Bacterial Transcription Factors against Reactive Oxygen Species

This is a curated page. Report corrections to Microbewiki.

Overview

By Jiayu Chen

NOX2 complex produces anti-microbial ROS, Yang et al.

Bacteria live in a world full of toxic reactive oxygen species (ROS), including hydrogen peroxide (H₂O₂), superoxide (O₂^-), and hydroxyl radicals. ROS can cause severe damage to all kinds of cell components, including nucleic acids, lipids and proteins . These highly reactive species are generated endogenously by accidental autoxidation of flavoproteins, a group of non-respiratory redox enzymes. Thus, aerobic bacteria have evolved to maintain high concentration of enzymes that keep intracellular ROS below a lethal level. However, various conditions are known to increase these species above such threshold, inflicting oxidative stress to the bacteria, a phenomenon called “oxidative burst.” One of the major scenario is when bacteria encounter ROS generated by the immune system of their host as a defensive weapon. In human, phagocytes including neutrophils use NADPH oxidase 2 (NOX2) complex as one of the major enzyme mediating the immune response by generating antimicrobial ROS. The resultant ROS would be secreted either outside of the cell as a trap to kill bacteria, or into phagolysosome where internalized bacteria would be degraded. Another situation that may happen during bacterial infection is the ROS generated by the mitochondria. Such defensive mechanism is mediated by many signaling molecule in the Toll pathways including several Toll-like receptors (TLRs), tumor necrosis factor receptor-associated factor 6 (TRAF6) and evolutionarily conserved signaling intermediate in Toll pathways (ECSIT).

Anti-bacterial Mitochondrial ROS, Yang et al.

The activation of Toll pathways resulted in increased cellular ROS as well as anti-bacterial pro-inflammatory cytokine reaction in the nucleus. Pro-inflammatory Th1 cytokine IFN-γ is also involved as a transcriptional regulator via activation of estrogen-related receptor α (ERRα) and PPARγ-coactivator-1β (PGC-1β) in the nucleus. Such interaction induces the genes that encode mitochondrial respiratory chain machinery and thus increase cellular ROS concentration.

Therefore, bacteria have developed defensive systems to deal with such situations. One mechanism is ROS inducible transcriptional factors that regulate gene expression of a variety of enzymes to respond to the oxidative stress. Three redox-sensitive transcription factors in bacteria have been intensively studied: OxyR and PerR against H₂O₂, and SoxR against O₂^- .
OxyR is induced by H₂O₂ and activated by reversible disulfide bond formation in its active site. OxyR is widely distributed in Gram-negative bacteria, such as E. coli. The binding of OxyR to DNA generally turns on the gene expression of a large range of oxidative stress defense enzymes. Recent research also has shown that Gram-negative bacteria Vibrio vulnificus, facultative aerobes, contain two OxyR-type regulators with similar sensory mechanism but different sensitivity in order to fine-tune the expression of genes against H₂O₂ pressure.
PerR is the equivalent H₂O₂ sensor protein in many Gram-positive bacteria, such as Staphylococcus aureus and Bacillus subtilis. When PerR is in its reduced form, its binding to DNA generally serves as an inhibitor of the expression of target genes. PerR is activated by oxidation of its regulatory metal, either Fe²⁺ or Mn²⁺, which leads to conformational change in the protein and dissociation from DNA, thus relieving the inhibition and initiating transcription of H₂O₂ defensive genes.
SoxR is conserved widely in both proteobacteria and actinobacteria with an O₂^- sensitive metal complex in its active site. In E. coli, The binding of SoxR to DNA induces the transcription of a secondary transcription factor, SoxS, which activates a group of enzymes suppressing the level of O₂^-.

Defending against hydrogen peroxide (H₂O₂)

Why is H₂O₂ poisonous?

Environmental H₂O₂ is primarily generated as a metabolism by-product and as defense mechanism of plants, animals and bacteria to other microbes. Because its chemical structure is similar to that of water and has no charge, H₂O₂ can easily penetrate the membrane. Once it enters the cell, H₂O₂ damages the cell components through the Fenton Reaction in which H₂O₂ oxidizes Fe²⁺ ion, generating one Fe³⁺ ion and one hydroxyl radical. Free hydroxyl radicals can further react with lipids, nucleic acids and the disulfide bonds in proteins, which are all critical cellular components. For example, guanine is one of the most vulnerable targets to hydroxyl radical because of its small reduction potential. The resulting product is often highly mutagenic 8-hydroxyguanine, because it is able to base pair with adenine such a way that allows it to escape from the DNA mismatch detection system. The Fenton reaction also impedes the function of Fe²⁺-dependent enzymes that are distributed in various cellular processes.

Defensive strategy for Gram-negative bacteria: OxyR

H₂O₂ sensory mechanism of OxyR

Crystal structures of OxyR in different bacteria have shown that it is a tetrameric protein. Each monomer contains a C-terminal regulatory domain and an N-terminal DNA-binding domain.The redox-switch mechanism in OxyR depends on two H₂O₂ sensory cysteine residues (Cys-199 and Cys-208) in its C-terminal. Cys-199 residue locates in a hydrophobic pocket, where it is stabilized by hydrophobic interaction of nearby Leucine residues. Cys-199 is rapidly oxidized by H₂O₂ and forms in a sulfenic acid intermediate (-SOH). This intermediate is not stable in the hydrophobic pocket, and thus lead to a conformational change that flips out the oxidized Cys-199. A flexible amino acid region between these two Cysteine residues then brings Cys-199 to the proximity of the Cys-208 to form a disulfide bond. This bond formation induces OxyR conformational change in its regulatory domain, recruiting RNA polymerase and thus stimulating the expression of genes against H₂O₂ stress. Furthermore, the interaction between OxyR and RNA polymerase seems to be able to reduce the disulfide bond and makes the transcriptional regulation reversible.

Activation of OxyR. (a) Oxidation of the H₂O₂ sensitive Cysteine residues activate gene transcription. (b) Reaction mechanism of OxyR H₂O₂ sensitivity

OxyR in E. coli

As the model organism in microbiology study, the OxyR of E. colihas been extensively studied in past decades. A genome-wide transcription profile of E. coli using DNA microarray has shown that 23 genes are significantly induced (>20-fold) under H₂O₂ stress. KatG, a catalase, and AphC, a NADH peroxidase, are activated, both of which directly reduce cellular peroxide. Several iron-related enzymes are also induced. Dps, a ferritin homolog, is most significantly upregulated (180-fold). Dps oxidizes cellular free Fe²⁺, thus inhibits the Fenton chemistry and protect DNA from hydroxyl radical damage. Similarly, in order to decrease free cellular Fe²⁺ concentration, a Ferric uptake regulator (Fur) protein is also induced, which serves as an inhibitor to iron import. Furthermore, MntH, a Mn importer, is induced, too. Mn2+ cannot be oxidized by H₂O₂and is able to serve as enzymatic cofactor. Thus, higher cellular Mn2+ concentration allows Mn²⁺ to substitute for Fe²⁺ and protect the iron-dependent enzymes from damage cause by the Fenton chemistry. There are other regulon members that are induced significantly under H₂O₂ pressure. However, the functions of some are still unknown, while the others’ roles seem to be less relevant after mutation assay. Notably, there is no direct DNA or lipid repair gene regulated by OxyR.

OxyRs in Vibrio vulnificus

Vibrio vulnificus is a pathogenic Gram-negative bacterium. Although the number of infected individual is relatively low compared with other member in the Vibrio genus, it could result in severe and usually fatal infections on immunocompromised individuals. The infection of V. vulnificus is usually related to consumption of raw shellfish, which engross the bacteria via filter feeding. V. vulnificus can lead to two kinds of diseases: septicemia and tissue necrosis, both of which develop very quickly and could lead to fatality. Since immune system defends against this kind of pathogenic bacteria by generating ROS, thorough characterization of the mechanism of V. vulnificus under such stress is crucial to develop better clinical treatment to its infection. In previous research, two distinct peroxiredoxins (Prx), Prx1 and Prx2, have been identified in gram-negative facultative aerobe V. vulnificus Prx1 is previously known as the homolog of NADH peroxidase AhpC in V. vulnificus. Prx1 is regulated by transcription factor OxyR in V. vulnificus and has similar function as that found in E. coli. However, the discovery and characterization of Prx2 led researchers to hypothesize the presence of another OxyR-type transcription factor in V. vulnificus. Prx2 shares low amino acid sequence identity with Prx1 and other bacterial AhpC. Prx2 is able to sense H₂O₂ at a level that is much lower than what Prx1 is able to do. Since V. vulnificus is facultative aerobic, two Prxs thus allow the bacteria to optimize the defendant process against different amount of H₂O₂. However, it is not clear how V. vulnificus regulate the gene expression of the second Prx.
Kim et al. successfully identified and characterized OxyR2, a new OxyR-type transcription factor in V. vulnificus. Sequence analysis shows that OxyR2 shares 34% amino acid sequence identity to the first OxyR (thereafter OxyR1) and contain the two Cysteine residues located corresponding to the redox-sensitive ones in OxyR1. Similarly, the two Cysteine residues in OxyR2 are subject to H₂O₂ oxidation to form a reversible disulfide bond. Prx2 expression is only induced by oxidized form of OxyR2 in vivo. The minimum concentration of H₂O₂ required to oxidize OxyR2 is much lower than that of OxyR1, which explains the high sensitivity of OxyR2 and thus that of Prx2. Compared to OxyR1, the enhanced H₂O₂ sensitivity could because of the fact that the microenvironment that the redox-active Cysteine residues locate are different. In OxyR1, Cys-199 is adjacent to a negatively charged Asp residue, while in OxyR2, the corresponding residue Cys-206 is adjacent to a positively charged Lys residue, which could enhance the nucleophilicity of Cys-206 by decreasing the thiol pKa, resulting in a higher reactivity to H₂O₂.
By possessing two OxyRs with different affinity to H₂O₂, V. vulnificus are able to fine-tune the detoxification of different amount of H₂O₂ they may encounter in natural environment. Such optimization could be an evolutionary advantage relevant to its pathogenesis. Furthermore, BLAST search reveals that there are many other bacteria containing homologs of both OxyR1 and OxyR2, suggesting that the coexistence of both OxyRs is widely distributed in bacteria.
From a medical perspective, mutation on the key Cysteine residues in OxyR2 resulted in lower concentration of cellular lactate dehydrogenase in the infected host cells, which indicated decreasing cytotoxicity caused by infection. In addition, during infection, the oxyR2 mutants showed decreased growth rate compared to the wild type. This could be explained by the fact that the lack of OxyR2 caused decreased Prx2 in the bacteria, so they were not able to fight against the ROS generated by the immune system of the host cells. These results implied that OxyR2 might be important for the virulence of V. vulnificus.

Defensive strategy for Gram-positive bacteria: PerR

H₂O₂ Sensory mechanism of PerR

PerR is the common H₂O₂-inducible transcription factor in many Gram-positive bacteria such as Bacillus subtilis and Staphylococcus aureus. It belongs to the Fur protein family and uses metal oxidation reaction to sense H₂O₂. PerR is a dimer and each subunit has a structural zinc-binding site that irreversibly binds to Zn²⁺. Each subunit also contains at least one regulatory metal binding site. The metal in the regulatory site varies from organisms, usually Fe²⁺ and/or Mn²⁺. When cellular H₂O₂ concentration increases, it will oxidize the regulatory metal. Contrast to cysteine thiol-dependent OxyR, the oxidized metal cause rapid oxidation of histidine residues nearby and leads to a conformational change of the protein. This conformational change causes a decreased binding affinity of PerR to DNA. Since PerR usually functions as a repressor, its detachment from DNA relieves the inhibition on the gene expression of the regulon members.

Structure of PerR in Bacillus subtilis. (a) holoenzyme (b) demetallated (Manganese removed) enzyme. The orange balls are Zn and the purple ones are Mn

PerR in Bacillus subtilis

PerR in B. subtilis (PerR_BS) is the most studied PerR in bacteria. In B. subtilis, the metal in the regulatory site is Fe²⁺. PerR_BS may also bind Mn²⁺ as an alternative, which, however, as mentioned before, is not sensitive to H₂O₂. PerR_BS shows same catalytic rate with H₂O₂ as that of OxyR in E. coli. Compared with the reversible cysteine oxidation in OxyR, the oxidation of histidine residues in PerR_BS seems to be irreversible, since there is no oxo-histidine reduction mechanism discovered yet. The PerR_BS regulon overlaps a lot with that of OxyR in Gram-negative bacteria, such as KatA, a KatG homolog, MrgA, a Dps homolog, and a Fur protein. However, contrary to OxyR, PerR_BS regulon does not include the enzymes related to maintain thiol and to reduce disulfide bonds.

PerR in Staphylococcus aureus

Staphylococcus aureus is a Gram-positive pathogenic bacterium. As an opportunistic pathogen, it is often found on normal skin microflora and in the nasal passage and respiratory tract. However, on susceptible individuals, it is the most common cause of mild skin infections as well as often-fatal infections including sepsis and toxic shock syndrome (Lowy, 1998). Current therapeutics of S. aureus infection is increasingly complicated because of the emergence and prevalence of methicillin-resistant S. aureus (MRSA) across the world (Klevens et al., 2007). It has been known that in the infected organisms, their neutrophils use phagosome containing ROS to defend against S. aureus (Reviewd by Spaan et al., 2013; van Kessel et al., 2014). Therefore, in order to develop enhanced treatment to S. aureus infection, it is important to characterize the mechanism(s) that S. aureus uses to defend oxidative stress.
PerR has been previously identified as the major transcription factor on S. aureus to response to oxidative burst (Horsburge et al., 2001a; Horsburge et al., 2001b). The characterization of PerR in S. aureus (PerR_SA) has shown that it is a H₂O₂-inducible translation factor using the same sensory mechanism as B. subtilis. Together with PerR found in other bacteria, Ji et al. suggest that PerR and PerR-type transcription factors may be a conserved mechanism for Gram-positive bacteria against H₂O₂ pressure (2015). However, under the same H₂O₂ pressure, the expression levels of PerR regulon were higher in S. aureus expressing PerR_SA than those expressing PerR_BS, which suggests that
PerR_SA was more sensitive to H₂O₂ oxidation and thus more resistant to H₂O₂. Furthermore, Ji et al. used Caenorhabditis elegans as the model to study the relationship between H₂O₂ sensitivity of PerR and pathogenesis of S. aureus. They observed that S. aureus expressing PerR_BS killed C. elegans faster than PerR_SA. This suggested that lower susceptibility of PerR to H₂O₂ oxidation actually increases the virulence of S. aureus.

Section 2

Include some current research, with at least one figure showing data.

Section 3

Include some current research, with at least one figure showing data.

Overall structure of the SoxR–DNA complex in E. coli. The SoxR dimer is shown in a ribbon representation and the DNA fragment appears in a stick model. The DNA-binding domain, dimerization helix, and Fe-S cluster binding domain are shown in blue, magenta, and yellow, respectively. The two iron and two sulfur atoms of the [2Fe-2S] cluster are represented with brown and green spheres, respectively. Watanabe et al., 2008.

References

Authored for BIOL 238 Microbiology, taught by Joan Slonczewski, 2016, Kenyon College.