VuuB and IutB reduce ferric-vulnibactin in Vibrio vulnificus M2799.

Vibrio vulnificus, a pathogenic bacterium that causes serious infections in humans, requires iron for growth. Clinical isolate, V. vulnificus M2799, secretes a catecholate siderophore, namely, vulnibactin, to capture iron (III) from the environment. Growth experiments using a deletion mutant indicated that VuuB, a member of the FAD-containing siderophore-interacting protein family, plays a crucial role in Fe3+-vulnibactin reduction. IutB, a member of the ferric-siderophore reductase family, stands a substitute for VuuB in its absence. It remained unclear why V. vulnificus M2799 has two proteins with relevant functions. Here we biochemically characterized VuuB and IutB using purified recombinant proteins. Purified VuuB, a flavoprotein, catalyzed the reduction of Fe3+-nitrilotriacetic acid as its electron acceptor, in the presence of NADH as its electron donor and FAD as its cofactor. IutB catalyzed the reduction of Fe3+-nitrilotriacetic acid, in the presence of NADH, NADPH, or reduced glutathione as its electron donor. The optimal pH values and temperatures of VuuB and IutB were 7.0 and 37 °C, and 8.5 and 45 °C, respectively. On analyzing their ferric-chelate reductase activities, both VuuB and IutB were found to catalyze the reduction of Fe3+-aerobactin, Fe3+-vibriobactin, and Fe3+-vulnibactin. When the biologically relevant substrate, Fe3+-vulnibactin, was used, the levels of ferric-chelate reductase activities were similar between VuuB and IutB. Finally, the mRNA levels were quantified by qRT-PCR in M2799 cells cultivated under low-iron conditions. The number of vuuB mRNA was 8.5 times greater than that of iutB. The expression ratio correlated with the growth of their mutants in the presence of vulnibactin.

Transcriptome sequencing reveals the virulence and environmental genetic programs of Vibrio vulnificus exposed to host and estuarine conditions.

Vibrio vulnificus is a natural inhabitant of estuarine waters worldwide and is of medical relevance due to its ability to cause grievous wound infections and/or fatal septicemia. Genetic polymorphisms within the virulence-correlated gene (vcg) serve as a primary feature to distinguish clinical (C-) genotypes from environmental (E-) genotypes. C-genotypes demonstrate superior survival in human serum relative to E-genotypes, and genome comparisons have allowed for the identification of several putative virulence factors that could potentially aid C-genotypes in disease progression. We used RNA sequencing to analyze the transcriptome of C-genotypes exposed to human serum relative to seawater, which revealed two divergent genetic programs under these two conditions. In human serum, cells displayed a distinct "virulence profile" in which a number of putative virulence factors were upregulated, including genes involved in intracellular signaling, substrate binding and transport, toxin and exoenzyme production, and the heat shock response. Conversely, the "environmental profile" exhibited by cells in seawater revealed upregulation of transcription factors such as rpoS, rpoN, and iscR, as well as genes involved in intracellular signaling, chemotaxis, adherence, and biofilm formation. This dichotomous genetic switch appears to be largely governed by cyclic-di-GMP signaling, and remarkably resembles the dual life-style of V. cholerae as it transitions from host to environment. Furthermore, we found a "general stress response" module, known as the stressosome, to be upregulated in seawater. This signaling system has been well characterized in Gram-positive bacteria, however its role in V. vulnificus is not clear. We examined temporal gene expression patterns of the stressosome and found it to be upregulated in natural estuarine waters indicating that this system plays a role in sensing and responding to the environment. This study advances our understanding of gene regulation in V. vulnificus, and brings to the forefront a number of previously overlooked genetic networks.

Genetic analysis and prevalence studies of the brp exopolysaccharide locus of Vibrio vulnificus.

Phase variation in the Gram-negative human pathogen Vibrio vulnificus involves three colonial morphotypes- smooth opaque colonies due to production of capsular polysaccharide (CPS), smooth translucent colonies as the result of little or no CPS expression, and rugose colonies due to production of a separate extracellular polysaccharide (EPS), which greatly enhances biofilm formation. Previously, it was shown that the brp locus, which consists of nine genes arranged as an operon, is up-regulated in rugose strains in a c-di-GMP-dependent manner, and that plasmid insertions into the locus resulted in loss of rugosity and efficient biofilm production. Here, we have used non-polar mutagenesis to assess the involvement of individual brp genes in production of EPS and related phenotypes. Inactivation of genes predicted to be involved in various stages of EPS biosynthesis eliminated both the rugose colonial appearance and production of EPS, while knockout of a predicted flippase function involved in EPS transport resulted in a dry, lightly striated phenotype, which was associated with a reduction of brp-encoded EPS on the cell surface. All brp mutants retained the reduced motility characteristic of rugose strains. Lastly, we provide evidence that the brp locus is highly prevalent among strains of V. vulnificus.

Ubiquitin reference technique and its use in ubiquitin-lacking prokaryotes.

In a pulse-chase assay, the in vivo degradation of a protein is measured through a brief labeling of cells with, for example, a radioactive amino acid, followed by cessation of labeling and analysis of cell extracts prepared at different times afterward ("chase"), using immunoprecipitation, electrophoresis and autoradiography of a labeled protein of interest. A conventional pulse-chase assay is fraught with sources of data scatter, as the efficacy of labeling and immunoprecipitation can vary, and sample volumes can vary as well. The ubiquitin reference technique (URT), introduced in 1996, addresses these problems. In eukaryotes, a DNA-encoded linear fusion of ubiquitin to another protein is cleaved by deubiquitylases at the ubiquitin-protein junction. A URT assay uses a fusion in which the ubiquitin moiety is located between a downstream polypeptide (test protein) and an upstream polypeptide (a long-lived reference protein). The cotranslational cleavage of a URT fusion by deubiquitylases after the last residue of ubiquitin produces, at the initially equimolar ratio, a test protein with a desired N-terminal residue and a reference protein containing C-terminal ubiquitin moiety. In addition to being more accurate than pulse-chases without a reference, URT makes it possible to detect and measure the degradation of a test protein during the pulse (before the chase). Because prokaryotes, including Gram-negative bacteria such as, for example, Escherichia coli and Vibrio vulnificus, lack the ubiquitin system, the use of URT in such cells requires ectopic expression of a deubiquitylase. We describe designs and applications of plasmid vectors that coexpress, in bacteria, both a URT-type fusion and Ubp1, a deubiquitylase of the yeast Saccharomyces cerevisiae. This single-plasmid approach extends the accuracy-enhancing URT assay to studies of protein degradation in prokaryotes.

In vitro and in vivo antimicrobial activity of water-soluble chitosan oligosaccharides against Vibrio vulnificus.

Vibrio vulnificus is a Gram-negative bacterium that induces severely rapid pathological progress. In this study, we evaluated the antibacterial activity of two water-soluble chitosan oligosaccharides, COS A (MW, 10,000 Da) and COS B (MW, 1,000 Da), from 90-95% deacetylated chitosan, against V. vulnificus in vitro and in vivo. Treatment with COS A resulted in significantly higher suppressive effects on the growth of V. vulnificus than treatment with COS B. The growth of V. vulnificus was inhibited within 1 h of treatment with water-soluble COS A in concentrations ranging from 0.5 to 10 mg/ml. Additionally, treatment with COS A completely inhibited V. vulnificus-induced cytotoxicity in human intestinal epithelial INT-407 cells, while COS B did not. Furthermore, the administration of COS A (0.1-0.5 mg per mouse) significantly increased the survival period of V. vulnificus-infected mice. The number of viable V. vulnificus in the spleen, liver, small intestine, and blood was significantly lower in COS A-treated mice than in untreated mice. Here, we clearly demonstrate that COS A is a potential agent for the prevention and treatment of infection with V. vulnificus.

Phospholipase A as a potent virulence factor of Vibrio vulnificus.

Vibrio vulnificus infection has attracted special interest because of its high mortality rate. However, the identification of its major pathogenic determinant still remains obscure. In this study, a cytolysin-negative mutant strain of V. vulnificus CVD707 was used to determine the role of phospholipase A (PLA) in the pathogenesis of this bacterial infection. The mutant strain caused the lysis of erythrocytes in vitro and elevated plasma hemoglobin during the infection in mice. Both the hemolytic and PLA activities were dependent on calcium. Inhibition of hemolysis by PLA inhibitors including tetracyclin and the PLA substrate phosphatidylcholine also supports the possibility of membranous PLA as a major hemolytic factor in the cytolysin-deficient mutant. To identify the role of PLA in the pathogenesis of V. vulnificus infection, the effects of tetracycline on bacteria-induced macrophage cytotoxicity and lethality were compared with those of penicillin, an antibiotic with no inhibitory effect on PLA. Both the macrophage cytotoxicity and the lethality of V. vulnificus CVD707 to mice were significantly attenuated by tetracycline, but not by penicillin. However, bacterial counts in culture medium and mouse blood revealed that penicillin was more effective than tetracycline in killing bacteria under our experimental conditions. These results indicate that PLA activity is important in V. vulnificus-induced cytotoxicity and lethality, suggesting a crucial role for PLA in the pathogenesis of V. vulnificus infection.

Evidence for an intermediate colony morphology of Vibrio vulnificus.

Vibrio vulnificus causes both food-borne disease and wound infections. Most V. vulnificus strains express capsular polysaccharide (CPS), which is required for the virulence of this organism. Under standard growth conditions, CPS expression is lost at a relatively high frequency (10(-3) to 10(-4)), resulting in a switch from an opaque (Op, CPS+) colony morphology to a translucent (Tr, CPS-) colony morphology. The wzb gene, which encodes a phosphatase required for CPS expression, has been proposed to be involved in this switch through a site-specific deletion of the entire gene. In an examination of five strains, we found that the frequency of wzb deletion in Tr colonies varies by strain and therefore does not account for all the Tr colonies that are seen. In addition, we have identified a third, intermediate (Int) colony morphotype, in which the colonies appear less opaque but are not fully translucent. PCR studies have demonstrated that Int colonies still contain the wzb gene, while reverse transcriptase PCR studies have shown that although Int strains retain expression of wzb, in some cases the transcription of wzb is reduced. Int strains switch to a true Tr (wzb negative) morphotype at a very high frequency (nearly 100%) under certain conditions. Finally, Int colonies, which in some cases can easily be mistaken for Tr colonies, have been observed to occasionally revert to Op, while Tr colonies containing a wzb deletion presumably are unable to revert to the encapsulated form.

Swarming differentiation of vibrio vulnificus downregulates the expression of the vvhBA hemolysin gene via the LuxS quorum-sensing system.

Swarming has proven to be a good in vitro model for bacterial surface adherence and colonization, and the swarming differentiation of a bacterium has been shown to be coupled with changes in the expression of virulence factors associated with its invasiveness, particularly in the early stages of infection. In this study, we attempted to determine whether the expression of vvhA, which encodes for hemolysin/cytolysin (VvhA), is either upregulated or downregulated during the swarming differentiation of V. vulnificus. The insertional inactivation of vvhA itself exerted no detectable effect on the expression of V. vulnificus swarming motility. However, in our lacZ-fused vvhA transcriptional reporter assay, vvhA expression decreased in swarming V. vulnificus as compared to non-swarming or planktonic V. vulnificus. The reduced expression of vvhA in swarming V. vulnificus increased as a result of the deletional inactivation of luxS, a gene associated with quorum sensing. These results show that vvhA expression in swarming V. vulnificus is downregulated via the activity of the LuxS quorum-sensing system, suggesting that VvhA performs no essential role in the invasiveness of V. vulnificus via the adherence to and colonization on the body surfaces required in the early stages of the infection. However, VvhA may play a significant role in the pathophysiological deterioration occurring after swarming V. vulnificus is differentiated into planktonic V. vulnificus.

In situ and in vitro gene expression by Vibrio vulnificus during entry into, persistence within, and resuscitation from the viable but nonculturable state.

Isolation of Vibrio vulnificus during winter months is difficult due to the entrance of these cells into the viable but nonculturable (VBNC) state. While several studies have investigated in vitro gene expression upon entrance into and persistence within the VBNC state, to our knowledge, no in situ studies have been reported. We incubated clinical and environmental isolates of V. vulnificus in estuarine waters during winter months to monitor the expression of several genes during the VBNC state and compared these to results from in vitro studies. katG (periplasmic catalase) was down-regulated during the VBNC state in vitro and in situ compared to the constitutively expressed gene tufA. Our results indicate that the loss of catalase activity we previously reported is a direct result of katG repression, which likely accounts for the VBNC response of this pathogen. While expression of vvhA (hemolysin) was detectable in environmental strains during in situ incubation, it ceased in all cases by ca. 1 h. These results suggest that the natural role of hemolysin in V. vulnificus may be in osmoprotection and/or the cold shock response. Differences in expression of the capsular genes wza and wzb were observed in the two recently reported genotypes of this species. Expression of rpoS, encoding the stress sigma factor RpoS, was continuous upon entry into the VBNC state during both in situ and in vitro studies. We found the half-life of mRNA to be less than 60 minutes, confirming that mRNA detection in these VBNC cells is a result of de novo RNA synthesis.

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.