Distinct H2O2-Scavenging System in Yersinia pseudotuberculosis: KatG and AhpC Act Together to Scavenge Endogenous Hydrogen Peroxide

Abstract

To colonize in the digestive tract of animals and humans, Yersinia pseudotuberculosis has to deal with reactive oxygen species (ROS) produced by host cells and microbiota. However, an understanding of the ROS-scavenging systems and their regulation in this bacterium remains largely elusive. In this study, we identified OxyR as the master transcriptional regulator mediating cellular responses to hydrogen peroxide (H₂O₂) in Y. pseudotuberculosis through genomics and transcriptomics analyses. OxyR activates transcription of diverse genes, especially the core members of its regulon, including those encoding catalases, peroxidases, and thiol reductases. The data also suggest that sulfur species and manganese may play a particular role in the oxidative stress response of Y. pseudotuberculosis. Among the three H₂O₂-scavenging systems in Y. pseudotuberculosis, catalase/peroxidase KatE functions as the primary scavenger for high levels of H₂O₂; NADH peroxidase alkyl hydroperoxide reductase (AhpR) and catalase KatG together are responsible for removing low levels of H₂O₂. The simultaneous loss of both AhpC (the peroxidatic component of AhpR) and KatG results in activation of OxyR. Moreover, we found that AhpC, unlike its well-characterized Escherichia coli counterpart, has little effect on protecting cells against toxicity of organic peroxides. These findings provide not only novel insights into the structural and functional diversity of bacterial H₂O₂-scavenging systems but also a basic understanding of how Y. pseudotuberculosis copes with oxidative stress.

Keywords: AhpC; OxyR; Yersinia; catalase; oxidative stresss response.

FIGURE 1

Characteristics of Y. pseudotuberculosis in response to H₂O₂. (A) Minimum inhibitory concentration (MIC) assay. Mid-exponential phase cultures (OD₆₀₀ of ∼0.4) were used to inoculate each well to an OD₆₀₀ of 0.01, and MIC was determined 16 h later. (B) Impact of H₂O₂ on growing cells. H₂O₂ was added to mid-exponential phase cultures of the wild type to the final concentrations as indicated. Growth was monitored by recording OD₆₀₀ values. (C) Volcano plot of the different expression genes in wild-type cells between before and after the H₂O₂ treatment (0.5 mM). (D) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of differentially expressed genes in wild-type cells between before and after the H₂O₂ treatment.

FIGURE 2

Characteristics of the Y. pseudotuberculosis oxyR mutant. (A) OxyR-binding motifs in YPIII derived from the top 20 members of the predicted regulon. The matrix for screening the YPIII genome was generated using most conserved members of OxyR regulons of diverse bacteria. Tetranucleotide sequences are underlined based on the E. coli and S. oneidensis OxyR consensus. (B) Disk diffusion assay for H₂O₂. Paper disks loaded with 10 μl of 5 M H₂O₂ were placed onto bacterial lawns (pregrown for 6 h). Results shown are from 24 h after the disks were placed. The sensitivity was represented by the diameter of the inhibition zone. poxyR represents a copy of oxyR to be expressed in trans for complementation. (C) Measuring H₂O₂-scavenging rates. Cells grown to the mid-exponential phase were collected, washed, and suspended to an OD₆₀₀ of ∼0.4. 5 min after the addition of 0.5 mM H₂O₂, the remaining H₂O₂ was determined. The data were normalized to the initial concentration. (D) Plating defects of the oxyR mutant. Mid-exponential phase cultures were adjusted to ∼10⁸ CFU/ml and diluted by dilution factor (10-fold serial dilution), and 5 μl of each dilution was spotted onto LB plates. Photos were taken 24 h after plating. CAT, catalase (2,000 Units/ml). Experiments were performed at least four times, and representative results were presented. (E) Disk diffusion assay for tert-butyl hydroperoxide (t-BHP). Paper disks loaded with 10 μl of 5 M t-BHP. In panels B, C, E, asterisks indicate statistically significant differences of the values compared (n = 4; ns, not significant; **p < 0.01; and ***p < 0.001).

FIGURE 3

Role of catalases in decomposition of H₂O₂. (A) Disk diffusion assay of catalase mutants. (B) Measuring H₂O₂-scavenging rate of catalase mutants. (C) qRT-PCR analysis of transcription differences upon oxyR deletion. Genes with high-confident (katE, trxB, dps, and grxA) and low-confident (acnA and zwf) OxyR-binding motifs and without (yfeC and tauA) were examined. Signal intensities were processed, and the fold changes [values of the H₂O₂-treated wild type (WT) and ΔoxyR/values of the untreated WT] were calculated according to the method described in Materials and Methods. (D) Impacts of OxyR on the expression of katE, katG, and ahpC. Cells of mid-exponential phase before and 20 min after the H₂O₂ treatment were harvested for measuring the β-galactosidase assays using an integrative lacZ reporter. The activity of β-galactosidase represents the activity of indicated promoters. In all panels, asterisks indicate statistically significant differences of the values compared (n = 4; ns, not significant; *p < 0.05; **p < 0.01; and ***p < 0.001).

FIGURE 4

Role of AhpC in decomposition of H₂O₂. (A) Measuring the scavenging rate of H₂O₂ in indicated strains. Asterisks indicate statistically significant differences of the values compared (n = 4; ns, not significant; *p < 0.05; **p < 0.01; and ***p < 0.001). (B) Plating defects of the indicated strains. S. oneidensis represents S. oneidensis, which is shown for comparison. Strains outside the green box are YPIII. Experiments were performed at least four times, and representative results were presented.

FIGURE 5

Simultaneous loss of AhpC and KatG increases H₂O₂ resistance. Efficiencies of AhpC, KatE, and KatG at different H₂O₂ concentrations. H₂O₂ was added at a final concentration of 2 μM (A) and 150 μM (B) to cultures of YPIII strains indicated. 2 min after addition of H₂O₂, the H₂O₂ concentration was measured. (C) KatE expression in strains indicated before and after the H₂O₂ treatment. In all panels, asterisks indicate statistically significant differences of the values compared (n = 4; ns, not significant; *p < 0.05; **p < 0.01; and ***p < 0.001).

FIGURE 6

Comparative analysis of AhpC proteins. (A) The domain structure of EcAhpC and YpAhpC. Both proteins are composed of two domains, thioredoxin (green) and peroxiredoxin (blue), with peroxidatic Cys47 (or Cys50) and resolving Cys166 (or Cys171) in red and yellow, respectively. Sequence alignment of four representative AhpCs shown below demonstrates the conservation of regions covering two active Cys residues (identical residues are in bold with star mark) and the C-terminal tail region. Conserved residues in three out of four AhpCs are shown in red and green; the identical residues in YpAhpC and HpAhpC are shown in blue. (B) Structural comparison of YpAhpC and EcAhpC (PDB accession number 4o5r). Shown is a superimposition of YpAhpC (cyan) and EcAhpC (green). The α helix formed by the C-terminal tail of YpAhpC is shown in yellow. Active Cys residues are labeled.