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Virstatin is a previously described small molecule inhibitor of Vibrio cholerae virulence. We have demonstrated that the molecule inhibits the activity of the transcriptional activator ToxT, thereby preventing elaboration of the toxin co-regulated pilus (TCP) and cholera toxin in vitro and in vivo in O1 strains of V. cholerae. While strains of the O1 and O139 serogroups are the cause of most epidemic and endemic cholera currently seen globally, sporadic disease caused by strains of non-O1/non-O139 serogroups suggests that understanding the pathogenic mechanisms of these unusual strains is relevant for disease. Although some non-O1/non-O139 strains have acquired the pathogenicity island that encodes the TCP, the role that this essential colonization factor of O1/O139 strains plays in the virulence of non-O1/non-O139 strains has not been determined. In this study, we utilize virstatin in a 'chemical genetic approach' to examine the role of ToxT, and thus by inference TCP, in the colonization of a panel of predominantly non-O1/non-O139 tcp+ strains. We identified nine strains whose colonization was resistant to virstatin inhibition in the infant mouse model. These strains presumably colonize by a TCP-independent mechanism or contain a naturally occurring virstatin-resistant ToxT. Four strains contained the typical toxT gene found in O1/O139 strains (toxT(EPI)) isolated from cholera epidemics. Interruption of toxT in one of these strains did not affect colonization of the infant mouse small intestine. The remaining five strains were found to contain a sequence divergent toxT gene that has been previously designated toxT(ENV) because of its occurrence in isolates of V. cholerae from the environment. We show that ToxT(ENV) is resistant to virstatin in two separate heterologous systems and is necessary for efficient colonization of the infant mouse small intestine. These results support the new concept that chemical genetic probes for the in vivo function or expression of virulence genes can be used to identify strains that express alternative virulence factors or novel regulatory systems that are functional in vivo.

Vibrio cholerae is a Gram-negative bacterium that causes the acute diarrhoeal disease cholera. After the bacterium is ingested, it passes through the digestive tract, encountering various environmental stresses including the acidic milieu of the stomach and the toxic effects of bile in the duodenum. While these stresses serve as part of a host defence system, V. cholerae has evolved resistance mechanisms that allow it to evade these defences and establish infection. We examined the expression profiles of V. cholerae in response to bile or bile acids and found an induction of biofilm genes. We found that V. cholerae shows significantly enhanced biofilm formation in response to bile acids, and that bacteria within the biofilm are more resistant to the toxicity of bile acids compared with planktonic cells. Bile acid induction of biofilms was found to be dependent on the vps genes (Vibrio polysaccharide synthesis) and their transcriptional activator VpsR, but VpsT is not required. These results contribute to the developing picture of a complex relationship between V. cholerae and its environment within the host during infection.

Summary: The Type VI secretion system (T6SS) is a bacterial nanomachine that delivers effector proteins into prokaryotic and eukaryotic preys. This secretion system has emerged as a key player in regulating the microbial diversity in a population. In the plant pathogen Agrobacterium tumefaciens, the signalling cascades regulating the activity of this secretion system are poorly understood. Here, we outline how the universal eubacterial second messenger cyclic di‐GMP impacts the production of T6SS toxins and T6SS structural components. We demonstrate that this has a significant impact on the ability of the phytopathogen to compete with other bacterial species in vitro and in planta. Our results suggest that, as opposed to other bacteria, c‐di‐GMP turns down the T6SS in A. tumefaciens thus impacting its ability to compete with other bacterial species within the rhizosphere. We also demonstrate that elevated levels of c‐di‐GMP within the cell decrease the activity of the Type IV secretion system (T4SS) and subsequently the capacity of A. tumefaciens to transform plant cells. We propose that such peculiar control reflects on c‐di‐GMP being a key second messenger that silences energy‐costing systems during early colonization phase and biofilm formation, while low c‐di‐GMP levels unleash T6SS and T4SS to advance plant colonization. [ABSTRACT FROM AUTHOR]

Summary: Contractile tail bacteriophages, or myobacteriophages, use a sophisticated biomolecular structure to inject their genome into the bacterial host cell. This structure consists of a contractile sheath enveloping a rigid tube that is sharpened by a spike‐shaped protein complex at its tip. The spike complex forms the centerpiece of a baseplate complex that terminates the sheath and the tube. The baseplate anchors the tail to the target cell membrane with the help of fibrous proteins emanating from it and triggers contraction of the sheath. The contracting sheath drives the tube with its spiky tip through the target cell membrane. Subsequently, the bacteriophage genome is injected through the tube. The structural transformation of the bacteriophage T4 baseplate upon binding to the host cell has been recently described in near‐atomic detail. In this review we discuss structural elements and features of this mechanism that are likely to be conserved in all contractile injection systems (systems evolutionary and structurally related to contractile bacteriophage tails). These include the type VI secretion system (T6SS), which is used by bacteria to transfer effectors into other bacteria and into eukaryotic cells, and tailocins, a large family of contractile bacteriophage tail‐like compounds that includes the P. aeruginosa R‐type pyocins. [ABSTRACT FROM AUTHOR]

Vibrio cholerae, the causative agent of the severe diarrheal disease cholera, has evolved signal transduction systems to control co-ordinately the expression of virulence determinants. It was previously shown that the presence of the bile salts glycocholate and taurocholate in the small intestine causes dimerization of the transmembrane transcription factor TcpP by inducing intermolecular disulphide bonds in the TcpP periplasmic domain. In this study, they further investigated the mechanism of how taurocholate affects V. cholerae virulence determinants. In vitro assay of TcpP oxidation by VcDsbA showed that VcDsbA induced TcpP dimerization in the presence of taurocholate. Taurocholate bound to VcDsbA with a KD of 40 ± 2.5 μM, and also bound other Dsb proteins, including EcDsbA, EcDsbC and VcDsbC. Taurocholate inhibited VcDsbA reductase activity without affecting VcDsbA secondary structure or thermostability. VcDsbA and its substrates were more extensively reduced in the presence of taurocholate, as compared with their redox state in the absence of taurocholate. The data presented here not only provide new insights into the mechanism by which bile salts induce V. cholerae virulence but also suggest a means by which to develop inhibitors against DsbA. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The bacterial type VI secretion system (T6SS) system is a contractile secretion apparatus that delivers proteins to neighboring bacterial or eukaryotic cells. Antibacterial effectors are mostly toxins that inhibit the growth of other species and help to dominate the niche. A broad variety of these toxins cause cell lysis of the prey cell by disrupting the cell envelope. Other effectors are delivered into the cytoplasm where they affect DNA integrity, cell division or exhaust energy resources. The modular nature of T6SS machinery allows different means of recruitment of toxic effectors to secreted inner tube and spike components that act as carriers. Toxic effectors can be translationally fused to the secreted components or interact with them through specialized structural domains. These interactions can also be assisted by dedicated chaperone proteins. Moreover, conserved sequence motifs in effector-associated domains are subject to genetic rearrangements and therefore engage in the diversification of the arsenal of toxic effectors. This review discusses the diversity of T6SS secreted toxins and presents current knowledge about their loading on the T6SS machinery., (© 2020 John Wiley & Sons Ltd.)

"CryoEM" has come of age, enabling considerable structural insights into many facets of molecular biology. Here, we present a primer for microbiologists to understand the capabilities and limitations of two complementary cryoEM techniques for studying bacterial secretion systems. The first, single particle analysis, determines the structures of purified protein complexes to resolutions sufficient for molecular modeling, while the second, electron cryotomography and subtomogram averaging, tends to determine more modest resolution structures of protein complexes in intact cells. We illustrate these abilities with examples of insights provided into how secretion systems work by cryoEM, with a focus on type III secretion systems., (© 2020 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Type VI secretion is critical for V ibrio cholerae to successfully combat phagocytic eukaryotes and to survive in the presence of competing bacterial species. V . cholerae type VI secretion system genes are encoded in one large and two small clusters. In V . cholerae, type VI secretion is controlled by quorum sensing, the cell-cell communication process that enables bacteria to orchestrate group behaviours. The quorum-sensing response regulator LuxO represses type VI secretion genes at low cell density and the quorum-sensing regulator HapR activates type VI secretion genes at high cell density. We demonstrate that the quorum regulatory small RNAs ( Qrr sRNAs) that function between LuxO and HapR in the quorum-sensing cascade are required for these regulatory effects. The Qrr sRNAs control type VI secretion via two mechanisms: they repress expression of the large type VI secretion system cluster through base pairing and they repress HapR, the activator of the two small type VI secretion clusters. This regulatory arrangement ensures that the large cluster encoding many components of the secretory machine is expressed prior to the two small clusters that encode the secreted effectors. Qrr sRNA-dependent regulation of the type VI secretion system is conserved in pandemic and non-pandemic V . cholerae strains. [ABSTRACT FROM AUTHOR]

The multicomponent type VI secretion system ( T6SS) mediates the transport of effector proteins by puncturing target membranes. T6SSs are suggested to form a contractile nanomachine, functioning similar to the cell-puncturing device of tailed bacteriophages. The T6SS members VipA/ VipB form tubular complexes and are predicted to function in analogy to viral tail sheath proteins by providing the energy for secretion via contraction. The ATPase ClpV disassembles VipA/ VipB tubules in vitro, but the physiological relevance of tubule disintegration remained unclear. Here, we show that VipA/ VipB tubules localize near-perpendicular to the inner membrane of Vibrio cholerae cells and exhibit repetitive cycles of elongation, contraction and disassembly. VipA/ VipB tubules are decorated by ClpV in vivo and become static in Δ clpV cells, indicating that ClpV is required for tubule removal. VipA/ VipB tubules mislocalize in Δ clpV cells and exhibit a reduced frequency of tubule elongation, indicating that ClpV also suppresses the spontaneous formation of contracted, non-productive VipA/ VipB tubules. ClpV activity is restricted to the contracted state of VipA/ VipB, allowing formation of functional elongated tubules at a T6SS assembly. Targeting of an unrelated ATPase to VipA/ VipB is sufficient to replace ClpV function in vivo, suggesting that ClpV activity is autonomously regulated by VipA/ VipB conformation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In Pseudomonas aeruginosa three type VI secretion systems ( T6SSs) coexist, called H1- to H3-T6SSs. Several T6SS components are proposed to be part of a macromolecular complex resembling the bacteriophage tail. The T6SS protein HsiE1 ( TagJ) is unique to the H1-T6SS and absent from the H2- and H3-T6SSs. We demonstrate that HsiE1 interacts with a predicted N-terminal α-helix in HsiB1 ( TssB) thus forming a novel subcomplex of the T6SS. HsiB1 is homologous to the Vibrio cholerae VipA component, which contributes to the formation of a bacteriophage tail sheath-like structure. We show that the interaction between HsiE1 and HsiB1 is specific and does not occur between HsiE1 and HsiB2. Proteins of the TssB family encoded in T6SS clusters lacking a gene encoding a TagJ-like component are often devoid of the predicted N-terminal helical region, which suggests co-evolution. We observe that a synthetic peptide corresponding to the N-terminal 20 amino acids of HsiB1 interacts with purified HsiE1 protein. This interaction is a common feature to other bacterial T6SSs that display a TagJ homologue as shown here with Serratia marcescens. We further show that hsiE1 is a non-essential gene for the T6SS and suggest that HsiE1 may modulate incorporation of HsiB1 into the T6SS. [ABSTRACT FROM AUTHOR]

Campylobacter jejuni, a prevalent cause of bacterial gastroenteritis, must adapt to different environments to be a successful pathogen. We previously identified a C. jejuni two-component regulatory system (Cj1226/7c) as upregulated during cell infections. Analyses described herein led us to designate the system CprRS ( ampylobacter lanktonic growth egulation). While the response regulator was essential, a cprS sensor kinase mutant was viable. The Δ cprS mutant displayed an apparent growth defect and formed dramatically enhanced and accelerated biofilms independent of upregulation of previously characterized surface polysaccharides. Δ cprS also displayed a striking dose-dependent defect for colonization of chicks and was modestly enhanced for intracellular survival in INT407 cells. Proteomics analyses identified changes consistent with modulation of essential metabolic genes, upregulation of stress tolerance proteins, and increased expression of MOMP and FlaA. Consistent with expression profiling, we observed enhanced motility and secretion in Δ cprS, and decreased osmotolerance and oxidative stress tolerance. We also found that C. jejuni biofilms contain a DNase I-sensitive component and that biofilm formation is influenced by deoxycholate and the metabolic substrate fumarate. These results suggest that CprRS influences expression of factors important for biofilm formation, colonization and stress tolerance, and also add to our understanding of C. jejuni biofilm physiology. [ABSTRACT FROM AUTHOR]

An author index for the December 2007 issue of "Molecular Microbiology" is presented.

Membrane-associated ATPase constitutes an essential element common to all secretion machineries in Gram-negative bacteria. How ATP hydrolysis by these ATPases is coupled to secretion process remains unclear. Here we identified R286 as a key residue in the type II secretion system (T2SS) ATPase XpsE of Xanthomonas campestris that plays a pivotal role in coupling ATP hydrolysis to protein translocation. Mutation of R286 to alanine made XpsE hydrolyse ATP at a rate five times that of the wild-type XpsE. Yet the mutant XpsE(R286A) is non-functional in protein secretion via T2SS. Detailed analyses indicated that the mutant XpsE(R286A) lost the ability co-ordinating the N- and C-domain of XpsE. Without significantly influencing XpsE binding affinity with ATP or its oligomerization, R286A mutation however, caused XpsE lose the ability to associate with the cytoplasmic membrane via XpsLN. As a consequence, ATP hydrolysis by XpsE was uncoupled from protein secretion. Because R286 is highly conserved among members of the secretion NTPase superfamily, we speculate that its equivalent in other homologues may also play a critical energy coupling role for T2SS, type IV pilus assembly and type IV secretion system. [ABSTRACT FROM AUTHOR]

Burkholderia mallei is a host-adapted pathogen and a category B biothreat agent. Although the B. mallei VirAG two-component regulatory system is required for virulence in hamsters, the virulence genes it regulates are unknown. Here we show with expression profiling that overexpression of virAG resulted in transcriptional activation of ∼60 genes, including some involved in capsule production, actin-based intracellular motility, and type VI secretion (T6S). The 15 genes encoding the major sugar component of the homopolymeric capsule were up-expressed > 2.5-fold, but capsule was still produced in the absence of virAG. Actin tail formation required virAG as well as bimB, bimC and bimE, three previously uncharacterized genes that were activated four- to 15-fold when VirAG was overproduced. Surprisingly, actin polymerization was found to be dispensable for virulence in hamsters. In contrast, genes encoding a T6S system were up-expressed as much as 30-fold and mutations in this T6S gene cluster resulted in strains that were avirulent in hamsters. SDS-PAGE and mass spectrometry demonstrated that BMAA0742 was secreted by the T6S system when virAG was overexpressed. Purified His-tagged BMAA0742 was recognized by glanders antiserum from a horse, a human and mice, indicating that this Hcp-family protein is produced in vivo during infection. [ABSTRACT FROM AUTHOR]

Successful colonization of a eukaryotic host by a microbe involves complex microbe–microbe and microbe–host interactions. Previously, we identified in Vibrio fischeri a putative sensor kinase, RscS, required for initiating symbiotic colonization of its squid host Euprymna scolopes. Here, we analysed the role of rscS by isolating an allele, rscS1, with increased activity. Multicopy rscS1 activated transcription of genes within the recently identified symbiosis polysaccharide ( syp) cluster. Wild-type cells carrying rscS1 induced aggregation phenotypes in culture, including the formation of pellicles and wrinkled colonies, in a syp-dependent manner. Colonies formed by rscS1-expressing cells produced a matrix not found in control colonies and largely lost in an rscS1-expressing sypN mutant. Finally, multicopy rscS1 provided a colonization advantage over control cells and substantially enhanced the ability of wild-type cells to aggregate on the surface of the symbiotic organ of E. scolopes; this latter phenotype similarly depended upon an intact syp locus. These results suggest that transcription induced by RscS-mediated signal transduction plays a key role in colonization at the aggregation stage by modifying the cell surface and increasing the ability of the cells to adhere to one another and/or to squid-secreted mucus. [ABSTRACT FROM AUTHOR]

Almost all aspects of protein traffic in bacteria were covered at the ASM-FEMS meeting on the topic in Iraklio, Crete in May 2006. The studies presented ranged from mechanistic analysis of specific events leading proteins to their final destinations to the physiological roles of the targeted proteins. Among the highlights from the meeting that are reviewed here are the molecular dynamics of SecA protein, membrane protein insertion, type III secretion needles and chaperones, type IV secretion, the two partner and autosecretion systems, the ‘secretion competent state’, and the recently discovered type VI secretion system. [ABSTRACT FROM AUTHOR]

An author index for the March 15, 2006 issue of the periodical "Molecular Microbiology" is presented.