Peroxide responsive regulator PerR of group A Streptococcus is required for the expression of phage-associated DNase Sda1 under oxidative stress.
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ABSTRACT:
SUBMITTER: Wang CH
PROVIDER: S-EPMC3849366 | biostudies-literature | 2013
REPOSITORIES: biostudies-literature
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Project description:Group A Streptococcus (GAS) has developed a broad arsenal of virulence factors that serve to circumvent host defense mechanisms. The virulence factor DNase Sda1 of the hyperinvasive M1T1 GAS clone degrades DNA-based neutrophil extracellular traps allowing GAS to escape extracellular killing. TLR9 is activated by unmethylated CpG-rich bacterial DNA and enhances innate immune resistance. We hypothesized that Sda1 degradation of bacterial DNA could alter TLR9-mediated recognition of GAS by host innate immune cells. We tested this hypothesis using a dual approach: loss and gain of function of DNase in isogenic GAS strains and presence and absence of TLR9 in the host. Either DNA degradation by Sda1 or host deficiency of TLR9 prevented GAS induced IFN-? and TNF-? secretion from murine macrophages and contributed to bacterial survival. Similarly, in a murine necrotizing fasciitis model, IFN-? and TNF-? levels were significantly decreased in wild type mice infected with GAS expressing Sda1, whereas no such Sda1-dependent effect was seen in a TLR9-deficient background. Thus GAS Sda1 suppressed both the TLR9-mediated innate immune response and macrophage bactericidal activity. Our results demonstrate a novel mechanism of bacterial innate immune evasion based on autodegradation of CpG-rich DNA by a bacterial DNase.
| S-EPMC3375267 | biostudies-literature
Project description:We have characterized group A Streptococcus (GAS) genome-wide responses to hydrogen peroxide and assessed the role of the peroxide response regulator (PerR) in GAS under oxidative stress. Comparison of transcriptome changes elicited by peroxide in wild-type bacteria with those in a perR deletion mutant showed that 76 out of 237 peroxide-regulated genes are PerR dependent. Unlike the PerR-mediated upregulation of peroxidases and other peroxide stress defense mechanisms previously reported in gram-positive species, PerR-dependent genes in GAS were almost exclusively downregulated and encoded proteins involved in purine and deoxyribonucleotide biosynthesis, heme uptake, and amino acid/peptide transport, but they also included a strongly activated putative transcriptional regulator (SPy1198). Of the 161 PerR-independent loci, repressed genes (86 of 161) encoded proteins with functions similar to those coordinated by PerR, in contrast to upregulated loci that encoded proteins that function in DNA damage repair, cofactor metabolism, reactive oxygen species detoxification, pilus biosynthesis, and hypothetical proteins. Complementation of the perR deletion mutant with wild-type PerR restored PerR-dependent regulation, whereas complementation with either one of two PerR variants carrying single mutations in two predicted metal-binding sites did not rescue the mutant phenotype. Metal content analyses of the recombinant wild type and respective PerR mutants, in addition to regulation studies in metal-supplemented and iron-depleted media, showed binding of zinc and iron by PerR and an iron requirement for optimal responses to peroxide. Our findings reveal a novel physiological contribution of PerR in coordinating DNA and protein metabolic functions in peroxide and identify GAS adaptive responses that may serve to enhance oxidative stress resistance and virulence in the host.
| S-EPMC3232902 | biostudies-literature
Project description:Survival in aerobic conditions is critical to the pathogenicity of many bacteria. To investigate the means of aerotolerance and resistance to oxidative stress in the catalase-negative organism Streptococcus pyogenes, we used a genomics-based approach to identify and inactivate homologues of two peroxidase genes, encoding alkyl hydroperoxidase (ahpC) and glutathione peroxidase (gpoA). Single and double mutants survived as well as the wild type under aerobic conditions. However, they were more susceptible than the wild type to growth suppression by paraquat and cumene hydroperoxide. In addition, we show that S. pyogenes demonstrates an inducible peroxide resistance response when treated with sublethal doses of peroxide. This resistance response was intact in ahpC and gpoA mutants but not in mutants lacking PerR, a repressor of several genes including ahpC and catalase (katA) in Bacillus subtilis. Because our data indicate that these peroxidase genes are not essential for aerotolerance or induced resistance to peroxide stress in S. pyogenes, genes for a novel mechanism of managing peroxide stress may be regulated by PerR in streptococci.
| S-EPMC110969 | biostudies-literature
Project description:Group A Streptococcus (GAS) causes a variety of diseases globally. The DNases in GAS promote GAS evasion of neutrophil killing by degrading neutrophil extracellular traps (NETs). Sda1 is a prophage-encoded DNase associated with virulent GAS strains. However, protective immunity against Sda1 has not been determined. In this study, we explored the potential of Sda1 as a vaccine candidate. Sda1 was used as a vaccine to immunize mice intranasally. The effect of anti-Sda1 IgG in neutralizing degradation of NETs was determined and the protective role of Sda1 was investigated with intranasal and systemic challenge models. Antigen-specific antibodies were induced in the sera and pharyngeal mucosal site after Sda1 immunization. The anti-Sda1 IgG efficiently prevented degradation of NETs by supernatant samples from different GAS serotypes with or without Sda1. Sda1 immunization promoted clearance of GAS from the nasopharynx independent of GAS serotypes but did not reduce lethality after systemic GAS challenge. Anti-Sda1 antibody can neutralize degradation of NETs by Sda1 and other phage-encoded DNases and decrease GAS colonization at the nasopharynx across serotypes. These results indicate that Sda1 can be a potential vaccine candidate for reduction in GAS reservoir and GAS tonsillitis-associated diseases.
| S-EPMC8779841 | biostudies-literature
Project description:Peroxide sensing is essential for bacterial survival during aerobic metabolism and host infection. Peroxide stress regulators (PerRs) are homodimeric transcriptional repressors with each monomer typically containing both structural and regulatory metal-binding sites. PerR binding to gene promoters is controlled by the presence of iron in the regulatory site, and iron-catalyzed oxidation of PerR by H2O2 leads to the dissociation of PerR from DNA. In addition to a regulatory metal, most PerRs require a structural metal for proper dimeric assembly. We present here a structural and functional characterization of the PerR from the pathogenic spirochete Leptospira interrogans, a rare example of PerR lacking a structural metal-binding site. In vivo studies showed that the leptospiral PerR belongs to the peroxide stimulon in pathogenic species and is involved in controlling resistance to peroxide. Moreover, a perR mutant had decreased fitness in other host-related stress conditions, including at 37 °C or in the presence of superoxide anion. In vitro, leptospiral PerR could bind to the perR promoter region in a metal-dependent manner. The crystal structure of the leptospiral PerR revealed an asymmetric homodimer, with one monomer displaying complete regulatory metal coordination in the characteristic caliper-like DNA-binding conformation and the second monomer exhibiting disrupted regulatory metal coordination in an open non-DNA-binding conformation. This structure showed that leptospiral PerR assembles into a dimer in which a metal-induced conformational switch can occur independently in the two monomers. Our study demonstrates that structural metal binding is not compulsory for PerR dimeric assembly and for regulating peroxide stress.
| S-EPMC5767856 | biostudies-literature
Project description:Metal homeostasis plays a critical role in antioxidative stress. Streptococcus oligofermentans, an oral commensal facultative anaerobe lacking catalase activity, produces and tolerates abundant H2O2, whereas Dpr (an Fe(2+)-chelating protein)-dependent H2O2 protection does not confer such high tolerance. Here, we report that inactivation of perR, a peroxide-responsive repressor that regulates zinc and iron homeostasis in Gram-positive bacteria, increased the survival of H2O2-pulsed S. oligofermentans 32-fold and elevated cellular manganese 4.5-fold. perR complementation recovered the wild-type phenotype. When grown in 0.1 to 0.25 mM MnCl2, S. oligofermentans increased survival after H2O2 stress 2.5- to 23-fold, and even greater survival was found for the perR mutant, indicating that PerR is involved in Mn(2+)-mediated H2O2 resistance in S. oligofermentans. Mutation of mntA could not be obtained in brain heart infusion (BHI) broth (containing ~0.4 ?M Mn(2+)) unless it was supplemented with ?2.5 ?M MnCl2 and caused 82 to 95% reduction of the cellular Mn(2+) level, while mntABC overexpression increased cellular Mn(2+) 2.1- to 4.5-fold. Thus, MntABC was identified as a high-affinity Mn(2+) transporter in S. oligofermentans. mntA mutation reduced the survival of H2O2-pulsed S. oligofermentans 5.7-fold, while mntABC overexpression enhanced H2O2-challenged survival 12-fold, indicating that MntABC-mediated Mn(2+) uptake is pivotal to antioxidative stress in S. oligofermentans. perR mutation or H2O2 pulsing upregulated mntABC, while H2O2-induced upregulation diminished in the perR mutant. This suggests that perR represses mntABC expression but H2O2 can release the suppression. In conclusion, this work demonstrates that PerR regulates manganese homeostasis in S. oligofermentans, which is critical to H2O2 stress defenses and may be distributed across all oral streptococci lacking catalase.
| S-EPMC3993191 | biostudies-literature
Project description:The ability of bacteria, such as the dental pathogen Streptococcus mutans, to coordinate a response against damage-inducing oxidants is a critical aspect of their pathogenicity. The oxidative stress regulator SpxA1 has been demonstrated to be a major player in the ability of S. mutans to withstand both disulfide and peroxide stresses. While studying spontaneously occurring variants of an S. mutans ΔspxA1 strain, we serendipitously discovered that our S. mutans UA159 host strain bore a single-nucleotide deletion within the coding region of perR, resulting in a premature truncation of the encoded protein. PerR is a metal-dependent transcriptional repressor that senses and responds to peroxide stress such that loss of PerR activity results in activation of oxidative stress responses. To determine the impact of loss of PerR regulation, we obtained a UA159 isolate bearing an intact perR copy and created a clean perR deletion mutant. Our findings indicate that loss of PerR activity results in a strain that is primed to tolerate oxidative stresses in the laboratory setting. Interestingly, RNA deep sequencing (RNA-Seq) and targeted transcriptional expression analyses reveal that PerR offers a minor contribution to the ability of S. mutans to orchestrate a transcriptional response to peroxide stress. Furthermore, we detected loss-of-function perR mutations in two other commonly used laboratory strains of S. mutans, suggesting that this may be not be an uncommon occurrence. This report serves as a cautionary tale regarding the so-called domestication of laboratory strains and advocates for the implementation of more stringent strain authentication practices.IMPORTANCE A resident of the human oral biofilm, Streptococcus mutans is one of the major bacterial pathogens associated with dental caries. This report highlights a spontaneously occurring mutation within the laboratory strain S. mutans UA159 found in the coding region of perR, a gene encoding a transcriptional repressor associated with peroxide tolerance. Though perR mutant strains of S. mutans showed a distinct growth advantage and enhanced tolerance toward H2O2, a ΔperR deletion strain showed a small number of differentially expressed genes compared to the parent strain, suggesting few direct regulatory targets. In addition to characterizing the role of PerR in S. mutans, our findings serve as a warning to laboratory researchers regarding bacterial adaptation to in vitro growth conditions.
| S-EPMC8088507 | biostudies-literature
Project description:Regulation of oxidative stress responses by the peroxide stress regulator (PerR) is critical for the in vivo fitness and virulence of group A Streptococcus. To elucidate the molecular mechanism of DNA binding, peroxide sensing, and gene regulation by PerR, we performed biochemical and structural characterization of PerR. Sequence-specific DNA binding by PerR does not require regulatory metal occupancy. However, metal binding promotes higher affinity PerR-DNA interactions. PerR metallated with iron directly senses peroxide stress and dissociates from operator sequences. The crystal structure revealed that PerR exists as a homodimer with two metal-binding sites per subunit as follows: a structural zinc site and a regulatory metal site that is occupied in the crystals by nickel. The regulatory metal-binding site in PerR involves a previously unobserved HXH motif located in its unique N-terminal extension. Mutational analysis of the regulatory site showed that the PerR metal ligands are involved in regulatory metal binding, and integrity of this site is critical for group A Streptococcus virulence. Interestingly, the metal-binding HXH motif is not present in the structurally characterized members of ferric uptake regulator (Fur) family but is fully conserved among PerR from the genus Streptococcus. Thus, it is likely that the PerR orthologs from streptococci share a common mechanism of metal binding, peroxide sensing, and gene regulation that is different from that of well characterized PerR from Bacillus subtilis. Together, our findings provide key insights into the peroxide sensing and regulation of the oxidative stress-adaptive responses by the streptococcal subfamily of PerR.
| S-EPMC3689973 | biostudies-literature
Project description:The peroxide stress response regulator PerR coordinates the oxidative-stress defenses of group A Streptococcus (GAS). We now show that PerR is expressed from an operon encoding a putative DNA polymerase I (PolA1), among other GAS products. A polA1 deletion mutant exhibited wild-type growth but showed reduced capacity to repair DNA damage caused by UV light or ciprofloxacin. Mutant bacteria were hypersensitive to H(2)O(2), compared with the wild type or a complemented mutant strain, and remained severely attenuated even after adaptation at sublethal H(2)O(2) levels, whereas wild-type bacteria could adapt to withstand peroxide challenge under identical conditions. The hypersensitivity of the mutant was reversed when bacteria were grown in iron-depleted medium and challenged in the presence of a hydroxyl radical scavenger, results that indicated sensitivity to hydroxyl radicals generated by Fenton chemistry. The peroxide resistance of a perR polA1 double mutant following adaptation at sublethal H(2)O(2) levels was decreased 9-fold relative to a perR single mutant, thus implicating PolA1 in PerR-mediated defenses against peroxide stress. Cultures of the polA1 mutant grown with or without prior H(2)O(2) exposure yielded considerably lower numbers of rifampin-resistant mutants than cultures of the wild type or the complemented mutant strain, a finding consistent with PolA1 lacking proofreading activity. We conclude that PolA1 promotes genome sequence diversity while playing an essential role in oxidative DNA damage repair mechanisms of GAS, dual functions predicted to confer optimal adaptive capacity and fitness in the host. Together, our studies reveal a unique genetic and functional relationship between PerR and PolA1 in streptococci.
| S-EPMC3562117 | biostudies-literature
Project description:SDA1 encodes a highly conserved protein that is widely distributed in eukaryotic organisms. SDA1 is essential for cell cycle progression and organization of the actin cytoskeleton in yeasts and humans. In this study, we identified a Phytophthora capsici orthologue of yeast SDA1, named PcSDA1. In P. capsici, PcSDA1 is strongly expressed in three asexual developmental states (mycelium, sporangia and germinating cysts), as well as late in infection. Silencing or overexpression of PcSDA1 in P. capsici transformants affected the growth of hyphae and sporangiophores, sporangial development, cyst germination and zoospore release. Phalloidin staining confirmed that PcSDA1 is required for organization of the actin cytoskeleton. Moreover, 4',6-diamidino-2-phenylindole (DAPI) staining and PcSDA1-green fluorescent protein (GFP) fusions revealed that PcSDA1 is involved in the regulation of nuclear distribution in hyphae and sporangia. Both silenced and overexpression transformants showed severely diminished virulence. Thus, our results suggest that PcSDA1 plays a similar role in the regulation of the actin cytoskeleton and nuclear division in this filamentous organism as in non-filamentous yeasts and human cells.
| S-EPMC6638425 | biostudies-literature