RESUMEN
The bacterial type VI secretion system (T6SS) is a dynamic organelle that bacteria use to target prey cells for inhibition via translocation of effector proteins. Time-lapse fluorescence microscopy has documented striking dynamics of opposed T6SS organelles in adjacent sister cells of Pseudomonas aeruginosa. Such cell-cell interactions have been termed "T6SS dueling" and likely reflect a biological process that is driven by T6SS antibacterial attack. Here, we show that T6SS dueling behavior strongly influences the ability of P. aeruginosa to prey upon heterologous bacterial species. We show that, in the case of P. aeruginosa, T6SS-dependent killing of either Vibrio cholerae or Acinetobacter baylyi is greatly stimulated by T6SS activity occurring in those prey species. Our data suggest that, in P. aeruginosa, T6SS organelle assembly and lethal counterattack are regulated by a signal that corresponds to the point of attack of the T6SS apparatus elaborated by a second aggressive T6SS(+) bacterial cell. PAPERFLICK:
Asunto(s)
Sistemas de Secreción Bacterianos , Bacterias Gramnegativas/metabolismo , Interacciones Microbianas , Pseudomonas aeruginosa/metabolismo , Acinetobacter/metabolismo , Proteínas Bacterianas/metabolismo , Escherichia coli/metabolismo , Microscopía Fluorescente , Transducción de Señal , Imagen de Lapso de Tiempo , Vibrio cholerae/metabolismoRESUMEN
Interbacterial competition is known to shape the microbial communities found in the host, however the interplay between this competition and host defense are less clear. Here, we use the zebrafish hindbrain ventricle (HBV) as an in vivo platform to investigate host responses to defined bacterial communities with distinct forms of interbacterial competition. We found that antibacterial activity of the type VI secretion system (T6SS) from both Vibrio cholerae and Acinetobacter baylyi can induce host inflammation and sensitize the host to infection independent of any individual effector. Chemical suppression of inflammation could resolve T6SS-dependent differences in host survival, but the mechanism by which this occurred differed between the two bacterial species. By contrast, colicin-mediated antagonism elicited by an avirulent strain of Shigella sonnei induced a negligible host response despite being a more potent bacterial killer, resulting in no impact on A. baylyi or V. cholerae virulence. Altogether, these results provide insight into how different modes of interbacterial competition in vivo affect the host in distinct ways.
Asunto(s)
Sistemas de Secreción Tipo VI , Vibrio cholerae , Pez Cebra , Animales , Pez Cebra/microbiología , Sistemas de Secreción Tipo VI/metabolismo , Vibrio cholerae/patogenicidad , Acinetobacter , Virulencia , Interacciones Huésped-Patógeno , Antibiosis/fisiología , Rombencéfalo/microbiología , Rombencéfalo/metabolismoRESUMEN
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.
Asunto(s)
Polisacáridos Bacterianos/metabolismo , Sistemas de Secreción Tipo VI/metabolismo , Vibrio cholerae/metabolismo , Polisacáridos Bacterianos/genética , Sistemas de Secreción Tipo VI/genética , Vibrio cholerae/genéticaRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Transporte de Proteínas/fisiología , Sistemas de Secreción Tipo VI/fisiología , Vibrio cholerae/fisiología , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión GénicaRESUMEN
There is an inseverable link between the size of a cell and the size of its subcellular components. The type 6 secretion system (T6SS) is no exception. In this issue, Stietz et al. report their analysis of the T6SS under conditions in which the cell size was altered to an extreme degree (M. S. Stietz, X. Liang, M. Wong, S. Hersch, and T. G. Dong, J Bacteriol 202:e00425-19, 2020, https://doi.org/10.1128/JB.00425-19). That study and others investigating the regulation of T6SS filament polymerization have provided insight into how the T6SS apparatus matches its size to the cell that contains it.
Asunto(s)
Sistemas de Secreción Tipo VI , Proteínas Bacterianas , ElasticidadRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sistemas de Secreción Bacterianos/genética , Repeticiones de Microsatélite/fisiología , Acinetobacter/genética , Acinetobacter/metabolismo , Unión Proteica , Vibrio cholerae/genética , Vibrio cholerae/metabolismoRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/genética , Sistemas de Secreción Bacterianos/genética , Interacciones Microbianas/genética , Vibrio cholerae/genética , Actinas/metabolismo , Proteínas Bacterianas/inmunología , Sistemas de Secreción Bacterianos/inmunología , Western Blotting , Elementos Transponibles de ADN/genética , Dictyostelium/crecimiento & desarrollo , Dictyostelium/microbiología , Vectores Genéticos/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Inmunoprecipitación , Lipasa/genética , Interacciones Microbianas/inmunología , Muramidasa/genética , Mutagénesis Sitio-Dirigida , Plásmidos/genética , Células Madre , Vibrio cholerae/inmunologíaRESUMEN
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.
Asunto(s)
Segregación Cromosómica/fisiología , Cromosomas Bacterianos/genética , Escherichia coli/genética , Segregación Cromosómica/genética , Hibridación Fluorescente in Situ , Modelos Genéticos , Factores de TiempoRESUMEN
Bacterial conjugation is a process by which DNA is transferred unidirectionally from a donor cell to a recipient cell. It is the main means by which antibiotic resistance genes spread among bacterial populations. It is crucially dependent upon the elaboration of an extracellular appendage, termed "pilus," by a large double-membrane-spanning secretion system termed conjugative "type IV secretion system." Here we present the structure of the conjugative pilus encoded by the R388 plasmid. We demonstrate that, as opposed to all conjugative pili produced so far for cryoelectron microscopy (cryo-EM) structure determination, the conjugative pilus encoded by the R388 plasmid is greatly stimulated by the presence of recipient cells. Comparison of its cryo-EM structure with existing conjugative pilus structures highlights a number of important differences between the R388 pilus structure and that of its homologs, the most prominent being the highly distinctive conformation of its bound lipid.
RESUMEN
Extensive drug resistance (XDR) is an escalating global problem. Escherichia coli strain Sanji was isolated from an outbreak of pheasant colibacillosis in Fujian province, China, in 2011. This strain has XDR properties, exhibiting sensitivity to carbapenems but no other classes of known antibiotics. Whole-genome sequencing revealed a total of 32 known antibiotic resistance genes, many associated with insertion sequence 26 (IS26) elements. These were found on the Sanji chromosome and 2 of its 6 plasmids, pSJ_255 and pSJ_82. The Sanji chromosome also harbors a type 2 secretion system (T2SS), a type 3 secretion system (T3SS), a type 6 secretion system (T6SS), and several putative prophages. Sanji and other ST167 strains have a previously uncharacterized O-antigen (O89b) that is most closely related to serotype O89 as determined on the basis of analysis of the wzm-wzt genes and in silico serotyping. This O89b-antigen gene cluster was also found in the genomes of a few other pathogenic sequence type 617 (ST617) and ST10 complex strains. A time-scaled phylogeny inferred from comparative single nucleotide variant analysis indicated that development of these O89b-containing lineages emerged about 30 years ago. Comparative sequence analysis revealed that the core genome of Sanji is nearly identical to that of several recently sequenced strains of pathogenic XDR E. coli belonging to the ST167 group. Comparison of the mobile elements among the different ST167 genomes revealed that each genome carries a distinct set of multidrug resistance genes on different types of plasmids, indicating that there are multiple paths toward the emergence of XDR in E. coli. IMPORTANCE E. coli strain Sanji is the first sequenced and analyzed genome of the recently emerged pathogenic XDR strains with sequence type ST167 and novel in silico serotype O89b:H9. Comparison of the genomes of Sanji with other ST167 strains revealed distinct sets of different plasmids, mobile IS elements, and antibiotic resistance genes in each genome, indicating that there exist multiple paths toward achieving XDR. The emergence of these pathogenic ST167 E. coli strains with diverse XDR capabilities highlights the difficulty of preventing or mitigating the development of XDR properties in bacteria and points to the importance of better understanding of the shared underlying virulence mechanisms and physiology of pathogenic bacteria.
RESUMEN
Vibrio cholerae is the causative agent of the diarrheal disease cholera. Although many V. cholerae virulence factors have been studied, the role of interbacterial interactions within the host gut and their influence on colonization are poorly understood. Here, we utilized the conjugative properties of a Vibrio-specific plasmid to serve as a quantifiable genetic marker for direct contact among V. cholerae cells in the infant rabbit model for cholera. In conjunction, we also quantified contact-dependent type 6 secretion system (T6SS)-mediated killing of co-infecting V. cholerae strains. Tracking these interbacterial interactions revealed that most contact-dependent cell-cell interactions among V. cholerae occur in specific intestinal microenvironments, notably the distal small intestine and cecum, and that the T6SS confers a competitive advantage within the middle small intestine. These results support a model for V. cholerae gut colonization, which includes microenvironments where critical microbial-host and bacterial-bacterial interactions occur to facilitate colonization by this pathogen.
Asunto(s)
Cólera/patología , Interacciones Huésped-Patógeno/fisiología , Intestinos/microbiología , Sistemas de Secreción Tipo VI/fisiología , Vibrio cholerae , Animales , Cólera/microbiología , Conjugación Genética/fisiología , Intestinos/patología , Plásmidos/genética , Conejos , Vibrio cholerae/genética , Vibrio cholerae/crecimiento & desarrollo , Vibrio cholerae/patogenicidadRESUMEN
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.
Asunto(s)
Bacterias/metabolismo , Proteínas Bacterianas/metabolismo , Sistemas de Secreción Bacterianos/genética , Regulación Bacteriana de la Expresión Génica , Proteínas de la Membrana/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Bacterias/genética , Bacterias/patogenicidad , Bacterias/ultraestructura , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Bacteriófagos/fisiología , Bacteriófagos/ultraestructura , Células Eucariotas/citología , Células Eucariotas/microbiología , Interacciones Huésped-Patógeno , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Percepción de Quorum , Transducción de SeñalRESUMEN
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.