A mutant that exhibited less cytotoxic activity toward INT-407 human intestinal epithelial cells than the wild type was screened from a random transposon mutant library of Vibrio vulnificus, and an open reading frame encoding an Fe-S cluster regulator, IscR, was identified using a transposon-tagging method. A mutational analysis demonstrated that IscR contributes to mouse mortality as well as cytotoxicity toward the INT-407 cells, indicating that IscR is essential for the pathogenesis of V. vulnificus. A whole-genome microarray analysis revealed that IscR influenced the expression of 67 genes, of which 52 were upregulated and 15 were downregulated. Among these, 12 genes most likely involved in motility and adhesion to host cells, hemolytic activity, and survival under oxidative stress of the pathogen during infection were selected and experimentally verified to be upregulated by IscR. Accordingly, the disruption of iscR resulted in a significant reduction in motility and adhesion to INT-407 cells, in hemolytic activity, and in resistance to reactive oxygen species (ROS) such as H 2 O 2 and tert-butyl hydroperoxide (t-BOOH). Furthermore, the present study demonstrated that iscR expression was induced by exposure of V. vulnificus to the INT-407 cells, and the induction appeared to be mediated by ROS generated by the host cells during infection. Consequently, the combined results indicated that IscR is a global regulator that contributes to the overall success in the pathogenesis of V. vulnificus by regulating the expression of various virulence and survival genes in addition to Fe-S cluster genes. Most of virulence factors of pathogenic bacteria act cooperatively to obtain maximum effectiveness during pathogenesis while their expression is coordinately regulated by common global regulators in response to environmental conditions, and this coordinated regulation facilitates the cooperation of virulence factors and is crucial to the overall success of the pathogens during infection (1, 2). Vibrio vulnificus is an opportunistic Gram-negative pathogen that frequently contaminates oysters. It has been proposed that numerous virulence factors account for the fulminating and destructive nature of V. vulnificus infections and contribute to not only disease development but also the survival and multiplication on or within the host (for a recent review, see reference 3). However, studies about global regulators involved in the regulation of V. vulnificus virulence factors are still very limited.Iron-sulfur proteins containing the Fe-S cluster as a cofactor carry out multiple important cellular processes such as electron transfer, metabolic reactions, and gene regulation and are widely distributed (for recent reviews, see references 4 and 5). A highly conserved isc operon, iscRSUA-hscBA-fdx, was discovered to encode all of the proteins required for the biogenesis of the majority, if not all, of the Fe-S cluster proteins in Escherichia coli (for recent reviews, see references 5 and 6). Expression of the isc operon is autoregu...
SummaryTwo peroxiredoxins, Prx1 and Prx2, were previously identified in Vibrio vulnificus. Besides OxyR1, a homologue of Escherichia coli OxyR (EcOxyR), OxyR2 that shares low homology with EcOxyR was first identified in V. vulnificus. OxyR2 activated prx2 during aerobic growth, while OxyR1 activated prx1 only when exposed to exogenous H2O2. OxyR2 was oxidized to form a reversible C206 to C215 disulphide bond by sensing low levels of H2O2, which were insufficient to oxidize OxyR1, and only the oxidized OxyR2 activated prx2. OxyR25CA, in which all cysteine residues except for C206 and C215 were replaced with alanines, and its mutants, OxyR25CA-C206S and OxyR25CA-C215S, were constructed. OxyR25CA and OxyR25CA-C215S directly bound to a specific binding sequence centred at −56.5 from the prx2 transcription start site, albeit with different binding affinities. The binding sequence consisted of four ATCGnt elements spaced by a helical turn and aligned in the twofold dyad symmetry, suggesting that OxyR2 binds DNA as a tetramer. OxyR25CA-C206S also directly bound to DNA comprising more extended sequences, indicating that oxidized and reduced OxyR2 adopt different conformational states, leading to altered DNA contacts. The oxyR2 mutation reduced cytotoxicity and growth during infection, indicating that OxyR2 is essential for the pathogenesis of V. vulnificus.
For successful infection of their hosts, pathogenic bacteria recognize host-derived signals that induce the expression of virulence factors in a spatiotemporal manner. The fulminating food-borne pathogen Vibrio vulnificus produces a cytolysin/hemolysin protein encoded by the vvhBA operon, which is a virulence factor preferentially expressed upon exposure to murine blood and macrophages. The Fe-S cluster containing transcriptional regulator IscR activates the vvhBA operon in response to nitrosative stress and iron starvation, during which the cellular IscR protein level increases. Here, electrophoretic mobility shift and DNase I protection assays revealed that IscR directly binds downstream of the vvhBA promoter PvvhBA, which is unusual for a positive regulator. We found that in addition to IscR, the transcriptional regulator HlyU activates vvhBA transcription by directly binding upstream of PvvhBA, whereas the histone-like nucleoid-structuring protein (H-NS) represses vvhBA by extensively binding to both downstream and upstream regions of its promoter. Of note, the binding sites of IscR and HlyU overlapped with those of H-NS. We further substantiated that IscR and HlyU outcompete H-NS for binding to the PvvhBA regulatory region, resulting in the release of H-NS repression and vvhBA induction. We conclude that concurrent antirepression by IscR and HlyU at regions both downstream and upstream of PvvhBA provides V. vulnificus with the means of integrating host-derived signal(s) such as nitrosative stress and iron starvation for precise regulation of vvhBA transcription, thereby enabling successful host infection.
Binding to mucin is the initial step for enteropathogens to establish pathogenesis. An open reading frame, gbpA, of Vibrio vulnificus was identified and characterized in this study. Compared with wild type, the gbpA mutant was impaired in binding to mucin-agar and the mucin-secreting HT29-methotrexate cells, and the impaired mucin binding was restored by the purified GbpA provided exogenously. The gbpA mutant had attenuated virulence and ability of intestinal colonization in a mouse model, indicating that GbpA is a mucin-binding protein and essential for pathogenesis of V. vulnificus. The gbpA transcription was growth phase-dependent, reaching a maximum during the exponential phase. The Fe-S cluster regulator (IscR) and the cyclic AMP receptor protein (CRP) coactivated, whereas SmcR, a LuxR homologue, repressed gbpA. The cellular levels of IscR, CRP, and SmcR were not significantly affected by one another, indicating that the regulator proteins function cooperatively to regulate gbpA rather than sequentially in a regulatory cascade. The regulatory proteins directly bind upstream of the gbpA promoter P gbpA . DNase I protection assays, together with the deletion analyses of P gbpA , demonstrated that IscR binds to two specific sequences centered at ؊164.5 and ؊106, and CRP and SmcR bind specifically to the sequences centered at ؊68 and ؊45, respectively. Furthermore, gbpA was induced by exposure to H 2 O 2 , and the induction appeared to be mediated by elevated intracellular levels of IscR. Consequently, the combined results indicated that IscR, CRP, and SmcR cooperate for precise regulation of gbpA during the V. vulnificus pathogenesis.
Background: ROS and iron availability influence [2Fe-2S] cluster occupancy in IscR.Results: Prx3 is a Grx3/glutathione-dependent 1-Cys peroxiredoxin essential for survival under oxidative stress and pathogenesis of Vibrio vulnificus, and IscR directly activates prx3 by sensing ROS and iron starvation. Conclusion: IscR-dependent prx3 expression contributes to the pathogenesis of V. vulnificus. Significance: This study elucidated the IscR-mediated regulation of an antioxidant enzyme.
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