Role of ectoine in Vibrio cholerae osmoadaptation

Vibrio cholerae is both an intestinal pathogen and a microbe in the estuarine community. To persist in the estuarine environment, V. cholerae must adjust to changes in ionic composition and osmolarity. These changes in the aquatic environment have been correlated with cholera epidemics. In this work, we study the response of V. cholerae to increases in environmental osmolarity. Optimal growth of V. cholerae in minimal medium requires supplementation with 200 mM NaCl and KCl. However, when the NaCl concentration is increased beyond 200 mM, a proportionate delay in growth is observed. During this delay in growth, osmotic equilibrium is reached by cytoplasmic accumulation of small, uncharged solutes that are compatible with growth. We show that synthesis of the compatible solute ectoine and transport of the compatible solute glycine betaine impact the length of the osmoadaptive growth delay. We also demonstrate that high-osmolarity-adapted V. cholerae displays a growth advantage when competed against unadapted cells in high-osmolarity medium. In contrast, low-osmolarity-adapted V. cholerae displays no growth advantage when competed against high-osmolarity-adapted cells in low-osmolarity medium. These results may have implications for V. cholerae population dynamics when seawater and freshwater and their attendant microbes mix.     

Role of sodium bioenergetics in Vibrio cholerae

The ability of the bacterium to use sodium in bioenergetic processes appears to play a key role in both the environmental and pathogenic phases of Vibrio cholerae. Aquatic environments, including fresh, brackish, and coastal waters, are an important factor in the transmission of cholera and an autochthonous source. The organism is considered to be halophilic and has a strict requirement for Na(+) for growth. Furthermore, expression of motility and virulence factors of V. cholerae is intimately linked to sodium bioenergetics and to each other. Several lines of evidence indicated that the activity of the flagellum of V. cholerae might have an impact on virulence gene regulation. As the V. cholerae flagellum is sodium-driven and the Na(+)-NQR enzyme is known to create a sodium motive force across the bacterial membrane, it was recently suggested that the increased toxT expression observed in a nqr-negative strain is mediated by affecting flagella activity. It was suggested that the V. cholerae flagellum might respond to changes in membrane potential and the resulting changes in flagellar rotation might serve as a signal for virulence gene expression. However, we recently demonstrated that although the flagellum of V. cholerae is not required for the effects of ionophores on virulence gene expression, changes in the sodium chemical potential are sensed and thus alternative mechanisms, perhaps involving the TcpP/H proteins, for the detection of these conditions must exist. Analyzing the underlying mechanisms by which bacteria respond to changes in the environment, such as their ability to monitor the level of membrane potential, will probably reveal complex interplays between basic physiological processes and virulence factor expression in a variety of pathogenic species.     

Role of Ectoine in Vibrio cholerae Osmoadaptation

Vibrio cholerae is both an intestinal pathogen and a microbe in the estuarine community. To persist in the estuarine environment, V. cholerae must adjust to changes in ionic composition and osmolarity. These changes in the aquatic environment have been correlated with cholera epidemics. In this work, we study the response of V. cholerae to increases in environmental osmolarity. Optimal growth of V. cholerae in minimal medium requires supplementation with 200 mM NaCl and KCl. However, when the NaCl concentration is increased beyond 200 mM, a proportionate delay in growth is observed. During this delay in growth, osmotic equilibrium is reached by cytoplasmic accumulation of small, uncharged solutes that are compatible with growth. We show that synthesis of the compatible solute ectoine and transport of the compatible solute glycine betaine impact the length of the osmoadaptive growth delay. We also demonstrate that high-osmolarity-adapted V. cholerae displays a growth advantage when competed against unadapted cells in high-osmolarity medium. In contrast, low-osmolarity-adapted V. cholerae displays no growth advantage when competed against high-osmolarity-adapted cells in low-osmolarity medium. These results may have implications for V. cholerae population dynamics when seawater and freshwater and their attendant microbes mix.     

The Function of the Na+-Driven Flagellum of Vibrio cholerae Is Determined by Osmolality and pH

Vibrio cholerae is motile by its polar flagellum, which is driven by a Na⁺-conducting motor. The stators of the motor, composed of four PomA and two PomB subunits, provide access for Na⁺ to the torque-generating unit of the motor. To characterize the Na⁺ pathway formed by the PomAB complex, we studied the influence of chloride salts (chaotropic, Na⁺, and K⁺) and pH on the motility of V. cholerae. Motility decreased at elevated pH but increased if a chaotropic chloride salt was added, which rules out a direct Na⁺ and H⁺ competition in the process of binding to the conserved PomB D23 residue. Cells expressing the PomB S26A/T or D42N variants lost motility at low Na⁺ concentrations but regained motility in the presence of 170 mM chloride. Both PomA and PomB were modified by N,N′-dicyclohexylcarbodiimide (DCCD), indicating the presence of protonated carboxyl groups in the hydrophobic regions of the two proteins. Na⁺ did not protect PomA and PomB from this modification. Our study shows that both osmolality and pH have an influence on the function of the flagellum from V. cholerae. We propose that D23, S26, and D42 of PomB are part of an ion-conducting pathway formed by the PomAB stator complex.     

Role of surface proteins in Vibrio cholerae attachment to chitin

The role of surface proteins in Vibrio cholerae attachment to chitin particles in vitro was studied. Treatment of V. cholerae O1 ATCC 14034 and ATCC 14035 with pronase E reduced the attachment of bacteria to chitin particles by 57 to 77%. A statistically significant reduction was also observed when the attachment to chitin was evaluated in the presence of homologous Sarkosyl-insoluble membrane proteins (MPs) (67 to 84%), N-acetylglucosamine (GlcNAc) (62%), the sugar that makes up chitin, and wheat germ agglutinin (40 to 56%), a lectin that binds GlcNAc. The soluble oligomers N,N'-diacetylchitobiose or N,N', N"-triacetylchitotriose caused an inhibition of 14 to 23%. Sarkosyl-insoluble MPs able to bind chitin particles were isolated and visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; two of these peptides (molecular sizes, 36 and 53 kDa) specifically bind GlcNAc.     

Role of rpoS in Stress Survival and Virulence of Vibrio cholerae

Vibrio cholerae is known to persist in aquatic environments under nutrient-limiting conditions. To analyze the possible involvement of the alternative sigma factor encoded by rpoS, which is shown to be important for survival during nutrient deprivation in several other bacterial species, a V. cholerae rpoS homolog was cloned by functional complementation of an Escherichia coli mutant by using a wild-type genomic library. Sequence analysis of the complementing clone revealed an 1.008-bp open reading frame which is predicted to encode a 336-amino-acid protein with 71 to 63% overall identity to other reported rpoS gene products. To determine the functional role of rpoS in V. cholerae, we inactivated rpoS by homologous recombination. V. cholerae strains lacking rpoS are impaired in the ability to survive diverse environmental stresses, including exposure to hydrogen peroxide, hyperosmolarity, and carbon starvation. These results suggest that rpoS may be required for the persistence of V. cholerae in aquatic habitats. In addition, the rpoS mutation led to reduced production or secretion of hemagglutinin/protease. However, rpoS is not critical for in vivo survival, as determined by an infant mouse intestinal competition assay.     

Role of ToxS in the proteolytic cascade of virulence regulator ToxR in Vibrio cholerae

Two of the primary virulence regulators of Vibrio cholerae, ToxR and TcpP, function together with cognate effector proteins. ToxR undergoes regulated intramembrane proteolysis (RIP) during late stationary phase in response to nutrient limitation at alkaline pH; however, the specific function of its cognate ToxS remains unresolved. In this work, we found that ToxR rapidly becomes undetectable in a ΔtoxS mutant when cultures are exposed to either starvation conditions or after alkaline pH shock individually. A ΔtoxS mutant enters into a dormant state associated with the proteolysis of ToxR at a faster rate than wild‐type, closely resembling a ΔtoxR mutant. Using a mutant with a periplasmic substitution in ToxS, we found that the proteases DegS and DegP function additively with VesC and a novel protease, TapA, to degrade ToxR in the mutant. Overall, the results shown here reveal a role for ToxS in the stabilization of ToxR by protecting the virulence regulator from premature proteolysis.     

Role of cyanobacteria in the persistence of Vibrio cholerae O139 in saline microcosms

Recently, a new strain of cholera, Vibrio cholerae O139, has emerged as an epidemic strain, but there is little information about its environmental reservoir. The present investigation was aimed to determine the role of cyanobacteria in the persistence of V. cholerae O139 in microcosms. An environmental isolate of V. cholerae O139 and three cyanobacteria (Anabaena sp., Nostoc sp., and Hapalosiphon sp.) were used in this study. Survival of culturable V. cholerae O139 in microcosms was monitored using taurocholate-tellurite gelatin agar medium. Viable but nonculturable V. cholerae O139 were detected using a fluorescent antibody technique. Vibrio cholerae O139 could be isolated for up to 12 days in a culturable form in association with cyanobacteria but could not be isolated in the culturable form after 2 days from control water without cyanobacteria. The viable but nonculturable V. cholerae O139 could be detected in association with cyanobacteria for up to 15 months. These results, therefore, suggest that cyanobacteria can act as a long-term reservoir of V. cholerae O139 in an aquatic environment.     

Toxins of Vibrio cholerae

Surveyed in the paper are published data on properties, biological activity, genetic determinants and action mechanisms of recently known toxins produced by different strains of Vibrio cholerae irrespectively of their capacity for the synthesis of choleric toxin--the main virulence factor. Their possible importance both for the general clinical pattern of cholera provoked by cholerogenic agents and as independent virulence factors causing diarrhea without cholera is elucidated. The sets and levels of expression of additional toxins can differ for different pathogenic clones and they can correspondingly condition degrees of their epidemic and etiological safety.     

The Zymovars of Vibrio cholerae: multilocus enzyme electrophoresis of Vibrio cholerae

Zymovars analysis also known as multilocus enzyme electrophoresis is applied here to investigate the genetic variation of Vibrio cholerae strains and characterise strains or group of strains of medical and epidemiological interest. Fourteen loci were analyzed in 171 strains of non-O1 non-O139, 32 classical and 61 El Tor from America, Africa, Europe and Asia. The mean genetic diversity was 0.339. It is shown that the same O antigen (both O1 and non-O1) may be present in several genetically diverse (different zymovars) strains. Conversely the same zymovar may contain more than one serogroup. It is confirmed that the South American epidemic strain differs from the 7th pandemic El Tor strain in locus LAP (leucyl leucyl aminopeptidase). Here it is shown that this rare allele is present in 1 V. mimicus and 4 non-O1 V. cholerae. Non toxigenic O1 strains from South India epidemic share zymovar 14A with the epidemic El Tor from the 7th pandemic, while another group have diverse zymovars. The sucrose negative epidemic strains isolated in French Guiana and Brazil have the same zymovar of the current American epidemic V. cholerae.     

Role of the Vibrio cholerae Matrix Protein Bap1 in Cross-Resistance to Antimicrobial Peptides

Outer membrane vesicles (OMVs) that are released from Gram-negative pathogenic bacteria can serve as vehicles for the translocation of effectors involved in infectious processes. In this study we have investigated the role of OMVs of the Vibrio cholerae O1 El Tor A1552 strain in resistance to antimicrobial peptides (AMPs). To assess this potential role, we grew V. cholerae with sub-lethal concentrations of Polymyxin B (PmB) or the AMP LL-37 and analyzed the OMVs produced and their effects on AMP resistance. Our results show that growing V. cholerae in the presence of AMPs modifies the protein content of the OMVs. In the presence of PmB, bacteria release OMVs that are larger in size and contain a biofilm-associated extracellular matrix protein (Bap1). We demonstrated that Bap1 binds to the OmpT porin on the OMVs through the LDV domain of OmpT. In addition, OMVs from cultures incubated in presence of PmB also provide better protection for V. cholerae against LL-37 compared to OMVs from V. cholerae cultures grown without AMPs or in presence of LL-37. Using a bap1 mutant we showed that cross-resistance between PmB and LL-37 involved the Bap1 protein, whereby Bap1 on OMVs traps LL-37 with no subsequent degradation of the AMP.     

Iron acquisition in Vibrio cholerae

Vibrio cholerae, the causative agent of cholera, has an absolute requirement for iron and must obtain this element in the human host as well as in its varied environmental niches. It has multiple systems for iron acquisition, including the TonB-dependent transport of heme, the endogenous siderophore vibriobactin and several siderophores that are produced by other microorganisms. There is also a Feo system for the transport of ferrous iron and an ABC transporter, Fbp, which transports ferric iron. There appears to be at least one additional high affinity iron transport system that has not yet been identified. In iron replete conditions, iron acquisition genes are repressed by Fur. Fur also represses the synthesis of a small, regulatory RNA, RyhB, which negatively regulates genes for iron-containing proteins involved in the tricarboxylic acid cycle and respiration as well as genes for motility and chemotaxis. The redundancy in iron transport systems has made it more difficult to determine the role of individual systems in vivo and in vitro, but it may reflect the overall importance of iron in the growth and survival of V. cholerae.     

Cloning of the Vibrio cholerae recA gene and construction of a Vibrio cholerae recA mutant

A recombinant plasmid carrying the recA gene of Vibrio cholerae was isolated from a V. cholerae genomic library, using complementation in Escherichia coli. The plasmid complements a recA mutation in E. coli for both resistance to the DNA-damaging agent methyl methanesulfonate and recombinational activity in bacteriophage P1 transductions. After determining the approximate location of the recA gene on the cloned DNA fragment, we constructed a defined recA mutation by filling in an XbaI site located within the gene. The 4-base pair insertion resulted in a truncated RecA protein as determined by minicell analysis. The mutation was spontaneously recombined onto the chromosome of a derivative of V. cholerae strain P27459 by screening for methyl methanesulfonate-sensitive variants. Southern blot analysis confirmed the presence of the inactivated XbaI site in the chromosome of DNA isolated from one of these methyl methanesulfonate-sensitive colonies. The recA V. cholerae strain was considerably more sensitive to UV light than its parent, was impaired in homologous recombination, and was deficient in induction of a temperate vibriophage upon exposure to UV light. We conclude that the V. cholerae RecA protein has activities which are analogous to those described for the RecA protein of E. coli.