Quorum sensing regulation confronts the development of a viable but non-culturable state in Vibrio cholerae

Vibrio cholerae can enter a viable but non-culturable (VBNC) state when it encounters unfavourable environments; VBNC cells serve as important reservoirs and still pose threats to public health. The genetic regulation of V. cholerae entering its VBNC state is not well understood. Here, we show a confrontation strategy adapted by V. cholerae O1 in which it utilizes a quorum sensing (QS) system to prevent transition into a VBNC state under low nutrition and temperature conditions. The upregulation of hapR resulted in a prolonged culturable state of V. cholerae in artificial sea water at 4°C, whereas the mutation of hapR led to fast entry into the VBNC state. We also observed that different V. cholerae O1 natural isolates with distinct QS functions present a variety of abilities to maintain culturability during the transition to a VBNC state. The strain groups with higher or constitutive expression of QS genes exhibit a greater tendency to maintain the culturable state during VBNC induction than those lacking QS functional groups. In summary, HapR-mediated QS regulation is associated with the transition to the VBNC state in V. cholerae. HapR expression causes V. cholerae to resist VBNC induction and become dominant over competitors in changing environments.     

Characterization of the Vibrio cholerae vexAB and vexCD efflux systems

Vibrio cholerae is an important human pathogen that causes the diarrheal disease cholera. Colonization of the human host is dependent upon coordinated expression of several virulence factors in response to as yet unknown environmental cues. Bile acids have been implicated in the in vitro regulation of several V. cholerae genes, including those involved in motility, chemotaxis, outer membrane protein production, and virulence factor production. Bile is toxic to bacteria and colonization of the intestinal tract is dependent upon bacterial resistance to bile acids. We have identified and characterized two bile-regulated RND-family efflux systems, named here vexAB and vexCD, that are involved in V. cholerae bile resistance. Mutational analysis revealed that the vexAB system is responsible for in vitro intrinsic resistance of V. cholerae to multiple antimicrobial compounds, including bile acids. In contrast, the vexCD efflux system was specific for certain bile acids and detergents and functioned in conjunction with the vexAB system to provide V. cholerae with high-level bile resistance. Mutants containing deletion of vexB, vexD, and vexB–vexD were able to efficiently colonize the infant mouse suggesting that these efflux systems were dispensable for V. cholerae growth in the small intestines of infant mice.     

The Vibrio cholerae type VI secretion system: toxins,regulators and consequences

The type VI secretion system (T6SS) is a proteinaceous weapon used by many Gram-negative bacteria to deliver toxins into adjacent target cells. Vibrio cholerae, the bacterium responsible for the fatal water-borne cholera disease, uses the T6SS to evade phagocytic eukaryotes, cause intestinal inflammation, and compete against other bacteria with toxins that disrupt lipid membranes, cell walls and actin cytoskeletons. The control of T6SS genes varies among V. cholerae strains and typically includes inputs from external signals and cues, such as quorum sensing and chitin availability. In the following review, we highlight the repertoire of toxic T6SS effectors and the diverse genetic regulation networks among different isolates of V. cholerae. Finally, we discuss the roles played by the T6SS of V. cholerae in both natural environments and hosts.     

Isolation,identification, and characterization of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> from the Danube River in Slovakia

The occurrence of Vibrio cholerae, an important aquatic pathogen, was assessed in the surface water of the Danube River near Bratislava. The isolates were distinguished by biochemical tests and grouped by ARDRA to three clusters corresponding to three species (V. cholerae, Vibrio metschnikovii, and Aeromonas spp.). The identification of V. cholerae was confirmed by multiplex PCR using primer pairs targeted to ompW gene (membrane protein), ctxA gene (toxicity gene), and toxR gene (regulatory gene). None from the isolated V. cholerae from surface water contained ctxA gene; seven of them possessed toxR gene. Serotyping of V. cholerae isolates with polyvalent O antiserum and O/139 antiserum was negative. All isolates of V. cholerae were susceptible to chloramphenicol, rifampicin, tetracycline, variable to ampicillin, and resistant to kanamycin and streptomycin.     

Functional RelBE-Family Toxin-Antitoxin Pairs Affect Biofilm Maturation and Intestine Colonization in Vibrio cholerae

Toxin–antitoxin (TA) systems are small genetic elements that typically encode a stable toxin and its labile antitoxin. These cognate pairs are abundant in prokaryotes and have been shown to regulate various cellular functions. Vibrio cholerae, a human pathogen that is the causative agent of cholera, harbors at least thirteen TA loci. While functional HigBA, ParDE have been shown to stabilize plasmids and Phd/Doc to mediate cell death in V. cholerae, the function of seven RelBE-family TA systems is not understood. In this study we investigated the function of the RelBE TA systems in V. cholerae physiology and found that six of the seven relBE loci encoded functional toxins in E. coli. Deletion analyses of each relBE locus indicate that RelBE systems are involved in biofilm formation and reactive oxygen species (ROS) resistance. Interestingly, all seven relBE loci are induced under the standard virulence induction conditions and two of the relBE mutants displayed a colonization defect, which was not due to an effect on virulence gene expression. Although further studies are needed to characterize the mechanism of action, our study reveals that RelBE systems are important for V. cholerae physiology.     

Competence and natural transformation in vibrios

Natural transformation is a major mechanism of horizontal gene transfer in bacteria. By incorporating exogenous DNA elements into chromosomes, bacteria are able to acquire new traits that can enhance their fitness in different environments. Within the past decade, numerous studies have revealed that natural transformation is prevalent among members of the Vibrionaceae, including the pathogen Vibrio cholerae. Four environmental factors: (i) nutrient limitation, (ii) availability of extracellular nucleosides, (iii) high cell density and (iv) the presence of chitin, promote genetic competence and natural transformation in Vibrio cholerae by co‐ordinating expression of the regulators CRP, CytR, HapR and TfoX respectively. Studies of other Vibrionaceae members highlight the general importance of natural transformation within this bacterial family.     

Culturable and VBNC <Emphasis Type="Italic">Vibrio cholerae</Emphasis>: Interactions with Chironomid Egg Masses and Their Bacterial Population

Vibrio cholerae, the etiologic agent of cholera, is autochthonous to various aquatic environments. Recently, it was found that chironomid (nonbiting midges) egg masses serve as a reservoir for the cholera bacterium and that flying chironomid adults are possible windborne carriers of V. cholerae non-O1 non-O139. Chironomids are the most widely distributed insect in freshwater. Females deposit egg masses at the water's edge, and each egg mass contains eggs embedded in a gelatinous matrix. Hemagglutinin/protease, an extracellular enzyme of V. cholerae, was found to degrade chironomid egg masses and to prevent them from hatching. In a yearly survey, chironomid populations and the V. cholerae in their egg masses followed phenological succession and interaction of host–pathogen population dynamics. In this report, it is shown via FISH technique that most of the V. cholerae inhabiting the egg mass are in the viable but nonculturable (VBNC) state. The diversity of culturable bacteria from chironomid egg masses collected from two freshwater habitats was determined. In addition to V. cholerae, representatives of the following genera were isolated: Acinetobacter, Aeromonas, Klebsiella, Shewanella, Pseudomonas, Paracoccus, Exiguobacterium, and unidentified bacteria. Three important human pathogens, Aeromonas veronii, A. caviae, and A. hydrophila, were isolated from chironomid egg masses, indicating that chironomid egg masses may be a natural reservoir for pathogenic Aeromonas species in addition to V. cholerae. All isolates of V. cholerae were capable of degrading chironomid egg masses. This may help explain their host–pathogen relationship with chironomids. In contrast, almost none of the other bacteria that were isolated from the egg masses possessed this ability. Studying the interaction between chironomid egg masses, the bacteria inhabiting them, and V. cholerae could contribute to our understanding of the nature of the V. cholerae–egg mass interactions.     

Characterization of Vibrio cholerae O139 Synonym Bengal Isolated from Patients with Cholera-Like Disease in Bangladesh

Vibrio cholerae O139 (synonym Bengal), a novel serovar of V. cholerae, is the causative agent of large outbreaks of cholera-like illness currently sweeping India and Bangladesh. Eight randomly selected V. cholerae O139 isolates were studied for their biological properties, which were compared with those of V. cholerae O1 and other V. cholerae non-O1. The V. cholerae O139 isolates were characterized by the production of large amount of cholera toxin, hemagglutination, weak hemolytic properties, resistance to polymyxin B, lysogeny with, and production of, kappa type phage (4/8 isolates only), and resistance to both classical and El Tor-specific phages. Thus, V. cholerae O139 isolates had an overall similarity with V. cholerae O1 El Tor.     

Occurrence and Expression of Luminescence in Vibrio cholerae

Several species of the genus Vibrio, including Vibrio cholerae, are bioluminescent or contain bioluminescent strains. Previous studies have reported that only 10% of V. cholerae strains are luminescent. Analysis of 224 isolates of non-O1/non-O139 V. cholerae collected from Chesapeake Bay, MD, revealed that 52% (116/224) were luminescent when an improved assay method was employed and 58% (130/224) of isolates harbored the luxA gene. In contrast, 334 non-O1/non-O139 V. cholerae strains isolated from two rural provinces in Bangladesh yielded only 21 (6.3%) luminescent and 35 (10.5%) luxA⁺ isolates. An additional 270 clinical and environmental isolates of V. cholerae serogroups O1 and O139 were tested, and none were luminescent or harbored luxA. These results indicate that bioluminescence may be a trait specific for non-O1/non-O139 V. cholerae strains that frequently occur in certain environments. Luminescence expression patterns of V. cholerae were also investigated, and isolates could be grouped based on expression level. Several strains with defective expression of the lux operon, including natural K variants, were identified.     

Cloning and expression in Escherichia coli of a recA-like gene from Vibrio cholerae

Summary A library containing more than 80% of the Vibrio cholerae genome was constructed by cloning BamH1 restriction fragments into pBR322. Using interspecific complementation of an Escherichia coli recA mutant with plasmids containing the gene bank of V. cholerae, a recA-like gene was identified. The recombinant plasmid, designated as pDP145, contained a 1.45 kb segment of V. cholerae DNA which codes for a protein of molecular weight 39,000. The product of this gene confers methyl methane sulphonate resistance on the E. coli recA mutant, suppresses its ultraviolet (UV) light sensitive phenotype and has proteolytic activity on the phage repressor. Induction of a 39,000 dalton protein in UV-irradiated V. cholerae cells was demonstrated.     

Hsp33 confers bleach resistance by protecting elongation factor Tu against oxidative degradation in Vibrio cholerae

The redox‐regulated chaperone Hsp33 protects bacteria specifically against stress conditions that cause oxidative protein unfolding, such as treatment with bleach or exposure to peroxide at elevated temperatures. To gain insight into the mechanism by which expression of Hsp33 confers resistance to oxidative protein unfolding conditions, we made use of Vibrio cholerae strain O395 lacking the Hsp33 gene hslO. We found that this strain, which is exquisitely bleach‐sensitive, displays a temperature‐sensitive (ts) phenotype during aerobic growth, implying that V. cholerae suffers from oxidative heat stress when cultivated at 43°C. We utilized this phenotype to select for Escherichia coli genes that rescue the ts phenotype of V. cholerae ΔhslO when overexpressed. We discovered that expression of a single protein, the elongation factor EF‐Tu, was sufficient to rescue both the ts and bleach‐sensitive phenotypes of V. cholerae ΔhslO. In vivo studies revealed that V. cholerae EF‐Tu is highly sensitive to oxidative protein degradation in the absence of Hsp33, indicating that EF‐Tu is a vital chaperone substrate of Hsp33 in V. cholerae. These results suggest an ‘essential client protein’ model for Hsp33's chaperone action in Vibrio in which stabilization of a single oxidative stress‐sensitive protein is sufficient to enhance the oxidative stress resistance of the whole organism.     

High genetic diversity of Vibrio cholerae in the European lake Neusiedler See is associated with intensive recombination in the reed habitat and the long‐distance transfer of strains

Coastal marine Vibrio cholerae populations usually exhibit high genetic diversity. To assess the genetic diversity of abundant V. cholerae non‐O1/non‐O139 populations in the Central European lake Neusiedler See, we performed a phylogenetic analysis based on recA, toxR, gyrB and pyrH loci sequenced for 472 strains. The strains were isolated from three ecologically different habitats in a lake that is a hot‐spot of migrating birds and an important bathing water. We also analyzed 76 environmental and human V. cholerae non‐O1/non‐O139 isolates from Austria and other European countries and added sequences of seven genome‐sequenced strains. Phylogenetic analysis showed that the lake supports a unique endemic diversity of V. cholerae that is particularly rich in the reed stand. Phylogenetic trees revealed that many V. cholerae isolates from European countries were genetically related to the strains present in the lake belonging to statistically supported monophyletic clades. We hypothesize that the observed phenomena can be explained by the high degree of genetic recombination that is particularly intensive in the reed stand, acting along with the long distance transfer of strains most probably via birds and/or humans. Thus, the Neusiedler See may serve as a bioreactor for the appearance of new strains with new (pathogenic) properties.