It is now well accepted that Vibrio cholerae, the causative agent of the water-borne disease cholera, is acquired from environmental sources where it persists between outbreaks of the disease. Recent advances in molecular technology have demonstrated that this bacterium could be detected in areas where it had not been isolated from before, indicating a much broader, global distribution of this bacterium rather than specifically within regions where cholera is endemic. The environmental persistence of V. cholerae in the aquatic environment can be attributed to multiple intra- and interspecific strategies such as responsive gene regulation and biofilm formation on biotic and abiotic surfaces, as well as interactions with a multitude of other organisms. This review will discuss some of the mechanisms that enable the persistence of the bacterium in the sometimes hostile environment. In particular, we will discuss how V. cholerae can survive stressors such as starvation, temperature and salinity fluctuations as well as how the organism persists under constant predation by heterotrophic protists.

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Environmental reservoirs and mechanisms of persistence of Vibrio cholerae.

Vibrio cholerae can switch between motile and biofilm lifestyles. The last decades have been marked by a remarkable increase in our knowledge of the structure, regulation, and function of biofilms formed under laboratory conditions. Evidence has grown suggesting that V. cholerae can form biofilm-like aggregates during infection that could play a critical role in pathogenesis and disease transmission. However, the structure and regulation of biofilms formed during infection, as well as their role in intestinal colonization and virulence, remains poorly understood. Here, we review (i) the evidence for biofilm formation during infection, (ii) the coordinate regulation of biofilm and virulence gene expression, and (iii) the host signals that favor V. cholerae transitions between alternative lifestyles during intestinal colonization, and (iv) we discuss a model for the role of V. cholerae biofilms in pathogenicity.

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Vibrio cholerae Biofilms and Cholera Pathogenesis

Vibrio cholerae is a human pathogen that causes the life-threatening diarrheal disease cholera. A type VI secretion system (T6SS) was recently shown to be required for full virulence in the O37 serogroup strain V52, which causes only sporadic human disease, but T6SS is not expressed in seventh pandemic O1 El Tor strains under standard laboratory conditions. In this study, we show that in the O1 El Tor strain C6706, T6SS is repressed by both quorum sensing and the uncharacterized protein VC0070 (TsrA). Disruption of TsrA and the quorum sensing regulator LuxO induces expression and secretion of the T6SS substrate Hcp, and this is dependent on the downstream regulator HapR, which directly binds to the promoter region of the T6SS genes hcp1 and hcp2 to induce expression. The activated T6SS in C6706 is functional and can translocate the effector protein VgrG-1 into macrophage cells, and T6SS activation leads to fecal diarrhea and intestinal inflammation in infant rabbits. Using an infant mouse infection model, we show that deletion of tsrA results in a 9.3-fold increase in intestinal colonization compared with wild type. TsrA functions as a global regulator to activate expression of hemagglutinin protease and repress cholera toxin and toxin coregulated pilus. Our findings provide significant insight into the molecular mechanism of T6SS and ToxT regulon gene regulation by quorum sensing and TsrA.

https://www.pnas.org/content/pnas/107/49/21128.full.pdf

Quorum sensing and a global regulator TsrA control expression of type VI secretion and virulence in Vibrio cholerae

About one quarter to one third of all bacterial genes encode proteins of the inner or outer bacterial membrane. These proteins perform essential physiological functions, such as the import or export of metabolites, the homeostasis of metal ions, the extrusion of toxic substances or antibiotics, and the generation or conversion of energy. The last years have witnessed completion of a plethora of whole-genome sequences of bacteria important for biotechnology or medicine, which is the foundation for proteome and other functional genome analyses. In this review, we discuss the challenges in membrane proteome analysis, starting from sample preparation and leading to MS-data analysis and quantification. The current state of available proteomics technologies as well as their advantages and disadvantages will be described with a focus on shotgun proteomics. Then, we will briefly introduce the most abundant proteins and protein families present in bacterial membranes before bacterial membrane proteomics studies of the last years will be presented. It will be shown how these works enlarged our knowledge about the physiological adaptations that take place in bacteria during fine chemical production, bioremediation, protein overexpression, and during infections. Furthermore, several examples from literature demonstrate the suitability of membrane proteomics for the identification of antigens and different pathogenic strains, as well as the elucidation of membrane protein structure and function.

Bacterial membrane proteomics

Vibrio fluvialis is a pathogen commonly found in coastal environs. Considering recent increase in numbers of diarrheal outbreaks and sporadic extraintestinal cases, V. fluvialis has been considered as an emerging pathogen. Though this pathogen can be easily isolated by existing culture methods, its identification is still a challenging problem due to close phenotypic resemblance either with Vibrio cholerae or Aeromonas spp. However, using molecular tools, it is easy to identify V. fluvialis from clinical and different environmental samples. Many putative virulence factors have been reported, but its mechanisms of pathogenesis and survival fitness in the environment are yet to be explored. This chapter covers some of the major discoveries that have been made to understand the importance of V. fluvialis.

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Vibrio fluvialis: an emerging human pathogen.

IEM101, a Vibrio cholerae O1 El Tor Ogawa strain naturally deficient in CTXPhi, was previously selected as a live cholera vaccine candidate. To make a better and safer vaccine that can induce protective immunity against both the bacteria and cholera toxin (CT), a new vaccine candidate, IEM108, was constructed by introducing a ctxB gene and an El Tor-derived rstR gene into IEM101. The ctxB gene codes for the protective antigen CTB subunit, and the rstR gene mediates phage immunity. The stable expression of the two genes was managed by a chromosome-plasmid lethal balanced system based on the housekeeping gene thyA. Immunization studies indicate that IEM108 generates good immune responses against both the bacteria and CT. After a single-dose intraintestinal vaccination with 10(9) CFU of IEM108, both anti-CTB immunoglobulin G and vibriocidal antibodies were detected in the immunized-rabbit sera. However, only vibriocidal antibodies are detected in rabbits immunized with IEM101. In addition, IEM108 but not IEM101 conferred full protection against the challenges of four wild-type toxigenic strains of V. cholerae O1 and 4 micro g of CT protein in a rabbit model. By introducing the rstR gene, the frequency of conjugative transfer of a recombinant El Tor-derived RS2 suicidal plasmid to IEM108 was decreased 100-fold compared to that for IEM101. This indicated that the El Tor-derived rstR cloned in IEM108 was fully functional and could effectively inhibit the El Tor-derived CTXPhi from infecting IEM108. Our results demonstrate that IEM108 is an efficient and safe live oral cholera vaccine candidate that induces antibacterial and antitoxic immunity and CTXPhi phage immunity.

https://iai.asm.org/content/iai/71/10/5498.full.pdf

Construction and Evaluation of a Safe, Live, Oral Vibrio cholerae Vaccine Candidate, IEM108

Production of the Zn-metalloprotease hemagglutinin (HA)/protease by Vibrio cholerae has been reported to enhance enterotoxicity in rabbit ileal loops and the reactogenicity of live cholera vaccine candidates. Expression of HA/protease requires the alternate sigma factor S (RpoS), encoded by rpoS. The histone-like nucleoid structuring protein (H-NS) has been shown to repress rpoS expression in Escherichia coli .I nV. cholerae strains of the classical biotype, H-NS has been reported to silence virulence gene expression. In this study we examined the role of H-NS in the expression of HA/protease and motility in an El Tor biotype strain by constructing a hns mutant. The hns mutant exhibited multiple phenotypes, such as production of cholera toxin in nonpermissive LB medium, reduced resistance to high osmolarity, enhanced resistance to low pH and hydrogen peroxide, and reduced motility. Depletion of H-NS by overexpression of a dominant-negative allele or by deletion of hns resulted in diminished expression of HA/protease. Epistasis analysis of HA/protease expression in hns, rpoS, and hns rpoS mutants, analysis of RpoS reporter fusions, quantitative reverse transcriptionPCR measurements, and ectopic expression of RpoS in rpoS and rpoS hns mutants showed that H-NS posttranscriptionally enhances RpoS expression. The hns mutant exhibited a lower degree of motility and lower levels of expression of flaA, flaC, cheR-2, and motX mRNAs than the wild type. Comparison of the mRNA abundances of these genes in wild-type, hns, rpoS, and hns rpoS strains revealed that deletion of rpoS had a more severe negative effect on their expression. Interestingly, deletion of hns in the rpoS background resulted in higher expression levels of flaA, flaC, and motX, suggesting that H-NS represses the expression of these genes in the absence of S . Finally, we show that the cyclic AMP receptor protein and H-NS act along the same pathway to positively affect RpoS expression.

Role of the Histone-Like Nucleoid Structuring Protein in the Regulation of rpoS and RpoS-Dependent Genes in Vibrio cholerae

The pathogen Vibrio cholerae causes severe diarrheal disease in humans. This environmental inhabitant has two distinct life cycles, in the environment and in the human small intestine, in which it differs in its multiplication behavior and virulence expression. Anaerobiosis, limitation of some nutrient elements, and excess burden from host metabolism reactants are the major stresses for V. cholerae living in intestine, in comparison to conditions in the environment and laboratory medium. For an insight into the response of V. cholerae to different microenvironments, we cultured the bacteria in aerobic and anaerobic conditions, and compared the whole cell proteome by two-dimensional electrophoresis. Among the protein spots identified, some protein species involved in aerobic respiration and the nutrient carbohydrate transporters were found to be more abundant in aerobic conditions, and some enzymes for anaerobic respiration and some stress response proteins were found more abundant in anaerobic culture. One spot corresponding to flagellin B subunit was decreased in anaerobic conditions, which suggests correlation with the meticulous regulation of bacterial motility during infection in the host intestine. This proteome analysis is the starting point for in-depth understanding of V. cholerae behavior in different environments.

Proteome comparison of Vibrio cholerae cultured in aerobic and anaerobic conditions

Quorum sensing (QS) is a process by which individual bacteria are able to communicate with one another, thereby enabling the population as a whole to coordinate gene regulation and subsequent phenotypic outcomes. Communication is accomplished through production and detection of small molecules in the extracellular milieu. In many bacteria, particularly Vibrio species, multiple QS systems result in multiple signals, as well as cross talk between systems. In this study, we identify two QS systems in the halophilic enteric pathogen Vibrio fluvialis: one acyl-homoserine lactone (AHL) based and one CAI-1/AI-2 based. We show that a LuxI homolog, VfqI, primarily produces 3-oxo-C10-HSL, which is sensed by a LuxR homolog, VfqR. VfqR-AHL is required to activate vfqI expression and autorepress vfqR expression. In addition, we have shown that similar to that in V. cholerae and V. harveyi, V. fluvialis produces CAI-1 and AI-2 signal molecules to activate the expression of a V. cholerae HapR homolog through LuxO. Although VfqR-AHL does not regulate hapR expression, HapR can repress vfqR transcription. Furthermore, we found that QS in V. fluvialis positively regulates production of two potential virulence factors, an extracellular protease and hemolysin. QS also affects cytotoxic activity against epithelial tissue cultures. These data suggest that V. fluvialis integrates QS regulatory pathways to play important physiological roles in pathogenesis.

Quorum sensing regulatory cascades control Vibrio fluvialis pathogenesis

Bacteriophage VP4 is a lytic phage of the Vibrio cholerae serogroup O1, and it is used in phage subtyping of V. cholerae biotype El Tor. Studies of phage infection mechanisms will promote the understanding of the basis of phage subtyping as well as the genetic differences between sensitive and resistant strains. In this study, we investigated the receptor that phage VP4 uses to bind to El Tor strains of V. cholerae and found that it infects strains through adsorbing the O antigen of V. cholerae O1. In some natural isolates that are resistant to VP4 infection, mutations were identified in the wb* cluster (O-antigen gene cluster), which is responsible for the biosynthesis of O antigen. Mutations in the manB, wbeE, and wbeU genes caused failure of adsorption of VP4 to these strains, whereas the observed amino acid residue mutations within wbeW and manC have no effect on VP4 infection. Additionally, although mutations in two resistant strains were found only in manB and wbeW, complementing both genes did not restore sensitivity to VP4 infection, suggesting that other resistance mechanisms may exist. Therefore, the mechanism of VP4 infection may provide a basis for subtyping the phage. Elaborate mutations of the O antigen may imbue V. cholerae strains with resistance to phage infection.

Weili Liang

Papers

Construction and Evaluation of a Safe, Live, Oral Vibrio cholerae Vaccine Candidate, IEM108

Role of the Histone-Like Nucleoid Structuring Protein in the Regulation of rpoS and RpoS-Dependent Genes in Vibrio cholerae

Proteome comparison of Vibrio cholerae cultured in aerobic and anaerobic conditions

Quorum sensing regulatory cascades control Vibrio fluvialis pathogenesis

O Antigen Is the Receptor of Vibrio cholerae Serogroup O1 El Tor Typing Phage VP4