To assess the role of the flagellum which was detected by immunoscreening of surface proteins of Vibrio vulnificus , an flgE -deleted mutant was constructed and tested for its pathogenicity. The ability of this nonmotile mutant to adhere to INT-407 cells and its role in biofilm were decreased, as was its lethality to mice.

Quorum sensing allows bacteria to sense and respond to changes in population density. Acyl-homoserine lactones serve as quorum-sensing signals for many Proteobacteria , and acyl-homoserine lactone signaling is known to control cooperative activities. Quorum-controlled activities vary from one species to another. Quorum-sensing controls a constellation of genes in the opportunistic pathogen Pseudomonas aeruginosa , which thrives in a number of habitats ranging from soil and water to animal hosts. We hypothesized that there would be significant variation in quorum-sensing regulons among strains of P. aeruginosa isolated from different habitats and that differences in the quorum-sensing regulons might reveal insights about the ecology of P. aeruginosa . As a test of our hypothesis we used RNA-seq to identify quorum-controlled genes in seven P. aeruginosa isolates of diverse origins. Although our approach certainly overlooks some quorum-sensing–regulated genes we found a shared set of genes, i.e., a core quorum-controlled gene set, and we identified distinct, strain-variable sets of quorum-controlled genes, i.e., accessory genes. Some quorum-controlled genes in some strains were not present in the genomes of other strains. We detected a correlation between traits encoded by some genes in the strain-variable subsets of the quorum regulons and the ecology of the isolates. These findings indicate a role for quorum sensing in extension of the range of habitats in which a species can thrive. This study also provides a framework for understanding the molecular mechanisms by which quorum-sensing systems operate, the evolutionary pressures by which they are maintained, and their importance in disparate ecological contexts.

Vibrio vulnificuswas found to produce a chemical that induced the expression ofVibrio fischeri luxgenes. Electron spray ionization-mass spectrometry and¹H nuclear magnetic resonance analyses indicated that the compound was cyclo(l-Phe-l-Pro) (cFP). The compound was produced at a maximal level when cell cultures reached the onset of stationary phase. Sodium dodecyl sulfate-polyacrylamide gel analysis of the total proteins ofV. vulnificusindicated that expression of OmpU was enhanced by exogenously added synthetic or purified cFP. AtoxR-null mutant failed to expressompUdespite the addition of cFP. The relatedVibriospp.V. cholerae,V. parahaemolyticus, andV. harveyialso produced cFP, which induced the expression of their ownompUgenes. cFP also enhanced the expression inV. choleraeof thectxgenes, which are known to be regulated by ToxR. Our results suggest that cFP is a signal molecule controlling the expression of genes important for the pathogenicity ofVibriospp.

Quorum sensing is a cell-to-cell communication system known to control many bacterial processes. In the present study, the functions of quorum sensing in the pathogenesis of Vibrio vulnificus , a food-borne pathogen, were assessed by evaluating the virulence of a mutant deficient in SmcR, a quorum-sensing regulator and homologue of LuxR. When biofilms were used as an inoculum, the smcR mutant was impaired in virulence and colonization capacity in the infection of mice. The lack of SmcR also resulted in decreased histopathological damage in mouse jejunum tissue. These results indicated that SmcR is essential for V. vulnificus pathogenesis. Moreover, the smcR mutant exhibited significantly reduced biofilm detachment. Upon exposure to INT-407 host cells, the wild type, but not the smcR mutant, revealed accelerated biofilm detachment. The INT-407 cells increased smcR expression by activating the expression of LuxS, an autoinducer-2 synthase, indicating that host cells manipulate the cellular level of SmcR through the quorum-sensing signaling of V. vulnificus . A whole-genome microarray analysis revealed that the genes primarily involved in biofilm detachment and formation are up- and downregulated by SmcR, respectively. Among the SmcR-regulated genes, vvpE encoding an elastolytic protease was the most upregulated, and the purified VvpE appeared to dissolve established biofilms directly in a concentration-dependent manner in vitro . These results suggest that the host cell-induced SmcR enhances the detachment of V. vulnificus biofilms entering the host intestine and thereby may promote the dispersal of the pathogen to new colonization loci, which is crucial for pathogenesis.

The putative virulence factors of Vibrio vulnificus include an elastase, the gene product of vvpE. We previously demonstrated that vvpE expression is differentially directed by two different promoters in a growth phase-dependent manner. The activity of the stationaryphase promoter (promoter S (PS)) is dependent on RpoS and is also under the positive control of cyclic AMP receptor protein (CRP). In this study, primer extension analyses revealed that SmcR, the Vibrio harveyi LuxR homolog, is also involved in the regulation of vvpE transcription by activating PS. Although the influence of CRP on PS is mediated by SmcR, the level of PS activity observed when CRP and SmcR function together was found to be greater than the sum of the PS activities achieved by each activator alone. Western blot analyses demonstrated that the cellular levels of RpoS, CRP, and SmcR were not significantly affected by one other, indicating that CRP and SmcR function cooperatively to activate PS rather than sequentially in a regulatory cascade. The binding sites for CRP and SmcR were mapped based on a deletion analysis of the vvpE promoter region and confirmed by in vitro DNase I protection assays. The binding sites for CRP and SmcR were juxtapositioned and centered 220 and 198 bp upstream of the transcription start site of PS, respectively. Accordingly, these results reveal that CRP and SmcR function synergistically to coactivate the expression of vvpE by the RpoS-dependent promoter (PS) and that the activators exert their effect by directly binding to the promoter in the stationary phase.