bacteria. In this study, we identified a T6SS in Vibrio anguillarum, a marine bacterium that causes a hemorrhagic septicemia in fish. A partial operon vtsA‐H (v ibrio t ype s ix secretion) was sequenced and shown to encode eight proteins. VtsE‐H are signature proteins found in other T6SSs, while VtsA‐D are not associated with T6SS studied so far. In‐frame deletions were made in each gene. Secretion of a haemolysin‐co‐regulated‐like protein (Hcp), a protein secreted by all studied T6SSs, was decreased in VtsE‐H. Unexpectedly, VtsA, VtsC and VtsD activated while VtsB and VtsE‐H repressed hcp expression. The T6SS proteins also regulated expression of two extracellular proteases, EmpA and PrtV, but inversely to Hcp expression. This regulation was indirect as T6S positively regulated expression of the stress‐response regulator RpoS and the quorum‐sensing regulator VanT, which positively regulate protease expression. Moreover, VtsA‐H proteins were not needed for virulence but did play a role in various stress responses. Thus, these data characterize a new role for T6S in the ecology of bacteria and we hypothesize this role to be a signal sensing mechanism that modulates the expression of regulators of the general stress response.     

Type VI secretion system in Pseudomonas aeruginosa: secretion and multimerization of VgrG proteins

Pseudomonas aeruginosa is a Gram-negative bacterium causing chronic infections in cystic fibrosis patients. Such infections are associated with an active type VI secretion system (T6SS), which consists of about 15 conserved components, including the AAA+ ATPase, ClpV. The T6SS secretes two categories of proteins, VgrG and Hcp. Hcp is structurally similar to a phage tail tube component, whereas VgrG proteins show similarity to the puncturing device at the tip of the phage tube. In P. aeruginosa, three T6SSs are known. The expression of H1-T6SS genes is controlled by the RetS sensor. Here, 10 vgrG genes were identified in the PAO1 genome, among which three are co-regulated with H1-T6SS, namely vgrG1a/b/c. Whereas VgrG1a and VgrG1c were secreted in a ClpV1-dependent manner, secretion of VgrG1b was ClpV1-independent. We show that VgrG1a and VgrG1c form multimers, which confirmed the VgrG model predicting trimers similar to the tail spike. We demonstrate that Hcp1 secretion requires either VgrG1a or VgrG1c, which may act independently to puncture the bacterial envelope and give Hcp1 access to the surface. VgrG1b is not required for Hcp1 secretion. Thus, VgrG1b does not require H1-T6SS for secretion nor does H1-T6SS require VgrG1b for its function. Finally, we show that VgrG proteins are required for secretion of a genuine H1-T6SS substrate, Tse3. Our results demonstrate that VgrG proteins are not only secreted components but are essential for secretion of other T6SS substrates. Overall, we emphasize variability in behavior of three P. aeruginosa VgrGs, suggesting that, although very similar, distinct VgrGs achieve specific functions.     

Remodelling of VipA/VipB tubules by ClpV‐mediated threading is crucial for type VI protein secretion

The recently identified type VI secretion systems (T6SS) have a crucial function in the virulence of various proteobacteria, including the human pathogen Vibrio cholerae. T6SS are encoded by a conserved gene cluster comprising approximately 15 open reading frames, mediating the appearance of Hcp and VgrG proteins in cell culture supernatants. Here, we analysed the function of the V. cholerae T6SS member ClpV, a specialized AAA+ protein. ClpV is crucial for a functional T6SS and interacts through its N‐terminal domain with the VipA/VipB complex that is composed of two conserved and essential members of T6SS. Transferring ClpV substrate specificity to a distinct AAA+ protein involved in proteolysis caused degradation of VipA but not Hcp or VgrG2, suggesting that VipA rather than Hcp/VgrG2 functions as a primary ClpV substrate. Strikingly, VipA/VipB form tubular, cogwheel‐like structures that are converted by a threading activity of ClpV into small complexes. ClpV‐mediated remodelling of VipA/VipB tubules represents a crucial step in T6S, illuminating an unexpected role of an ATPase component in protein secretion.     

Hcp2, a Secreted Protein of the Phytopathogen Pseudomonas syringae pv. Tomato DC3000, Is Required for Fitness for Competition against Bacteria and Yeasts

When analyzing the secretome of the plant pathogen Pseudomonas syringae pv. tomato DC3000, we identified hemolysin-coregulated protein (Hcp) as one of the secreted proteins. Hcp is assumed to be an extracellular component of the type VI secretion system (T6SS). Two copies of hcp genes are present in the P. syringae pv. tomato DC3000 genome, hcp1 (PSPTO_2539) and hcp2 (PSPTO_5435). We studied the expression patterns of the hcp genes and tested the fitness of hcp knockout mutants in host plant colonization and in intermicrobial competition. We found that the hcp2 gene is expressed most actively at the stationary growth phase and that the Hcp2 protein is secreted via the T6SS and appears in the culture medium as covalently linked dimers. Expression of hcp2 is not induced in planta and does not contribute to virulence in or colonization of tomato or Arabidopsis plants. Instead, hcp2 is required for survival in competition with enterobacteria and yeasts, and its function is associated with the suppression of the growth of these competitors. This is the first report on bacterial T6SS-associated genes functioning in competition with yeast. Our results suggest that the T6SS of P. syringae may play an important role in bacterial fitness, allowing this plant pathogen to survive under conditions where it has to compete with other microorganisms for resources.     

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.     

Quorum sensing and alternative sigma factor RpoN regulate type VI secretion system I (T6SSVA1) in fish pathogen <Emphasis Type="Italic">Vibrio alginolyticus</Emphasis>

Type VI secretion system (T6SS) is a highly conserved bacterial protein secretion system and is precisely regulated in Gram-negative pathogens. In Vibrio alginolyticus, an important fish pathogen, two complete T6SS gene clusters (T6SSVA1 and T6SSVA2) were identified. In this study, expression of a hemolysin coregulated protein (Hcp1), which is one of the hallmarks of T6SS, was found to be strictly regulated in this bacterium. We showed that the expression of Hcp1 was growth phase-dependent and the production of Hcp1 reached a maximum in the exponential phase. The expression of Hcp1 was positively and negatively regulated by quorum sensing regulators LuxO and LuxR, respectively. In addition, we observed that Hcp1 expression required the alternative sigma factor RpoN and the enhancer-binding protein VasH, which is encoded in T6SSVA1 gene cluster. Moreover, LuxR, RpoN, and VasH could positively regulate the expression of other T6SS genes. Taken together, we demonstrated that the expression of T6SS in V. alginolyticus was under the regulation of quorum sensing and alternative sigma factor.     

Genetic analysis of anti-amoebae and anti-bacterial activities of the type VI secretion system in Vibrio cholerae

A type VI secretion system (T6SS) was recently shown to be required for full virulence of Vibrio cholerae O37 serogroup strain V52. In this study, we systematically mutagenized each individual gene in T6SS locus and characterized their functions based on expression and secretion of the hemolysin co-regulated protein (Hcp), virulence towards amoebae of Dictyostelium discoideum and killing of Escherichia coli bacterial cells. We group the 17 proteins characterized in the T6SS locus into four categories: twelve (VipA, VipB, VCA0109-VCA0115, ClpV, VCA0119, and VasK) are essential for Hcp secretion and bacterial virulence, and thus likely function as structural components of the apparatus; two (VasH and VCA0122) are regulators that are required for T6SS gene expression and virulence; another two, VCA0121 and valine-glycine repeat protein G 3 (VgrG-3), are not essential for Hcp expression, secretion or bacterial virulence, and their functions are unknown; the last group is represented by VCA0118, which is not required for Hcp expression or secretion but still plays a role in both amoebae and bacterial killing and may therefore be an effector protein. We also showed that the clpV gene product is required for Dictyostelium virulence but is less important for killing E. coli. In addition, one vgrG gene (vgrG-2) outside of the T6SS gene cluster was required for bacterial killing but another (vgrG-1) was not. However, a bacterial killing defect was observed when vgrG-1 and vgrG-3 were both deleted. Several genes encoded in the same putative operon as vgrG-1 and vgrG-2 also contribute to virulence toward Dictyostelium but have a smaller effect on bacterial killing. Our results provide new insights into the functional requirements of V. cholerae's T6SS in the context of secretion as well as killing of bacterial and eukaryotic phagocytic cells.     

Systematic Dissection of the Agrobacterium Type VI Secretion System Reveals Machinery and Secreted Components for Subcomplex Formation

The type VI secretion system (T6SS) is widely distributed in pathogenic Proteobacteria. Sequence and structural analysis of T6SS reveals a resemblance to the T4 bacteriophage tail, in which an outer sheath structure contracts an internal tube for injecting nucleic acid into bacterial cells. However, the molecular details of how this phage tail-like T6SS structure is assembled in vivo and executed for exoprotein or effector secretion remain largely unknown. Here, we used a systematic approach to identify T6SS machinery and secreted components and investigate the interaction among the putative sheath and tube components of Agrobacterium tumefaciens. We showed that 14 T6SS components play essential roles in the secretion of the T6SS hallmark exoprotein Hcp. In addition, we discovered a novel T6SS exoprotein, Atu4347, that is dispensable for Hcp secretion. Interestingly, Atu4347 and the putative tube components, Hcp and VgrG, are mainly localized in the cytoplasm but also detected on the bacterial surface. Atu4342 (TssB) and Atu4341 (TssC₄₁) interact with and stabilize each other, which suggests that they are functional orthologs of the sheath components TssB (VipA) and TssC (VipB), respectively. Importantly, TssB interacts directly with the three exoproteins (Hcp, VgrG, and Atu4347), in which Hcp also interacts directly with VgrG-1 on co-purification from Escherichia coli. Further co-immunoprecipitation and pulldown assays revealed these subcomplex(es) in A. tumefaciens and thereby support T6SS functioning as a contractile phage tail-like structure.     

Structural basis for the secretion of EvpC: a key type VI secretion system protein from Edwardsiella tarda

The recently identified type VI secretion system (T6SS) is implicated in the virulence of many Gram-negative bacteria. Edwardsiella tarda is an important cause of hemorrhagic septicemia in fish and also gastro- and extra-intestinal infections in humans. The E . tarda virulent protein (EVP) gene cluster encodes a conserved T6SS which contains 16 open reading frames. EvpC is one of the three major EVP secreted proteins and shares high sequence similarity with Hcp1, a key T6SS virulence factor from Pseudomonas aeruginosa. EvpC contributes to the virulence of E. tarda by playing an essential role in functional T6SS. Here, we report the crystal structure of EvpC from E. tarda PPD130/91 at a 2.8 Å resolution, along with functional studies of the protein. EvpC has a β-barrel domain with extended loops. The β-barrel consists of 11 anti-parallel β-strands with an α-helix located on one side. In solution, EvpC exists as a dimer at low concentration and as a hexamer at higher concentration. In the crystal, the symmetry related EvpC molecules form hexameric rings which stack together to form a tube similar to Hcp1. Structure based mutagenesis revealed that N-terminal negatively charged residues, Asp4, Glu15 and Glu26, and C-terminal positively charged residues, Lys161, Lys162 and Lys163, played crucial roles in the secretion of EvpC. Moreover, the localization study indicates the presence of wild type EvpC in cytoplasm, periplasm and secreted fractions, whereas the N-terminal and C-terminal mutants were found mostly in the periplasmic region and was completely absent in the secreted fraction. Results reported here provide insight into the structure, assembly and function of EvpC. Further, these findings can be extended to other EvpC homologs for understanding the mechanism of T6SS and targeting T6SS mediated virulence in Gram-negative pathogens.     

Dissection of a type VI secretion system in Edwardsiella tarda

Bacterial pathogens use different protein secretion systems to deliver virulence factors. Recently, a novel secretion system was discovered in several Gram-negative bacterial pathogens, and was designated as the type VI secretion system (T6SS). In Edwardsiella tarda, a partial E. tardavirulent protein (EVP) gene cluster was implicated in protein secretion. Here, we identified the entire EVP cluster as a T6SS and two additional secreted proteins (EvpI, a homologue of VgrG, and EvpP) were found. We systematically mutagenized all the 16 EVP genes and found that the secretion of EvpP was dependent on 13 EVP proteins including EvpC (a homologue of Hcp) and EvpI but not EvpD and EvpJ. All EVP mutants except DeltaevpD were attenuated in blue gourami fish. The 16 EVP proteins can be grouped according to their functions and cellular locations. The first group comprises 11 non-secreted and possibly intracellular apparatus proteins. Among them, EvpO, a putative ATPase which contained a Walker A motif, showed possible interactions with three EVP proteins (EvpA, EvpL and EvpN). The second group includes three secreted proteins (EvpC, EvpI and EvpP). The secretion of EvpC and EvpI is mutually dependent, and they are required for the secretion of EvpP. The interaction between EvpC and EvpP was demonstrated. Lastly, two proteins (EvpD and EvpJ) are not required for the T6SS-dependent secretion.     

Threonine phosphorylation post-translationally regulates protein secretion in Pseudomonas aeruginosa

Secreted proteins are crucial to the arsenal of bacterial pathogens. Although optimal activity of these proteins is likely to require precise regulation of release, the signalling events that trigger secretion are poorly understood. Here, we identify a threonine phosphorylation event that post-translationally regulates the Hcp secretion island-I-encoded type VI secretion system of Pseudomonas aeruginosa (H-T6SS). We show that a serine-threonine kinase, PpkA, is required for assembly of the H-T6SS and for secretion of Hcp1. PpkA activity is antagonized by PppA, a Ser-Thr phosphatase. These proteins exhibit reciprocal effects on the H-T6SS by acting on an FHA domain-containing protein, termed Fha1. Colocalization experiments with the T6S AAA+ family protein, ClpV1, indicate that Fha1 is a core scaffolding protein of the H-T6SS. Mutations affecting this H-T6S regulatory pathway provide a molecular explanation for the variation in Hcp1 secretion among clinical P. aeruginosa isolates. This mechanism of triggering secretion may be general, as many T6SSs contain orthologues of these proteins. Post-translational regulation of protein secretion by Thr phosphorylation is unprecedented in bacteria, and is likely to reflect the requirement for T6S to respond rapidly and reversibly to its environment.     

BcsKC Is an Essential Protein for the Type VI Secretion System Activity in Burkholderia cenocepacia That Forms an Outer Membrane Complex with BcsLB

The type VI secretion system (T6SS) contributes to the virulence of Burkholderia cenocepacia, an opportunistic pathogen causing serious chronic infections in patients with cystic fibrosis. BcsK_C is a highly conserved protein among the T6SSs in Gram-negative bacteria. Here, we show that BcsK_C is required for Hcp secretion and cytoskeletal redistribution in macrophages upon bacterial infection. These two phenotypes are associated with a functional T6SS in B. cenocepacia. Experiments employing a bacterial two-hybrid system and pulldown assays demonstrated that BcsK_C interacts with BcsL_B, another conserved T6SS component. Internal deletions within BcsK_C revealed that its N-terminal domain is necessary and sufficient for interaction with BcsL_B. Fractionation experiments showed that BcsK_C can be in the cytosol or tightly associated with the outer membrane and that BcsK_C and BcsL_B form a high molecular weight complex anchored to the outer membrane that requires BcsF_H (a ClpV homolog) to be assembled. Together, our data show that BcsK_C/BcsL_B interaction is essential for the T6SS activity in B. cenocepacia.     

Fha Interaction with Phosphothreonine of TssL Activates Type VI Secretion in Agrobacterium tumefaciens

The type VI secretion system (T6SS) is a widespread protein secretion system found in many Gram-negative bacteria. T6SSs are highly regulated by various regulatory systems at multiple levels, including post-translational regulation via threonine (Thr) phosphorylation. The Ser/Thr protein kinase PpkA is responsible for this Thr phosphorylation regulation, and the forkhead-associated (FHA) domain-containing Fha-family protein is the sole T6SS phosphorylation substrate identified to date. Here we discovered that TssL, the T6SS inner-membrane core component, is phosphorylated and the phosphorylated TssL (p-TssL) activates type VI subassembly and secretion in a plant pathogenic bacterium, Agrobacterium tumefaciens. Combining genetic and biochemical approaches, we demonstrate that TssL is phosphorylated at Thr 14 in a PpkA-dependent manner. Further analysis revealed that the PpkA kinase activity is responsible for the Thr 14 phosphorylation, which is critical for the secretion of the T6SS hallmark protein Hcp and the putative toxin effector Atu4347. TssL phosphorylation is not required for the formation of the TssM-TssL inner-membrane complex but is critical for TssM conformational change and binding to Hcp and Atu4347. Importantly, Fha specifically interacts with phosphothreonine of TssL via its pThr-binding motif in vivo and in vitro and this interaction is crucial for TssL interaction with Hcp and Atu4347 and activation of type VI secretion. In contrast, pThr-binding ability of Fha is dispensable for TssM structural transition. In conclusion, we discover a novel Thr phosphorylation event, in which PpkA phosphorylates TssL to activate type VI secretion via its direct binding to Fha in A. tumefaciens. A model depicting an ordered TssL phosphorylation-induced T6SS assembly pathway is proposed.     

Pyramiding unmarked deletions in Ralstonia solanacearum shows that secreted proteins in addition to plant cell-wall-degrading enzymes contribute to virulence

Ralstonia solanacearum, like many phytopathogenic bacteria, makes multiple extracellular plant cell-wall-degrading enzymes (CWDE), some of which contribute to its ability to cause wilt disease. CWDE and many other proteins are secreted to the milieu via the highly conserved type II protein secretion system (T2SS). R. solanacearum with a defective T2SS is weakly virulent, but it is not known whether this is due to absence of all the CWDE or the loss of other secreted proteins that contribute to disease. These alternatives were investigated by creating mutants of wild-type strain GMI1000 lacking either the T2SS or up to six CWDE and comparing them for virulence on tomato plants. To create unmarked deletions, genomic regions flanking the target gene were polymerase chain reaction (PCR)-amplified, were fused using splice overlap extension PCR, were cloned into a suicide plasmid harboring the sacB counter-selectable marker, and then, were site-specifically introduced into the genome. Various combinations of five deletions (delta pehA, delta pehB, delta B, PehC, and Pme) was not statistically different from GMI1000, but all the mutants lacking one or both cellulolytic enzymes (Egl or CbhA) wilted plants significantly more slowly than did the wild type. The GMI-6 mutant that lacks all six CWDE was more virulent than the mutant lacking only its two cellulolytic enzymes, and both were significantly more virulent than the T2SS mutant (GMI-D). Very similar results were observed in wounded-petiole inoculation assays, so GMI-6 and GMI-D appear to be less capable of colonizing tomato tissues after invasion. Because the T2SS mutant was much less virulent than the sixfold CWDE mutant, we conclude that other secreted proteins contribute substantially to the ability of R. solanacearum GMI1000 to systemically colonize tomato plants.     

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.