How Microbes Defend Themselves From Incoming Hydrogen Peroxide

Abstract

Microbes rely upon iron as a cofactor for many enzymes in their central metabolic processes. The reactive oxygen species (ROS) superoxide and hydrogen peroxide react rapidly with iron, and inside cells they can generate both enzyme and DNA damage. ROS are formed in some bacterial habitats by abiotic processes. The vulnerability of bacteria to ROS is also apparently exploited by ROS-generating host defense systems and bacterial competitors. Phagocyte-derived ${\text{O}}_2^{-}$ can toxify captured bacteria by damaging unidentified biomolecules on the cell surface; it is unclear whether phagocytic H₂O₂, which can penetrate into the cell interior, also plays a role in suppressing bacterial invasion. Both pathogenic and free-living microbes activate defensive strategies to defend themselves against incoming H₂O₂. Most bacteria sense the H₂O₂via OxyR or PerR transcription factors, whereas yeast uses the Grx3/Yap1 system. In general these regulators induce enzymes that reduce cytoplasmic H₂O₂ concentrations, decrease the intracellular iron pools, and repair the H₂O₂-mediated damage. However, individual organisms have tailored these transcription factors and their regulons to suit their particular environmental niches. Some bacteria even contain both OxyR and PerR, raising the question as to why they need both systems. In lab experiments these regulators can also respond to nitric oxide and disulfide stress, although it is unclear whether the responses are physiologically relevant. The next step is to extend these studies to natural environments, so that we can better understand the circumstances in which these systems act. In particular, it is important to probe the role they may play in enabling host infection by microbial pathogens.

Keywords: OxyR regulator; Yap1p; nitric oxide; peroxide sensing repressor (PerR); reactive oxygen species.

Figure 1

(A) The reduction potential of oxygen and reactive oxygen species. The standard reduction potentials (pH 7) indicate that unlike O₂, superoxide, hydrogen peroxide, and hydroxyl radicals are potent univalent oxidants. The standard concentration of oxygen is regarded as 1 M. (B) The classes of damage caused by intracellular ${\text{O}}_2^{-}$ and H₂O₂. The transfer of electrons from redox enzymes to oxygen generates superoxide and hydrogen peroxide. Both species can oxidize the solvent-exposed iron centers of mononuclear iron enzymes and [4Fe-4S] dehydratases. Additionally, H₂O₂ directly reacts with the intracellular iron pool, which is loosely associated with biomolecules, including DNA. The reaction generates hydroxyl radicals, which can damage DNA.

Figure 2

H₂O₂-scavenging enzymes in E. coli. Environmental H₂O₂ gradually diffuses into the cytoplasm, where it is degraded by NADH peroxidase (AhpCF) and catalase (KatG). Both are induced by OxyR. Cytoplasmic H₂O₂ is therefore substantially lower in concentration than is extracellular H₂O₂. Under hypoxic conditions OxyR also induces the periplasmic cytochrome c peroxidase (Ccp), which allows the respiratory chain to employ H₂O₂ as a terminal oxidant. Because H₂O₂ rapidly crosses through OM porins, and Ccp activity is moderate, the periplasmic H₂O₂ concentration is likely equivalent to that outside the cell.

Figure 3

The damage caused to mononuclear iron proteins by hydrogen peroxide. H₂O₂ directly oxidizes the solvent-exposed Fe(II) cofactor, which then dissociates. The ferryl (FeO²⁺) species that is formed in this reaction can directly oxidize the polypeptide ligands to the iron atom, irreversibly inactivating the enzyme. However, if a cysteine residue coordinates the iron, it will quench the ferryl radical (as shown). The enzyme activity can then be restored by reduction of the cysteine sulfenate residue, probably by thioredoxins. OxyR induces the MntH manganese importer, allowing the proteins to be metallated with Mn(II), which provides activity and does not react with H₂O₂.

Figure 4

The damage caused to [4Fe-4S]²⁺ cluster enzymes by hydrogen peroxide. The catalytic Fe atom of the dehydratase enzyme reacts with H₂O₂ and dissociates, leaving behind an inactive [3Fe-4S]⁺ cluster. That cluster can be reactivated by reduction and remetallation. In some dehydratases the cluster completely disintegrates to form an apoenzyme. OxyR induces the Suf system to rebuild a functional holoenzyme.

Figure 5

Formation of ROS by phagosomes. NADPH oxidase (Nox) generates superoxide which cannot penetrate the cytoplasmic membranes of the engulfed bacteria. It is believed that either superoxide or its protonated form injures extracytoplasmic targets. Additionally, membrane-permeable H₂O₂ is generated through dismutation. Calculations suggested that the levels of ${\text{O}}_2^{-}$, HO₂• and H₂O₂ in isolated macrophages range from 10–50 μM, 0.1–4 μM and 1–4 μM, respectively, depending upon phagosomal pH. Modeling predicts a similar H₂O₂ concentration inside neutrophils. The H₂O₂ levels would rise, however, if it accumulates it the surrounding tissue.

Figure 6

OxyR activation in E. coli. The oxidation of the sensory C199 cysteine by H₂O₂ leads to the formation of a disulfide bond between C199 and C208. The resulting conformational change causes OxyR to bind as a tetramer to the promoter regions, which recruits RNA polymerase, and results in the transcription of genes in the OxyR regulon. In many other bacteria the reduced form also binds DNA, albeit in an elongated conformation that represses transcription; oxidation again converts it to a transcriptional activator.

Figure 7

OxyR control of the intracellular iron pool. In order to minimize DNA damage, OxyR decreases the intracellular iron pool by inducing Dps, YaaA, and Fur. The Clp system maintains a small residual iron pool to enable synthesis of iron-dependent enzymes. The H₂O₂-responsive PerR regulon in Bacillus subtilis also controls Fur and MrgA, which is a Dps homolog.

Figure 8

Yap1p activation. H₂O₂ oxidizes the C36 residue of glutathione peroxidase (Gpx3). The resulting sulfenate interacts with C598 in the C-terminal domain of Yap1p to form a intermolecular disulfide bond. Subsequent thiol-disulfide exchange reactions produce C303-C598 and C310-C629 disulfide bonds in Yap1p. Yap1p accumulates in the nucleus, leading to the activation of the Yap1p regulated genes. When H₂O₂ diminishes, Yap1p is reduced by the thioredoxin system; this change makes its nuclear export signal accessible to Crm, causing Yap1p to be transported back out of the nucleus.

Figure 9

PerR activation. PerR is a dimeric DNA-binding protein, and it binds two metal ions per monomer. The first ion is a structural Zn²⁺ that is necessary for dimerization and structural integrity. The second metal ion enables DNA binding, and can either be Fe²⁺ or Mn²⁺. Only PerR bound to Fe²⁺ is responsive to H₂O₂. The oxidation of Fe²⁺ by H₂O₂ generates a localized hydroxyl/ferryl radical, which irreversibly oxidizes either of two His ligands (H37 or H91) to form 2-oxo-histidine. Metal binding is blocked, PerR dissociates from promoter sites, and the regulon is induced.