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.

Nitric oxide (NO) and its derivatives are important effectors of host innate immunity, disrupting cellular function of infecting pathogens. Transcriptome analysis of Vibrio vulnificus, an opportunistic human pathogen, identified a set of genes induced upon exposure to NO. Among them, VvhmpA (V. vulnificus hmpA), encoding a multidomain NO dioxygenase, was the most greatly induced upon exposure to NO and was thus further characterized. Absorption spectra demonstrated that VvHmpA is a heme protein in which the heme iron can exist in either reduced, NO-bound, or oxidized state. Biochemical studies revealed that VvHmpA is a flavohemoglobin containing equimolar amounts of heme and FAD as cofactors. The KM and kcat values of VvHmpA for NO at 37°C, the temperature encountered by V. vulnificus in the host, were greater than those at 30°C, indicating that VvHmpA detoxifies high levels of NO effectively during infection. Compared with the wild type, the VvhmpA mutant exhibited a lower NO-decomposition activity and impaired growth in the presence of NO in vitro. Also, the cytotoxicity and survival of the VvhmpA mutant infecting the NO-producing murine macrophage cells were lower than those of the wild type. Furthermore, the mouse lethality of the VvhmpA mutant was reduced compared to that of the parental wild type. The combined results revealed that VvHmpA is a potent virulence factor that is induced upon exposure to NO and important for the survival and pathogenesis of V. vulnificus during infection.

The bacterial transcriptional regulator OxyR is known to function as a two-state redox switch. OxyR senses cellular levels of H 2 O 2 via a "sensing cysteine" that switches from the reduced to a disulfide state upon H 2 O 2 exposure, inducing the expression of antioxidant genes. The reduced and disulfide states of OxyR, respectively, bind to extended and compact regions of DNA, where the reduced state blocks and the oxidized state allows transcription and further induces target gene expression by interacting with RNA polymerase. Vibrio vulnificus OxyR2 senses H 2 O 2 with high sensitivity and induces the gene encoding the antioxidant Prx2. In this study, we used mass spectrometry to identify a third redox state of OxyR2, in which the sensing cysteine was overoxidized to S-sulfonated cysteine (Cys-SO 3 H) by high H 2 O 2 in vitro and in vivo, where the modification deterred the transcription of prx2. The DNA binding preferences of OxyR2 5CA -C206D, which mimics overoxidized OxyR2, suggested that overoxidized OxyR2 binds to the extended DNA site, masking the ؊35 region of the prx2 promoter. These combined results demonstrate that OxyR2 functions as a three-state redox switch to tightly regulate the expression of prx2, preventing futile production of Prx2 in cells exposed to high levels of H 2 O 2 sufficient to inactivate Prx2. We further provide evidence that another OxyR homolog, OxyR1, displays similar three-state behavior, inviting further exploration of this phenomenon as a potentially general regulatory mechanism.

Most Gram-negative bacteria respond to excessive levels of HO using the peroxide-sensing transcriptional regulator OxyR, which can induce the expression of antioxidant genes to restore normality. has two distinct OxyRs (OxyR1 and OxyR2), which are sensitive to different levels of HO and induce expression of two different peroxidases, Prx1 and Prx2. Although OxyR1 has both high sequence similarity and HO sensitivity comparable with that of other OxyR proteins, OxyR2 exhibits limited sequence similarity and is more sensitive to HO To investigate the basis for this difference, we determined crystal structures and carried out biochemical analyses of OxyR2. The determined structure of OxyR2 revealed a flipped conformation of the peptide bond before Glu-204, a position occupied by glycine in other OxyR proteins. Activity assays showed that the sensitivity to HO was reduced to the level of other OxyR proteins by the E204G mutation. We solved the structure of the OxyR2-E204G mutant with the same packing environment. The structure of the mutant revealed a dual conformation of the peptide bond before Gly-204, indicating the structural flexibility of the region. This structural duality extended to the backbone atoms of Gly-204 and the imidazole ring of His-205, which interact with HO and invariant water molecules near the peroxidatic cysteine, respectively. Structural comparison suggests that Glu-204 in OxyR2 provides rigidity to the region that is important in HO sensing, compared with the E204G structure or other OxyR proteins. Our findings provide a structural basis for the higher sensitivity of OxyR2 to HO and also suggest a molecular mechanism for bacterial regulation of expression of antioxidant genes at divergent concentrations of cellular HO.

Nitric oxide (NO) is an important antimicrobial effector produced by the host innate immune system to counteract invading pathogens. To survive and establish a successful infection, a fulminating human pathogen Vibrio vulnificus expresses the hmpA gene encoding an NO dioxygenase in an NO-responsive manner. In this study, we identified an Rrf2-family transcriptional regulator NsrR that is predicted to contain the Fe-S cluster coordinated by three cysteine residues. Transcriptome analysis showed that NsrR controls the expression of multiple genes potentially involved in nitrosative stress responses. Particularly, NsrR acts as a strong repressor of hmpA transcription and relieves the repression of hmpA upon exposure to NO. Notably, nsrR and hmpA are transcribed divergently, and their promoter regions overlap with each other. Molecular biological analyses revealed that NsrR directly binds to this overlapping promoter region, which is alleviated by loss of the Fe-S cluster, leading to the subsequent derepression of hmpA under nitrosative stress. We further found that a leucine-responsive regulatory protein (Lrp) negatively regulates hmpA in an NsrR-dependent manner by directly binding to the promoter region, presumably resulting in a DNA conformation change to support the repression by NsrR. Meanwhile, a cyclic AMP receptor protein (CRP) positively regulates hmpA probably through repression of nsrR and lrp by directly binding to each promoter region in a sequential cascade. Altogether, this collaborative regulation of NsrR along with Lrp and CRP enables an elaborate control of hmpA transcription, contributing to survival under host-derived nitrosative stress and thereby the pathogenesis of V. vulnificus.