Differential gene expression in response to hydrogen peroxide and the putative PerR regulon of Synechocystis sp. strain PCC 6803

We utilized a full genome cDNA microarray to identify the genes that comprise the peroxide stimulon in the cyanobacterium Synechocystis sp. strain PCC 6803. We determined that a gene (slr1738) encoding a protein similar to PerR in Bacillus subtilis was induced by peroxide. We constructed a PerR knockout strain and used it to help identify components of the PerR regulon, and we found that the regulatory properties were consistent with the hypothesis that PerR functions as a repressor. This effort was guided by finding putative PerR boxes in positions upstream of specific genes and by careful statistical analysis. PerR and sll1621 (ahpC), which codes for a peroxiredoxin, share a divergent promoter that is regulated by PerR. We found that isiA, encoding a Chl protein that is induced under low-iron conditions, was strongly induced by a short-term peroxide stress. Other genes that were strongly induced by peroxide included sigD, sigB, and genes encoding peroxiredoxins and Dsb-like proteins that have not been studied yet in this strain. A gene (slr1894) that encoded a protein similar to MrgA in B. subtilis was upregulated by peroxide, and a strain containing an mrgA knockout mutation was highly sensitive to peroxide. A number of genes were downregulated, including key genes in the chlorophyll biosynthesis pathway and numerous regulatory genes, including those encoding histidine kinases. We used PerR mutants and a thioredoxin mutant (TrxA1) to study differential expression in response to peroxide and determined that neither PerR nor TrxA1 is essential for the peroxide protective response.     

Catalase (KatA) and alkyl hydroperoxide reductase (AhpC) have compensatory roles in peroxide stress resistance and are required for survival, persistence, and nasal colonization in Staphylococcus aureus

Oxidative-stress resistance in Staphylococcus aureus is linked to metal ion homeostasis via several interacting regulators. In particular, PerR controls the expression of a regulon of genes, many of which encode antioxidants. Two PerR regulon members, ahpC (alkylhydroperoxide reductase) and katA (catalase), show compensatory regulation, with independent and linked functions. An ahpC mutation leads to increased H2O2 resistance due to greater katA expression via relief of PerR repression. Moreover, AhpC provides residual catalase activity present in a katA mutant. Mutation of both katA and ahpC leads to a severe growth defect under aerobic conditions in defined media (attributable to lack of catalase activity). This results in the inability to scavenge exogenous or endogenously produced H2O2, resulting in accumulation of H2O2 in the medium. This leads to DNA damage, the likely cause of the growth defect. Surprisingly, the katA ahpC mutant is not attenuated in two independent models of infection, which implies reduced oxygen availability during infection. In contrast, both AhpC and KatA are required for environmental persistence (desiccation) and nasal colonization. Thus, oxidative-stress resistance is an important factor in the ability of S. aureus to persist in the hospital environment and so contribute to the spread of human disease.     

Campylobacter jejuni Contains Two Fur Homologs: Characterization of Iron-Responsive Regulation of Peroxide Stress Defense Genes by the PerR Repressor

Expression of the peroxide stress genes alkyl hydroperoxide reductase (ahpC) and catalase (katA) of the microaerophile Campylobacter jejuni is repressed by iron. Whereas iron repression in gram-negative bacteria is usually carried out by the Fur protein, previous work showed that this is not the case in C. jejuni, as these genes are still iron repressed in a C. jejuni fur mutant. An open reading frame encoding a Fur homolog (designated PerR for "peroxide stress regulator") was identified in the genome sequence of C. jejuni. The perR gene was disrupted by a kanamycin resistance cassette in C. jejuni wild-type and fur mutant strains. Subsequent characterization of the C. jejuni perR mutants showed derepressed expression of both AhpC and KatA at a much higher level than that obtained by iron limitation, suggesting that expression of these genes is controlled by other regulatory factors in addition to the iron level. Other iron-regulated proteins were not affected by the perR mutation. The fur perR double mutant showed derepressed expression of known iron-repressed genes. Further phenotypic analysis of the perR mutant, fur mutant, and the fur perR double mutant showed that the perR mutation made C. jejuni hyperresistant to peroxide stress caused by hydrogen peroxide and cumene hydroperoxide, a finding consistent with the high levels of KatA and AhpC expression, and showed that these enzymes were functional. Quantitative analysis of KatA expression showed that both the perR mutation and the fur mutation had profound effects on catalase activity, suggesting additional non-iron-dependent regulation of KatA and, by inference, AhpC. The PerR protein is a functional but nonhomologous substitution for the OxyR protein, which regulates peroxide stress genes in other gram-negative bacteria. Regulation of peroxide stress genes by a Fur homolog has recently been described for the gram-positive bacterium Bacillus subtilis. C. jejuni is the first gram-negative bacterium where non-OxyR regulation of peroxide stress genes has been described and characterized.     

A Xanthomonas alkyl hydroperoxide reductase subunit C (ahpC) mutant showed an altered peroxide stress response and complex regulation of the compensatory response of peroxide detoxification enzymes

Alkyl hydroperoxide reductase subunit C (AhpC) is the catalytic subunit responsible for alkyl peroxide metabolism. A Xanthomonas ahpC mutant was constructed. The mutant had increased sensitivity to organic peroxide killing, but was unexpectedly hyperresistant to H(2)O(2) killing. Analysis of peroxide detoxification enzymes in this mutant revealed differential alteration in catalase activities in that its bifunctional catalase-peroxidase enzyme and major monofunctional catalase (Kat1) increased severalfold, while levels of its third growth-phase-regulated catalase (KatE) did not change. The increase in catalase activities was a compensatory response to lack of AhpC, and the phenotype was complemented by expression of a functional ahpC gene. Regulation of the catalase compensatory response was complex. The Kat1 compensatory response increase in activity was mediated by OxyR, since it was abolished in an oxyR mutant. In contrast, the compensatory response increase in activity for the bifunctional catalase-peroxidase enzyme was mediated by an unknown regulator, independent of OxyR. Moreover, the mutation in ahpC appeared to convert OxyR from a reduced form to an oxidized form that activated genes in the OxyR regulon in uninduced cells. This complex regulation of the peroxide stress response in Xanthomonas differed from that in other bacteria.     

Compensatory functions of two alkyl hydroperoxide reductases in the oxidative defense system of Legionella pneumophila

Legionella pneumophila expresses two catalase-peroxidase enzymes that exhibit strong peroxidatic but weak catalatic activities, suggesting that other enzymes participate in decomposition of hydrogen peroxide (H2O2). Comparative genomics revealed that L. pneumophila and its close relative Coxiella burnetii each contain two peroxide-scavenging alkyl hydroperoxide reductase (AhpC) systems: AhpC1, which is similar to the Helicobacter pylori AhpC system, and AhpC2 AhpD (AhpC2D), which is similar to the AhpC AhpD system of Mycobacterium tuberculosis. To establish a catalatic function for these two systems, we expressed L. pneumophila ahpC1 or ahpC2 in a catalase/peroxidase mutant of Escherichia coli and demonstrated restoration of H2O2 resistance by a disk diffusion assay. ahpC1::Km and ahpC2D::Km chromosomal deletion mutants were two- to eightfold more sensitive to H2O2, tert-butyl hydroperoxide, cumene hydroperoxide, and paraquat than the wild-type L. pneumophila, a phenotype that could be restored by trans-complementation. Reciprocal strategies to construct double mutants were unsuccessful. Mutant strains were not enfeebled for growth in vitro or in a U937 cell infection model. Green fluorescence protein reporter assays revealed expression to be dependent on the stage of growth, with ahpC1 appearing after the exponential phase and ahpC2 appearing during early exponential phase. Quantitative real-time PCR showed that ahpC1 mRNA levels were approximately 7- to 10-fold higher than ahpC2D mRNA levels. However, expression of ahpC2D was significantly increased in the ahpC1 mutant, whereas ahpC1 expression was unchanged in the ahpC2D mutant. These results indicate that AhpC1 or AhpC2D (or both) provide an essential hydrogen peroxide-scavenging function to L. pneumophila and that the compensatory activity of the ahpC2D system is most likely induced in response to oxidative stress.     

Evaluation of the roles that alkyl hydroperoxide reductase and Ohr play in organic peroxide-induced gene expression and protection against organic peroxides in Xanthomonas campestris

Alkyl hydroperoxide reductase (ahpC) and organic hydroperoxide resistance (ohr) are distinct genes, structurally and regulatory, but have similar physiological functions. In Xanthomonas campestris pv. phaseoli inactivation of either gene results in increased sensitivity to killing with organic peroxides. An ahpC1-ohr double mutant was highly sensitive to both growth inhibition and killing treatment with organic peroxides. High level expression of ahpC or ohr only partially complemented the phenotype of the double mutant, suggesting that these genes function synergistically, but through different pathways, to protect Xanthomonas from organic peroxide toxicity. Functional analyses of Ohr and AhpC abilities to degrade organic hydroperoxides revealed that both Ohr and AhpC could degrade tert-butyl hydroperoxide (tBOOH) while the former was more efficient at degrading cumene hydroperoxide (CuOOH). Expression analysis of these genes in the mutants showed no compensatory alterations in the levels of AhpC or Ohr. However, CuOOH induced expression of these genes in the mutants was affected. CuOOH induced ahpC expression was higher in the ohr mutant than in the parental strain; in contrast, the ahpC mutation has no effect on the level of induced ohr expression. These analyses reveal complex physiological roles and expression patterns of seemingly functionally similar genes.     

Oxidative stress response and its role in sensitivity to isoniazid in mycobacteria: characterization and inducibility of ahpC by peroxides in Mycobacterium smegmatis and lack of expression in M. aurum and M. tuberculosis.

Mycobacterium tuberculosis is a natural mutant with inactivated oxidative stress regulatory gene oxyR. This characteristic has been linked to the exquisite sensitivity of M. tuberculosis to isonicotinic acid hydrazide (INH). In the majority of mycobacteria tested, including M. tuberculosis, oxyR is divergently transcribed from ahpC, a gene encoding a homolog of the subunit of alkyl hydroperoxide reductase that carries out substrate peroxide reduction. Here we compared ahpC expression in Mycobacterium smegmatis, a mycobacterium less sensitive to INH, with that in two highly INH sensitive species, M. tuberculosis and Mycobacterium aurum. The ahpC gene of M. smegmatis was cloned and characterized, and the 5' ends of ahpC mRNA were mapped by S1 nuclease protection analysis. M. smegmatis AhpC and eight other polypeptides were inducible by exposure to H2O2 or organic peroxides, as determined by metabolic labeling and Western blot (immunoblot) analysis. In contrast, M. aurum displayed differential induction of only one 18-kDa polypeptide when exposed to organic peroxides. AhpC could not be detected in this organism by immunological means. AhpC was also below detection levels in M. tuberculosis H37Rv. These observations are consistent with the interpretation that ahpC expression and INH sensitivity are inversely correlated in the mycobacterial species tested. In further support of this conclusion, the presence of plasmid-borne ahpC reduced M. smegmatis susceptibility to INH. Interestingly, mutations in the intergenic region between oxyR and ahpC were identified and increased ahpC expression observed in deltakatG M. tuberculosis and Mycobacterium bovis INH(r) strains. We propose that mutations activating ahpC expression may contribute to the emergence of INH(r) strains.     

Aerotolerance and peroxide resistance in peroxidase and PerR mutants of Streptococcus pyogenes

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.     

Differential secretomics of Streptococcus pyogenes reveals a novel peroxide regulator (PerR)-regulated extracellular virulence factor mitogen factor 3 (MF3)

Streptococcus pyogenes is a human pathogen that causes various diseases. Numerous virulence factors secreted by S. pyogenes are involved in pathogenesis. The peroxide regulator (PerR) is associated with the peroxide resistance response and pathogenesis, but little is known about the regulation of the secretome involved in virulence. To investigate how PerR regulates the expression of the S. pyogenes secretome involved in virulence, a perR deficient mutant was used for comparative secretomic analysis with a wild-type strain. The conditioned medium containing secreted proteins of a wild-type strain and a perR deficient mutant at the stationary phase were collected for two-dimensional gel electrophoresis analysis, where protease inhibitors were applied to avoid the degradation of extracellular proteins. Differentially expressed protein spots were identified by liquid chromatography electrospray ionization tandem MS. More than 330 protein spots were detected on each gel. We identified 25 unique up-regulated proteins and 13 unique down-regulated proteins that were directly or indirectly controlled by the PerR regulator. Among these identified proteins, mitogen factor 3 (MF3), was selected to verify virulence and the expression of gene products. The data showed that MF3 protein levels in conditioned medium, as measured by immunoblot analysis, correlated well with protein levels determined by two-dimensional gel electrophoresis analysis. We also demonstrated that PerR bound to the promoter region of the mf3 gene. The result of an infection model showed that virulence was attenuated in the mf3 deficient mutant. Additional growth data of the wild-type strain and the mf3 deficient mutant suggested that MF3 played a role in digestion of exogenous DNA for promoting growth. To summarize, we conclude that PerR can positively regulate the expression of the secreted protein MF3 that contributes to the virulence in S. pyogenes. The analysis of the PerR-regulated secretome provided key information for the elucidation of the host-pathogen interactions and might assist in the development of potential chemotherapeutic strategies to prevent or treat streptococcal diseases.     

Peroxide Responsive Regulator PerR of group A Streptococcus Is Required for the Expression of Phage-Associated DNase Sda1 under Oxidative Stress

The peroxide regulator (PerR) is a ferric uptake repressor-like protein, which is involved in adaptation to oxidative stress and iron homeostasis in group A streptococcus. A perR mutant is attenuated in surviving in human blood, colonization of the pharynx, and resistance to phagocytic clearance, indicating that the PerR regulon affects both host environment adaptation and immune escape. Sda1 is a phage-associated DNase which promotes M1T1 group A streptococcus escaping from phagocytic cells by degrading DNA-based neutrophil extracellular traps. In the present study, we found that the expression of sda1 is up-regulated under oxidative conditions in the wild-type strain but not in the perR mutant. A gel mobility shift assay showed that the recombinant PerR protein binds the sda1 promoter. In addition, mutation of the conserved histidine residue in the metal binding site of PerR abolished sda1 expression under hydrogen peroxide treatment conditions, suggesting that PerR is directly responsible for the sda1 expression under oxidative stress. Our results reveal PerR-dependent sda1 expression under oxidative stress, which may aid innate immune escape of group A streptococcus.     

Molecular and physiological effects of mycobacterial oxyR inactivation

The majority of slow-growing mycobacteria have a functional oxyR, the central regulator of the bacterial oxidative stress response. In contrast, this gene has been inactivated during the evolution of Mycobacterium tuberculosis. Here we inactivated the oxyR gene in Mycobacterium marinum, an organism used to model M. tuberculosis pathogenesis. Inactivation of oxyR abrogated induction of ahpC, a gene encoding alkylhydroperoxide reductase, normally activated upon peroxide challenge. The absence of oxyR also resulted in increased sensitivity to the front-line antituberculosis drug isoniazid. Inactivation of oxyR in M. marinum did not affect either virulence in a fish infection model or survival in human macrophages. Our findings demonstrate, at the genetic and molecular levels, a direct role for OxyR in ahpC regulation in response to oxidative stress. Our study also indicates that oxyR is not critical for virulence in M. marinum. However, oxyR inactivation confers increased sensitivity to isonicotinic acid hydrazide, suggesting that the natural loss of oxyR in the tubercle bacillus contributes to the unusually high sensitivity of M. tuberculosis to isoniazid.     

AhpC, oxidative stress and drug resistance in Mycobacterium tuberculosis

The Mycobacterium tuberculosis AhpC is similar to a family of bacterial and eukaryotic antioxidant proteins with alkylhydroperoxidase (Ahp) and thioredoxin-dependent peroxidase (TPx) activities. AhpC expression is associated with resistance to the front-line antitubercular drug isoniazid in the naturally resistant organisms E. coli and M. smegmatis. We identified several isoniazid-resistant M. tuberculosis isolates with ahpC promoter mutations resulting in AhpC overexpression. These strains were more resistant to cumene hydroperoxide than were wild-type strains. However, these strains were unchanged in their sensitivity to isoniazid, refuting a role for AhpC in detoxification of this drug. All the isoniazid-resistant, AhpC-overexpressing strains were also deficient in activity of the mycobacterial catalase-peroxidase KatG. KatG, the only known catalase in M. tuberculosis, is required for activation of isoniazid. We propose that compensatory ahpC promoter mutations are selected from KatG-deficient, isoniazid-resistant M. tuberculosis during infections, to mitigate the added burden imposed by organic peroxides on these strains.