Epithelial retinoic acid receptor ß regulates serum amyloid A expression and vitamin A-dependent intestinal immunity.

Vitamin A is a dietary component that is essential for the development of intestinal immunity. Vitamin A is absorbed and converted to its bioactive derivatives retinol and retinoic acid by the intestinal epithelium, yet little is known about how epithelial cells regulate vitamin A-dependent intestinal immunity. Here we show that epithelial cell expression of the transcription factor retinoic acid receptor ß (RARß) is essential for vitamin A-dependent intestinal immunity. Epithelial RARß activated vitamin A-dependent expression of serum amyloid A (SAA) proteins by binding directly to Saa promoters. In accordance with the known role of SAAs in regulating Th17 cell effector function, epithelial RARß promoted IL-17 production by intestinal Th17 cells. More broadly, epithelial RARß was required for the development of key vitamin A-dependent adaptive immune responses, including CD4+ T-cell homing to the intestine and the development of IgA-producing intestinal B cells. Our findings provide insight into how the intestinal epithelium senses dietary vitamin A status to regulate adaptive immunity, and highlight the role of epithelial cells in regulating intestinal immunity in response to diet.

The hydrogen peroxide hypersensitivity of OxyR2 in Vibrio vulnificus depends on conformational constraints.

Most Gram-negative bacteria respond to excessive levels of H2O2 using the peroxide-sensing transcriptional regulator OxyR, which can induce the expression of antioxidant genes to restore normality. Vibrio vulnificus has two distinct OxyRs (OxyR1 and OxyR2), which are sensitive to different levels of H2O2 and induce expression of two different peroxidases, Prx1 and Prx2. Although OxyR1 has both high sequence similarity and H2O2 sensitivity comparable with that of other OxyR proteins, OxyR2 exhibits limited sequence similarity and is more sensitive to H2O2 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 H2O2 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 H2O2 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 H2O2 sensing, compared with the E204G structure or other OxyR proteins. Our findings provide a structural basis for the higher sensitivity of OxyR2 to H2O2 and also suggest a molecular mechanism for bacterial regulation of expression of antioxidant genes at divergent concentrations of cellular H2O2.

OxyR2 Functions as a Three-state Redox Switch to Tightly Regulate Production of Prx2, a Peroxiredoxin of Vibrio vulnificus.

The bacterial transcriptional regulator OxyR is known to function as a two-state redox switch. OxyR senses cellular levels of H2O2 via a "sensing cysteine" that switches from the reduced to a disulfide state upon H2O2 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 H2O2 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-SO3H) by high H2O2 in vitro and in vivo, where the modification deterred the transcription of prx2 The DNA binding preferences of OxyR25CA-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 H2O2 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.

Characterization of the Vibrio vulnificus 1-Cys peroxiredoxin Prx3 and regulation of its expression by the Fe-S cluster regulator IscR in response to oxidative stress and iron starvation.

Peroxiredoxins (Prxs) are ubiquitous antioxidant enzymes that reduce toxic peroxides. A new Vibrio vulnificus Prx, named Prx3, was identified and characterized in this study. Biochemical and mutational analyses revealed that Prx3 reduces H2O2, utilizing glutaredoxin 3 (Grx3) and glutathione (GSH) as reductants, and requires only N-terminal peroxidatic cysteine for its catalysis. These results, combined with the monomeric size of Prx3 observed under non-reducing conditions, suggested that Prx3 is a Grx3/GSH-dependent 1-Cys Prx and oxidized without forming intermolecular disulfide bonds. The prx3 mutation impaired growth in the medium containing peroxides and reduced virulence in mice, indicating that Prx3 is essential for survival under oxidative stress and pathogenesis of V. vulnificus. The Fe-S cluster regulator IscR activates prx3 by direct binding to a specific binding sequence centered at -44 from the transcription start site. The binding sequence was homologous to the Type 2 IscR-binding sequence, most likely recognized by the Fe-S clusterless apo-IscR in Escherichia coli. The iscR3CA mutant, chromosomally encoding the apo-locked IscR, exhibited 3-fold higher levels of activation of prx3 than the wild type and accumulated more IscR3CA protein in cells. The IscR-dependent activation of prx3 by aerobic growth and iron starvation was also associated with the increase in cellular levels of IscR protein. Taken together, the results suggested that IscR senses iron starvation as well as reactive oxygen species and shifts to the apo-form, which leads to the increase of cellular IscR and in turn prx3 expression, contributing to the survival and virulence of V. vulnificus during pathogenesis.

Distinct characteristics of OxyR2, a new OxyR-type regulator, ensuring expression of Peroxiredoxin 2 detoxifying low levels of hydrogen peroxide in Vibrio vulnificus.

Two 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.

Distinct characteristics of two 2-Cys peroxiredoxins of Vibrio vulnificus suggesting differential roles in detoxifying oxidative stress.

Peroxiredoxins (Prxs) are ubiquitous antioxidant enzymes reducing toxic peroxides. Two distinct 2-Cys Prxs, Prx1 and Prx2, were identified in Vibrio vulnificus, a facultative aerobic pathogen. Both Prxs have two conserved catalytic cysteines, C(P) and C(R), but Prx2 is more homologous in amino acid sequences to eukaryotic Prx than to Prx1. Prx2 utilized thioredoxin A as a reductant, whereas Prx1 required AhpF. Prx2 contained GGIG and FL motifs similar to the motifs conserved in sensitive Prxs and exhibited sensitivity to overoxidation. MS analysis and C(P)-SO(3)H specific immunoblotting demonstrated overoxidation of C(P) to C(P)-SO(2)H (or C(P)-SO(3)H) in vitro and in vivo, respectively. In contrast, Prx1 was robust and C(P) was not overoxidized. Discrete expression of the Prxs implied that Prx2 is induced by trace amounts of H(2)O(2) and thereby residential in cells grown aerobically. In contrast, Prx1 was occasionally expressed only in cells exposed to high levels of H(2)O(2). A mutagenesis study indicated that lack of Prx2 accumulated sufficient H(2)O(2) to induce Prx1. Kinetic properties indicated that Prx2 effectively scavenges low levels of peroxides because of its high affinity to H(2)O(2), whereas Prx1 quickly degrades higher levels of peroxides because of its high turnover rate and more efficient reactivation. This study revealed that the two Prxs are differentially optimized for detoxifying distinct ranges of H(2)O(2), and proposed that Prx2 is a residential scavenger of peroxides endogenously generated, whereas Prx1 is an occasional scavenger of peroxides exogenously encountered. Furthermore, genome sequence database search predicted widespread coexistence of the two Prxs among bacteria.