Brian T. Ho

A View to a Kill: The Bacterial Type VI Secretion System

The bacterial type VI secretion system (T6SS) is an organelle that is structurally and mechanistically analogous to an intracellular membrane-attached contractile phage tail. Recent studies determined that a rapid conformational change in the structure of a sheath protein complex propels T6SS spike and tube components along with antibacterial and antieukaryotic effectors out of predatory T6SS(+) cells and into prey cells. The contracted organelle is then recycled in an ATP-dependent process. T6SS is regulated at transcriptional and posttranslational levels, the latter involving detection of membrane perturbation in some species. In addition to directly targeting eukaryotic cells, the T6SS can also target other bacteria coinfecting a mammalian host, highlighting the importance of the T6SS not only for bacterial survival in environmental ecosystems, but also in the context of infection and disease. This review highlights these and other advances in our understanding of the structure, mechanical function, assembly, and regulation of the T6SS.

PAAR-repeat proteins sharpen and diversify the type VI secretion system spike.

The bacterial type VI secretion system (T6SS) is a large multicomponent, dynamic macromolecular machine that has an important role in the ecology of many Gram-negative bacteria. T6SS is responsible for translocation of a wide range of toxic effector molecules, allowing predatory cells to kill both prokaryotic as well as eukaryotic prey cells. The T6SS organelle is functionally analogous to contractile tails of bacteriophages and is thought to attack cells by initially penetrating them with a trimeric protein complex called the VgrG spike. Neither the exact protein composition of the T6SS organelle nor the mechanisms of effector selection and delivery are known. Here we report that proteins from the PAAR (proline-alanine-alanine-arginine) repeat superfamily form a sharp conical extension on the VgrG spike, which is further involved in attaching effector domains to the spike. The crystal structures of two PAAR-repeat proteins bound to VgrG-like partners show that these proteins sharpen the tip of the T6SS spike complex. We demonstrate that PAAR proteins are essential for T6SS-mediated secretion and target cell killing by Vibrio cholerae and Acinetobacter baylyi. Our results indicate a new model of the T6SS organelle in which the VgrG–PAAR spike complex is decorated with multiple effectors that are delivered simultaneously into target cells in a single contraction-driven translocation event.

Identification of T6SS-dependent effector and immunity proteins by Tn-seq in Vibrio cholerae

Type VI protein secretion system (T6SS) is important for bacterial competition through contact-dependent killing of competitors. T6SS delivers effectors to neighboring cells and corresponding antagonistic proteins confer immunity against effectors that are delivered by sister cells. Although T6SS has been found in more than 100 gram-negative bacteria including many important human pathogens, few T6SS-dependent effector and immunity proteins have been experimentally determined. Here we report a high-throughput approach using transposon mutagenesis and deep sequencing (Tn-seq) to identify T6SS immunity proteins in Vibrio cholerae. Saturating transposon mutagenesis was performed in wild type and a T6SS null mutant. Genes encoding immunity proteins were predicted to be essential in the wild type but dispensable in the T6SS mutant. By comparing the relative abundance of each transposon mutant in the mutant library using deep sequencing, we identified three immunity proteins that render protection against killing by T6SS predatory cells. We also identified their three cognate T6SS-secreted effectors and show these are important for not only antibacterial and antieukaryotic activities but also assembly of T6SS apparatus. The lipase and muramidase T6SS effectors identified in this study underscore the diversity of T6SS-secreted substrates and the distinctly different mechanisms that target these for secretion by the dynamic T6SS organelle.

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. choleraes T6SS in the context of secretion as well as killing of bacterial and eukaryotic phagocytic cells.

Escherichia coli sister chromosome separation includes an abrupt global transition with concomitant release of late-splitting intersister snaps

The basis for segregation of sister chromosomes in bacteria is not established. We show here that two discrete ~150-kb regions, both located early in the right replichore, exhibit prolonged juxtaposition of sister loci, for 20 and 30 min, respectively, after replication. Flanking regions, meanwhile, separate. Thus, the two identified regions comprise specialized late-splitting intersister connections or snaps. Sister snap loci separate simultaneously in both snap regions, concomitant with a major global nucleoid reorganization that results in emergence of a bilobed nucleoid morphology. Split snap loci move rapidly apart to a separation distance comparable with one-half the length of the nucleoid. Concomitantly, at already split positions, sister loci undergo further separation to a comparable distance. The overall consequence of these and other effects is that thus far replicated sister chromosomes become spatially separated (individualized) into the two nucleoid lobes, while the terminus region (and likely, all unreplicated portions of the chromosome) moves to midcell. These and other findings imply that segregation of Escherichia coli sister chromosomes is not a smooth continuous process but involves at least one and likely, two major global transition(s). The presented patterns further suggest that accumulation of internal intranucleoid forces and constraining of these forces by snaps play central roles in global chromosome dynamics. They are consistent with and supportive of our previous proposals that individualization of sisters in E. coli is driven primarily by internally generated pushing forces and is directly analogous to sister individualization at the prophase to prometaphase transition of the eukaryotic cell cycle.

Type 6 Secretion System–Mediated Immunity to Type 4 Secretion System–Mediated Gene Transfer

Bacterial Détente? Type VI secretion systems (T6SS) correspond to dynamic intracellular organelles that are functionally analogous to contractile bacteriophage tails. The T6SS of several bacteria species have been found to be responsible for antagonistic behavior that likely reflects the translocation of toxic proteins (effectors) between cells. Pseudomonas aeruginosa is able to sense exogenous T6SS attack and assemble its own T6SS apparatus to launch a retaliatory attack aimed directly at the attacker. Now, Ho et al. (p. 250) describe how exogenous attack is sensed in a process that involves membrane disruption and suggest that the T6SS provides a general cellular defense mechanism against not only T6SS but also conjugative DNA elements delivered via the type IV secretion system involved in mating pair formation. A general bacterial defense mechanism suppresses the movement of horizontally transferred DNA in bacterial populations. Gram-negative bacteria use the type VI secretion system (T6SS) to translocate toxic effector proteins into adjacent cells. The Pseudomonas aeruginosa H1-locus T6SS assembles in response to exogenous T6SS attack by other bacteria. We found that this lethal T6SS counterattack also occurs in response to the mating pair formation (Mpf) system encoded by broad-host-range IncPα conjugative plasmid RP4 present in adjacent donor cells. This T6SS response was eliminated by disruption of Mpf structural genes but not components required only for DNA transfer. Because T6SS activity was also strongly induced by membrane-disrupting natural product polymyxin B, we conclude that RP4 induces “donor-directed T6SS attacks” at sites corresponding to Mpf-mediated membrane perturbations in recipient P. aeruginosa cells to potentially block acquisition of parasitic foreign DNA.

Vibrio cholerae type 6 secretion system effector trafficking in target bacterial cells

Significance This work demonstrates that it is possible to alter the prey target range of the antibacterial activity of the type 6 secretion system (T6SS). Being able to change the T6SS target specificity is the first step toward using the T6SS as an antipathogen or commensal therapeutic, prophylactic, or probiotic. This work also uncovers a cryptic secretion mechanism(s) for delivering protein substrates from the bacterial cytosol to the periplasm. This mechanism is exploited by some T6SS effectors as an alternative pathway for reaching periplasmic targets when they are by chance delivered into the cytosol of target cells. The type 6 secretion system (T6SS) is used by many Gram-negative bacterial species to deliver toxic effector proteins into nearby bacteria prey cells to kill or inhibit their growth. VgrG proteins are core conserved secretion substrates of the T6SS and one subset of T6SS effectors consists of VgrG proteins with C-terminal extension domains carrying various enzymatic activities. In Vibrio cholerae, VgrG3 has a hydrolase extension domain and degrades peptidoglycan in the periplasm of target bacteria. In this study, we replaced this domain with a nuclease domain from Salmonella enterica subsp. arizonae. This modified V. cholerae strain was able to kill its parent using its T6SS. This result also demonstrated that V. cholerae T6SS is capable of delivering effectors that could attack substrates found either in the periplasm or cytosol of target bacteria. Additionally, we found that effectors VgrG3 and TseL, despite lacking a classical Sec or TAT secretion signal, were able to reach the periplasm when expressed in the bacterial cytosol. This effector trafficking likely represents an evolutionary strategy for T6SS effectors to reach their intended substrates regardless of which subcellular compartment in the target cell they happen to be delivered to by the T6SS apparatus.

Exopolysaccharide protects Vibrio cholerae from exogenous attacks by the type 6 secretion system

Significance This work demonstrates that exopolysaccharide (EPS) produced by Vibrio cholerae can protect against exogenous type 6 secretion system (T6SS) attacks from different bacterial species. This protection does not affect the ability of the EPS-producing cell to use their own T6SS to attack other bacteria, indicating that EPS does not simply increase the physical distance between cells to afford protection. Furthermore, this protective effect does not depend on other genes involved in biofilm biogenesis, suggesting that EPSs can determine the outcomes of antagonistic bacterial–bacterial interactions independent of their typical function in biofilm formation. Thus, extracellular biopolymers of bacteria may play a role in modulating resistance to exogenous attacks by other bacteria as well as providing potential resistance to host innate immune mechanisms. The type 6 secretion system (T6SS) is a nanomachine used by many Gram-negative bacteria, including Vibrio cholerae, to deliver toxic effector proteins into adjacent eukaryotic and bacterial cells. Because the activity of the T6SS is dependent on direct contact between cells, its activity is limited to bacteria growing on solid surfaces or in biofilms. V. cholerae can produce an exopolysaccharide (EPS) matrix that plays a role in adhesion and biofilm formation. In this work, we investigated the effect of EPS production on T6SS activity between cells. We found that EPS produced by V. cholerae cells functions as a unidirectional protective armor that blocks exogenous T6SS attacks without interfering with its own T6SS functionality. This EPS armor is effective against both same-species and heterologous attackers. Mutations modulating the level of EPS biosynthesis gene expression result in corresponding modulation in V. cholerae resistance to exogenous T6SS attack. These results provide insight into the potential role of extracellular biopolymers, including polysaccharides, capsules, and S-layers in protecting bacterial cells from attacks involving cell-associated macromolecular protein machines that cannot readily diffuse through these mechanical defenses.