Vibrio vulnificus AphB is involved in interleukin-8 production via an NF-κB-dependent pathway in human intestinal epithelial cells

We previously reported that the aphB gene mutant of Vibrio vulnificus had significantly impaired motility and adherence to host cells. In this study, we investigated the role of V. vulnificus AphB on the production of interleukin-8 (IL-8), a proinflammatory cytokine, as well as its underlying mechanism in human intestinal epithelial INT-407 cells. The aphB gene mutation significantly reduced the ability of V. vulnificus to stimulate IL-8 production and IL-8 gene promoter activation in INT-407 cells. The V. vulnificusaphB mutant also induced lower levels of NF-κB DNA binding activity and NF-κB minimal promoter activation than did the wild-type of V. vulnificus. Importantly, the observed reductions in IL-8 production, IL-8 gene promoter activation and NF-κB DNA binding activity were significantly restored by complementing the aphB gene into the V. vulnificusaphB mutant. These results indicate that V. vulnificus AphB is involved in the IL-8 production via an NF-κB dependent pathway in human intestinal epithelial cells.     

Vibrio vulnificus RTX toxin plays an important role in the apoptotic death of human intestinal epithelial cells exposed to Vibrio vulnificus

During Vibrio vulnificus infection, V. vulnificus reaches the intestine and then invades the bloodstream by crossing the intestinal mucosal barrier of the host, which results in systemic septicemia. Previously, we reported that the RtxA toxin secreted through the RtxE transporter contributes to the cytotoxicity of V. vulnificus against intestinal epithelial cells. Here, we used gene mutants of rtxE and rtxA to determine the role that V. vulnificus RtxA toxin plays in the apoptotic death of human intestinal epithelial cells. The levels of DNA fragmentation were lower in human epithelial cells infected with an rtxE mutant of V. vulnificus than in those that were infected with the wild type. In addition, the rtxE mutant was found to induce lower levels of TUNEL positive cells and cell cycle arrest at the subG(1) than the wild type V. vulnificus. Furthermore, the decreased levels of DNA fragmentation, TUNEL positive cells and subG(1) arrest by the rtxE gene mutation were restored by the complementation of an rtxE gene into the rtxE mutant V. vulnificus. Finally, the rtxA mutant induced significantly lower levels of apoptotic cell death than the wild type. The levels of the PARP, cytochrome c, caspase-3, and mitochondrial membrane depolarization were lower in human epithelial cells infected with the rtxE and rtxA mutants, compared with the wild type and rtxE gene-complemented strains of V. vulnificus. Taken together, these results indicate that V. vulnificus RtxA toxin induces the apoptotic death through a mitochondria-dependent pathway in human intestinal epithelial cells exposed to V. vulnificus.     

Essential role of an adenylate cyclase in regulating Vibrio vulnificus virulence

Vibrio vulnificus, a halophilic estuarine bacterium, causes a fatal septicemia and necrotizing wound infection. To investigate the role of cAMP in V. vulnificus virulence regulation, an in-frame deletion mutant of the cya gene encoding adenylate cyclase was constructed. The cya null mutation resulted in a pleiotropic change of virulence phenotypes. The production of hemolysin and protease, the motility, and the cytotoxicity were decreased by the cya mutation. The defects in the cya mutant were functionally complemented in trans by a plasmid carrying the wild type cya allele. The V. vulnificus cya mutant exhibited a 100-fold increase in LD50 to mice. The result indicates that cAMP plays an essential role in the global regulation of V. vulnificus virulence.     

Isolation and characterization of a cold-induced nonculturable suppression mutant of Vibrio vulnificus

The viable but nonculturable (VBNC) suppression mutant formed platable cells at low temperature stress after inoculation in artificial seawater (ASW). Suppression subtractive hybridization was used to identify differentially expressed genes among cDNAs of the VBNC suppression mutant and the wild-type Vibrio vulnificus strain. Glutathione S-transferase was identified as a responsive gene of the VBNC suppression mutant in our assay, and was highly expressed from the VBNC suppression mutant at low temperature stress. Culturability tests revealed that the wild-type cells were sensitive to oxidative stress in the hydrogen peroxide (H(2)O(2)) and to 1-chloro-2,4-dinitrobenzene (CDNB) compared with the VBNC suppression mutant cells. Adding glutathione showed that many wild-type V. vulnificus cells maintained culturability in cold ASW. These results suggest that non-nutritional growth inhibitors, such as peroxide that accumulates at low temperatures, influence VBNC in V. vulnificus cells.     

Identification of the Vibrio vulnificus htpG gene and its influence on cold shock recovery

An htpG gene encoding the heat shock protein HtpG was identified and cloned from Vibrio vulnificus. The deduced amino acid sequence of HtpG from V. vulnificus exhibited 71 and 85% identity to those reported from Escherichia coli and V. cholera, respectively. Functions of HtpG were assessed by the construction of an isogenic mutant whose htpG gene was deleted and by evaluating its phenotype changes during and after cold shock. The results demonstrated that recovery of the wild type from cold shock was significantly faster (p<0.05) than that of the htpG mutant, and indicated that the chaperone protein HtpG contributes to cold shock recovery, rather than cold shock tolerance, of V. vulnificus.     

Identification and functional analysis of vibrio vulnificus SmcR, a novel global regulator

Recently, quorum sensing has been implicated as an important global regulator controlling the production of numerous virulence factors such as capsular polysaccharides in bacterial pathogens. The nucleotide and deduced amino acid sequences of smcR, a homolog of V. harveyi luxR identified from V. vulnificus ATCC29307, were analyzed. The amino acid sequence of SmcR from V. vulnificus was 72 to 92% similar to those of LuxR homologs from Vibrio spp. Functions of SmcR were assessed by the construction of an isogenic mutant, whose smcR gene was inactivated by allelic exchanges, and by evaluating its phenotype changes in vitro and in mice. The disruption of smcR resulted in a significant alteration in biofilm formation, in type of colony morphology, and in motility. When compared with the wild-type, the smcR mutant exhibited reduced survival under adverse conditions, such as acidic pH and hyperosmotic stress. The smcR mutant exhibited decreased cytotoxic activity toward INT 407 cells in vitro. Furthermore, the intraperitoneal LD50 of the smcR mutant was approximately 10(2) times higher than that of parental wild-type. Therefore, it appears that SmcR is a novel global regulator, controlling numerous genes contributing to the pathogenesis as well as survival of V. vulnificus.     

Cloning and characterization of a periplasmic nuclease of Vibrio vulnificus and its role in preventing uptake of foreign DNA

We have cloned a nuclease gene, vvn, from Vibrio vulnificus, an estuarine bacterium that causes wound infections and septicemia in humans and eels. The gene contained a 696-bp open reading frame encoding 232 amino acids (aa), including a signal sequence of 18 aa. The deduced amino acid sequence of the mature nuclease predicted a molecular mass of 25 kDa, which was confirmed by vital stain, and a pI of 8.6. Vvn was produced in the periplasm of either V. vulnificus or recombinant Escherichia coli strains and was active in the oxidized (but not the reduced) form. This nuclease was able to digest DNA and RNA, with differential thermostability in DNase and RNase activities. Expression of Vvn in E. coli DH5alpha reduced the frequencies of transformation with the divalent ion-treated cells and electroporation by about 6 and 2 logs, respectively. In addition, the transformation frequency of a Vvn-deficient V. vulnificus mutant (ND) was 10-fold higher than that of the parent strain. These data suggested that Vvn may be involved in preventing uptake of foreign DNA by transformation. However, Vvn expressed in the recipients had little effect on the conjugation frequency in either E. coli or V. vulnificus. Some other DNase(s) may be present in the periplasm and responsible for a residual DNase activity, which was about one-fourth of that of the parent strain, detected in the ND mutant. We also demonstrated that Vvn was not required for the virulence of V. vulnificus mice.     

RpoS-dependent stress response and exoenzyme production in Vibrio vulnificus

Vibrio vulnificus is an estuarine bacterium capable of causing rapidly fatal infections through both ingestion and wound infection. Like other opportunistic pathogens, V. vulnificus must adapt to potentially stressful environmental changes while living freely in seawater, upon colonization of the oyster gut, and upon infection of such diverse hosts as humans and eels. In order to begin to understand the ability of V. vulnificus to respond to such stresses, we examined the role of the alternate sigma factor RpoS, which is important in stress response and virulence in many pathogens. An rpoS mutant of V. vulnificus strain C7184o was constructed by homologous recombination. The mutant strain exhibited a decreased ability to survive diverse environmental stresses, including exposure to hydrogen peroxide, hyperosmolarity, and acidic conditions. The most striking difference was a high sensitivity of the mutant to hydrogen peroxide. Albuminase, caseinase, and elastase activity were detected in the wild type but not in the mutant strain, and an additional two hydrolytic activities (collagenase and gelatinase) were reduced in the mutant strain compared to the wild type. Additionally, the motility of the rpoS mutant was severely diminished. Overall, these studies suggest that rpoS in V. vulnificus is important for adaptation to environmental changes and may have a role in virulence.     

Genetic analysis of spontaneous lactose-utilizing mutants from vibrio vulnificus

Wild-type V. vulnificus cannot grow using lactose as the sole carbon source or take up the sugar. However, prolonged culture of this species in media containing lactose as the sole carbon source leads to the generation of a spontaneous lactose-utilizing (LU) mutant. This mutant showed strong beta- galactosidase activity, whereas the wild-type strain showed a barely detectable level of the activity. A mutant with a lesion in a gene homologous to the lacZ of E. coli in the bacterium no longer showed beta-galactosidase activity or generated spontaneous LU mutants, suggesting that the lacZ homolog is responsible for the catabolism of lactose, but the expression of the gene and genes for transport of lactose is tightly regulated. Genetic analysis of spontaneous LU mutants showed that all the mutations occur in a lacI homolog, which is located downstream to the lacZ and putative ABC-type lac permease genes. Consistent with this, a genomic library clone containing the lacI gene, when present in trans, made the spontaneous LU mutants no longer able to utilize lactose as the sole carbon source. Taken together with the observation that excessive amounts of exogenously supplemented possible catabolic products of lactose have negative effects on the growth and survivability of V. vulnificus, we suggest that V. vulnificus has evolved to carry a repressor that tightly regulates the expression of lacZ to keep the intracellular toxic catabolic intermediates at a sublethal level.     

Identification and characterization of a novel serine protease, VvpS, that contains two functional domains and is essential for autolysis of Vibrio vulnificus

Little is known about the molecular mechanism for autolysis of Gram-negative bacteria. In the present study, we identified the vvpS gene encoding a serine protease, VvpS, from Vibrio vulnificus, a Gram-negative food-borne pathogen. The amino acid sequence predicted that VvpS consists of two functional domains, an N-terminal protease catalytic domain (PCD) and a C-terminal carbohydrate binding domain (CBD). A null mutation of vvpS significantly enhanced viability during stationary phase, as measured by enumerating CFU and differentially staining viable cells. The vvpS mutant reduced the release of cytoplasmic β-galactosidase and high-molecular-weight extracellular chromosomal DNA into the culture supernatants, indicating that VvpS contributes to the autolysis of V. vulnificus during stationary phase. VvpS is secreted via a type II secretion system (T2SS), and it exerts its effects on autolysis through intracellular accumulation during stationary phase. Consistent with this, a disruption of the T2SS accelerated intracellular accumulation of VvpS and thereby the autolysis of V. vulnificus. VvpS also showed peptidoglycan-hydrolyzing activity, indicating that the autolysis of V. vulnificus is attributed to the self-digestion of the cell wall by VvpS. The functions of the VvpS domains were assessed by C-terminal deletion analysis and demonstrated that the PCD indeed possesses a proteolytic activity and that the CBD is required for hydrolyzing peptidoglycan effectively. Finally, the vvpS mutant exhibited reduced virulence in the infection of mice. In conclusion, VvpS is a serine protease with a modular structure and plays an essential role in the autolysis and pathogenesis of V. vulnificus.     

Regulation of fatty acid metabolism by FadR is essential for Vibrio vulnificus to cause infection of mice

The opportunistic bacterial pathogen Vibrio vulnificus causes severe wound infection and fatal septicemia. We used alkaline phosphatase insertion mutagenesis in a clinical isolate of V. vulnificus to find genes necessary for virulence, and we identified fadR, which encodes a regulator of fatty acid metabolism. The fadR::mini-Tn5Km2phoA mutant was highly attenuated in a subcutaneously inoculated iron dextran-treated mouse model of V. vulnificus disease, was hypersensitive to the fatty acid synthase inhibitor cerulenin, showed aberrant expression of fatty acid biosynthetic (fab) genes and fatty acid oxidative (fad) genes, produced smaller colonies on agar media, and grew slower in rich broth than did the wild-type parent. Deletion of fadR essentially recapitulated the phenotypes of the insertion mutant, and the DeltafadR mutation was complemented in trans with the wild-type gene. Further characterization of the DeltafadR mutant showed that it was not generally hypersensitive to envelope stresses but had decreased motility and showed an altered membrane lipid profile compared to that of the wild type. Supplementation of broth with the unsaturated fatty acid oleate restored wild-type growth in vitro, and infection with oleate in the inoculum increased the ability of the DeltafadR mutant to infect mice. We conclude that fadR and regulation of fatty acid metabolism are essential for V. vulnificus to be able to cause disease in mammalian hosts.     

The role of quorum sensing and the effect of environmental conditions on biofilm formation by strains of Vibrio vulnificus

It has been suggested that Vibrio vulnificus attaches to plankton and algae and is found in large numbers in the environment. Factors affecting attachment, biofilm formation and morphology of V. vulnificus have not been thoroughly investigated. This study evaluated the role of quorum sensing (QS) and environmental conditions on biofilm development of V. vulnificus. It was found that biofilm development by V. vulnificus was affected by nutrient and glucose concentration, but not by NaCl concentration or temperature under the conditions used here. Moreover, biofilm development of a QS mutant strain proceeded rapidly and sloughing occurred earlier than for the isogenic parent strain. There was a significant loss of viability for the QS mutant biofilm early in development. Hence, it is hypothesised that factors regulated by the QS system play a role in proper biofilm development and maintenance of V. vulnificus. Furthermore, it is shown that biofilm development varied among isolates.     

Proteomic analysis of pathogenic bacterium Vibrio vulnificus

In this study we have constructed a proteome reference map of the pathogenic bacterium Vibrio vulnificus. From the reference map, we identified several virulence-related proteins, such as ToxR and ToxS, as well as numerous proteins involved in diverse cellular functions. To search for additional virulence-related proteins, we compared the whole proteomes from the wild-type and toxR mutant of V. vulnificus and found that several proteins were up- or down-regulated in the toxR mutant. We suggest that these differentially regulated proteins whose expression is coordinately controlled by a virulence regulator ToxR, some of which are already implicated in virulence, play roles in the pathogenesis of V. vulnificus.