Sarah T. Miyata

The type VI secretion system: translocation of effectors and effector-domains

A number of prominent Gram-negative bacteria use the type VI secretion system (T6SS) to transport proteins across the bacterial envelope. Rapid progress is being made in elucidating the structural components of the T6SS apparatus, and a few effectors have been reported to pass through it. However, this is not the complete story: a family of T6SS proteins, the VgrGs, share structural features with the cell-puncturing device of the T4 bacteriophage, and may be used in a similar fashion by bacteria to puncture host cell membranes and insert the T6SS apparatus into the host cytosol. Interestingly, a number of VgrGs contain C-terminal extensions with effector-domains. Thus, the T6SS may translocate soluble effectors, as well as VgrG effector-domains.

The Vibrio cholerae type VI secretion system displays antimicrobial properties

The acute diarrheal disease cholera is caused by the marine bacterium Vibrio cholerae. A type VI secretion system (T6SS), which is structurally similar to the bacteriophage cell-puncturing device, has been recently identified in V. cholerae and is used by this organism to confer virulence toward phagocytic eukaryotes, such as J774 murine macrophages and Dictyostelium discoideum. We tested the interbacterial virulence of V. cholerae strain V52, an O37 serogroup with a constitutively active T6SS. V52 was found to be highly virulent toward multiple Gram-negative bacteria, including Escherichia coli and Salmonella Typhimurium, and caused up to a 100,000-fold reduction in E. coli survival. Because the T6SS-deficient mutants V52ΔvasK and V52ΔvasH showed toxicity defects that could be complemented, virulence displayed by V. cholerae depends on a functional T6SS. V. cholerae V52 and strains of the O1 serogroup were resistant to V52, suggesting that V. cholerae has acquired immunity independently of its serogroup. We hypothesize that the T6SS, in addition to targeting eukaryotic host cells, confers toxicity toward other bacteria, providing a means of interspecies competition to enhance environmental survival. Thus, the V. cholerae T6SS may enhance the survival of V. cholerae in its aquatic ecosystem during the transmission of cholera and between epidemics.

Antibiotic resistance mechanisms of Vibrio cholerae

As the causative agent of cholera, the bacterium Vibrio cholerae represents an enormous public health burden, especially in developing countries around the world. Cholera is a self-limiting illness; however, antibiotics are commonly administered as part of the treatment regimen. Here we review the initial identification and subsequent evolution of antibiotic-resistant strains of V. cholerae. Antibiotic resistance mechanisms, including efflux pumps, spontaneous chromosomal mutation, conjugative plasmids, SXT elements and integrons, are also discussed. Numerous multidrug-resistant strains of V. cholerae have been isolated from both clinical and environmental settings, indicating that antibiotic use has to be restricted and alternative methods for treating cholera have to be implemented.

Vibrio cholerae Requires the Type VI Secretion System Virulence Factor VasX To Kill Dictyostelium discoideum

ABSTRACT The type VI secretion system (T6SS) is recognized as an important virulence mechanism in several Gram-negative pathogens. In Vibrio cholerae, the causative agent of the diarrheal disease cholera, a minimum of three gene clusters—one main cluster and two auxiliary clusters—are required to form a functional T6SS apparatus capable of conferring virulence toward eukaryotic and prokaryotic hosts. Despite an increasing understanding of the components that make up the T6SS apparatus, little is known about the regulation of these genes and the gene products delivered by this nanomachine. VasH is an important regulator of the V. cholerae T6SS. Here, we present evidence that VasH regulates the production of a newly identified protein, VasX, which in turn requires a functional T6SS for secretion. Deletion of vasX does not affect export or enzymatic function of the structural T6SS proteins Hcp and VgrG-1, suggesting that VasX is dispensable for the assembly of the physical translocon complex. VasX localizes to the bacterial membrane and interacts with membrane lipids. We present VasX as a novel virulence factor of the T6SS, as a V. cholerae mutant lacking vasX exhibits a phenotype of attenuated virulence toward Dictyostelium discoideum.

The Vibrio cholerae type VI secretion system employs diverse effector modules for intraspecific competition

Vibrio cholerae is a Gram-negative bacterial pathogen that consists of over 200 serogroups with differing pathogenic potential. Only strains that express the virulence factors cholera toxin (CT) and toxin-coregulated pilus (TCP) are capable of pandemic spread of cholera diarrhoea. Regardless, all V. cholerae strains sequenced to date harbour genes for the type VI secretion system (T6SS) that translocates effectors into neighbouring eukaryotic and prokaryotic cells. Here we report that the effectors encoded within these conserved gene clusters differ widely among V. cholerae strains, and that immunity proteins encoded immediately downstream from the effector genes protect their host from neighbouring bacteria producing corresponding effectors. As a consequence, strains with matching effector-immunity gene sets can coexist, while strains with different sets compete against each other. Thus, the V. cholerae T6SS contributes to the competitive behaviour of this species.

Dual Expression Profile of Type VI Secretion System Immunity Genes Protects Pandemic Vibrio cholerae

The Vibrio cholerae type VI secretion system (T6SS) assembles as a molecular syringe that injects toxic protein effectors into both eukaryotic and prokaryotic cells. We previously reported that the V. cholerae O37 serogroup strain V52 maintains a constitutively active T6SS to kill other Gram-negative bacteria while being immune to attack by kin bacteria. The pandemic O1 El Tor V. cholerae strain C6706 is T6SS-silent under laboratory conditions as it does not produce T6SS structural components and effectors, and fails to kill Escherichia coli prey. Yet, C6706 exhibits full resistance when approached by T6SS-active V52. These findings suggested that an active T6SS is not required for immunity against T6SS-mediated virulence. Here, we describe a dual expression profile of the T6SS immunity protein-encoding genes tsiV1, tsiV2, and tsiV3 that provides pandemic V. cholerae strains with T6SS immunity and allows T6SS-silent strains to maintain immunity against attacks by T6SS-active bacterial neighbors. The dual expression profile allows transcription of the three genes encoding immunity proteins independently of other T6SS proteins encoded within the same operon. One of these immunity proteins, TsiV2, protects against the T6SS effector VasX which is encoded immediately upstream of tsiV2. VasX is a secreted, lipid-binding protein that we previously characterized with respect to T6SS-mediated virulence towards the social amoeba Dictyostelium discoideum. Our data suggest the presence of an internal promoter in the open reading frame of vasX that drives expression of the downstream gene tsiV2. Furthermore, VasX is shown to act in conjunction with VasW, an accessory protein to VasX, to compromise the inner membrane of prokaryotic target cells. The dual regulatory profile of the T6SS immunity protein-encoding genes tsiV1, tsiV2, and tsiV3 permits V. cholerae to tightly control T6SS gene expression while maintaining immunity to T6SS activity.

Genomic and Functional Analysis of the Type VI Secretion System in Acinetobacter

The genus Acinetobacter is comprised of a diverse group of species, several of which have raised interest due to potential applications in bioremediation and agricultural purposes. In this work, we show that many species within the genus Acinetobacter possess the genetic requirements to assemble a functional type VI secretion system (T6SS). This secretion system is widespread among Gram negative bacteria, and can be used for toxicity against other bacteria and eukaryotic cells. The most studied species within this genus is A. baumannii, an emerging nosocomial pathogen that has become a significant threat to healthcare systems worldwide. The ability of A. baumannii to develop multidrug resistance has severely reduced treatment options, and strains resistant to most clinically useful antibiotics are frequently being isolated. Despite the widespread dissemination of A. baumannii, little is known about the virulence factors this bacterium utilizes to cause infection. We determined that the T6SS is conserved and syntenic among A. baumannii strains, although expression and secretion of the hallmark protein Hcp varies between strains, and is dependent on TssM, a known structural protein required for T6SS function. Unlike other bacteria, A. baumannii ATCC 17978 does not appear to use its T6SS to kill Escherichia coli or other Acinetobacter species. Deletion of tssM does not affect virulence in several infection models, including mice, and did not alter biofilm formation. These results suggest that the T6SS fulfils an important but as-yet-unidentified role in the various lifestyles of the Acinetobacter spp.

Constitutive Type VI Secretion System Expression Gives Vibrio cholerae Intra- and Interspecific Competitive Advantages

The type VI secretion system (T6SS) mediates protein translocation across the cell membrane of Gram-negative bacteria, including Vibrio cholerae – the causative agent of cholera. All V. cholerae strains examined to date harbor gene clusters encoding a T6SS. Structural similarity and sequence homology between components of the T6SS and the T4 bacteriophage cell-puncturing device suggest that the T6SS functions as a contractile molecular syringe to inject effector molecules into prokaryotic and eukaryotic target cells. Regulation of the T6SS is critical. A subset of V. cholerae strains, including the clinical O37 serogroup strain V52, express T6SS constitutively. In contrast, pandemic strains impose tight control that can be genetically disrupted: mutations in the quorum sensing gene luxO and the newly described regulator gene tsrA lead to constitutive T6SS expression in the El Tor strain C6706. In this report, we examined environmental V. cholerae isolates from the Rio Grande with regard to T6SS regulation. Rough V. cholerae lacking O-antigen carried a nonsense mutation in the gene encoding the global T6SS regulator VasH and did not display virulent behavior towards Escherichia coli and other environmental bacteria. In contrast, smooth V. cholerae strains engaged constitutively in type VI-mediated secretion and displayed virulence towards prokaryotes (E. coli and other environmental bacteria) and a eukaryote (the social amoeba Dictyostelium discoideum). Furthermore, smooth V. cholerae strains were able to outcompete each other in a T6SS-dependent manner. The work presented here suggests that constitutive T6SS expression provides V. cholerae with an advantage in intraspecific and interspecific competition.

VasH Is a Transcriptional Regulator of the Type VI Secretion System Functional in Endemic and Pandemic Vibrio cholerae

The gram-negative bacterium Vibrio cholerae is the etiological agent of cholera, a disease characterized by the release of high volumes of watery diarrhea. Many medically important proteobacteria, including V. cholerae, carry one or multiple copies of the gene cluster that encodes the bacterial type VI secretion system (T6SS) to confer virulence or interspecies competitiveness. Structural similarity and sequence homology between components of the T6SS and the cell-puncturing device of T4 bacteriophage suggest that the T6SS functions as a molecular syringe to inject effector molecules into prokaryotic and eukaryotic target cells. Although our understanding of how the structural T6SS apparatus assembles is developing, little is known about how this system is regulated. Here, we report on the contribution of the activator of the alternative sigma factor 54, VasH, as a global regulator of the V. cholerae T6SS. Using bioinformatics and mutational analyses, we identified domains of the VasH polypeptide that are essential for its ability to initiate transcription of T6SS genes and established a universal role for VasH in endemic and pandemic V. cholerae strains.

Type VI secretion system regulation as a consequence of evolutionary pressure.

The type VI secretion system (T6SS) is a mechanism evolved by Gram-negative bacteria to negotiate interactions with eukaryotic and prokaryotic competitors. T6SSs are encoded by a diverse array of bacteria and include plant, animal, human and fish pathogens, as well as environmental isolates. As such, the regulatory mechanisms governing T6SS gene expression vary widely from species to species, and even from strain to strain within a given species. This review concentrates on the four bacterial genera that the majority of recent T6SS regulatory studies have been focused on: Vibrio, Pseudomonas, Burkholderia and Edwardsiella.