INTRODUCTION {#sec1}
============

Reactive oxygen species (ROS) are highly reactive oxygen-containing molecules such as oxygen ions and peroxide generated from the metabolism of oxygen. Exogenous ROS can be formed following exposure to physical or chemical agents such as ionizing radiation (Dainton [@bib49]), ultraviolet (UV) radiation (Jurkiewicz and Buettner [@bib112]), mitomycin C (Tomasz [@bib197]) or desiccation (Potts [@bib165]). Depending on the setting, and the concentration, ROS can be either essential for, or detrimental to, cellular survival. ROS is required for mammalian cell survival, growth, proliferation and differentiation (Droge [@bib54]), notably through its role in intracellular signaling (Finkel [@bib63]; Rhee [@bib171]); however, ROS can also be highly injurious to cells, causing damage to proteins and DNA (Brawn and Fridovich [@bib23]), provoking lipid peroxidation (Niki [@bib151]) or oxidizing enzyme cofactors. ROS play a role in the development of a number of human diseases, including chronic inflammation, age-related disorders and cancers (Liou and Storz [@bib128]). Host phagocytic cells, primarily neutrophils and monocytes generate endogenous ROS to facilitate clearance of bacteria at the infection site.

Bacterial cells synthesize a number of enzymes to detoxify ROS, including alpha-1-microglobulin (Olsson *et al.*, [@bib154]), superoxide dismutases (Sods) (Lynch and Kuramitsu [@bib131]), catalases (Mishra and Imlay [@bib141]), glutathione peroxidases (Moore and Sparling [@bib145]) and peroxiredoxins (generally referred to as AhpC in bacteria) (Poole [@bib161]; Dubbs and Mongkolsuk [@bib55]). Antioxidants, including ascorbic acid (vitamin C), tocopherol (vitamin E), uric acid and glutathione, enhance resistance to oxidative stress. Reactive nitrogen species (RNS) are another group of antimicrobials produced by certain immune cells, resulting in nitrosative stress. RNS are most often derived from nitric oxide and superoxide. Like ROS, RNS have diverse functions within the host, and the importance of RNS in health and disease has been reviewed elsewhere (Nathan and Shiloh [@bib147]; Fang [@bib60]) and is not a focus of this review.

Bacterial pathogens such as *Streptococcus pyogenes*, also known as group A *Streptococcus* (GAS), encounter ROS during the innate immune response driven by host phagocytic cells. GAS is a Gram-positive facultative anaerobe, currently listed as a 'top 10' human pathogen, responsible for 700 million infections annually (Carapetis *et al.*, [@bib32]). Of these infections, 1.8 million are severe invasive infections (bacteremia, toxic shock syndrome and necrotizing fasciitis) with a mortality rate of 25%. The remaining infections caused by GAS are benign and self-limiting and include pharyngitis and impetigo (Walker *et al.*, [@bib210]). Currently, there is no safe and efficacious vaccine to protect against GAS infection (Henningham, Gillen and Walker [@bib95]). A better understanding of GAS pathogenesis and the complex interplay between GAS and host proteins, molecules and tissues, may lead to the development of novel therapeutic agents or effective vaccines. One group of host defense factors of particular interest in this regard is ROS. GAS encounters ROS at various stages of infection, and consequently GAS are well equipped to deal with oxidative stress and express an array of proteins and virulence factors to survive under these harsh conditions. In contrast to the human pathogen *Staphylococcus aureus*, which produces a similar spectrum of skin and invasive infections, GAS lack catalase, a heme-containing peroxidase that degrades hydrogen peroxide (H~2~O~2~) to H~2~O and oxygen (O~2~), and do not express an antioxidant carotenoid pigment to withstand ROS-mediated killing (Liu *et al.*, [@bib129]). Despite this, GAS can tolerate the high oxygen concentrations within the human host and has evolved multiple mechanisms to resist the toxic effects of ROS produced from the reduction of atmospheric oxygen or the activation of host phagocytic cells (Table [1](#tbl1){ref-type="table"}). Two fundamental mechanisms are employed by GAS to counter ROS: 1) suppression of ROS (indirect), and 2) expression of enzymes to detoxify ROS (direct). This review focuses on the ROS resistance mechanisms of GAS including the expression of surface-associated ROS resistance factors, intracellular and secreted enzymes involved in ROS detoxification or repair of ROS-damaged proteins, metal transporters important for metal ion homeostasis and oxidative stress resistance, and the transcription factors involved in the coordinated regulation of gene expression for GAS survival under conditions of oxidative stress.

###### 

Resistance mechanisms employed by GAS to thwart ROS.

  Resistance mechanism       GAS factor   Function
  -------------------------- ------------ --------------------------------------------------------------------------------------
  Surface/secreted protein   M protein    Impaired fusion of azurophilic granules with phagolysosome
                             HA capsule   H~2~O~2~ resistance by aggregation
                             Mac-1        Reduced phagocytosis and ROS production
                             Mac-2        Reduced phagocytosis and ROS production
  Enzymatic detoxification   SodA         Superoxide resistance
                             AhpC         H~2~O~2~ resistance
                             GpoA         Superoxide resistance
                             NoxA         Superoxide resistance and H~2~O~2~ resistance
  Enzymatic repair           HtrA         Protein repair upon ROS damage
                             PolA1        DNA repair upon ROS damage, H~2~O~2~ resistance and inhibition of Fenton reaction
  Metal ion binding          PmtA         Metal transporter and H~2~O~2~ resistance
                             Dpr          Iron sequestration and H~2~O~2~ resistance
                             MtsABC       Metal transporter, superoxide and H~2~O~2~ resistance, inhibition of Fenton reaction
                             Shr          Iron sequestration and H~2~O~2~ resistance

BIOLOGICAL ROLES OF ROS {#sec2}
=======================

ROS are generated during the metabolism of O~2~ and consist of highly toxic and reactive oxygen-containing molecules such as peroxide and oxygen ions. ROS are produced either via the transfer of electrons to O~2~, leading to the production of superoxide (O~2~•^−^), H~2~O~2~ and highly reactive hydroxyl radicals (OH•), or via the transfer of energy to O~2~, which leads to the formation of singlet oxygen (O~2~\*) (Yesilkaya *et al.*, [@bib217]). Superoxide damages proteins by oxidizing iron--sulfur clusters within enzymes (Kuo, Mashino and Fridovich [@bib119]; Flint, Tuminello and Emptage [@bib66]). The membrane-permeable H~2~O~2~ can target sulfur atoms in cysteine (Winterbourn and Metodiewa [@bib214]) or methionine residues in proteins (Griffiths and Cooney [@bib86]), and cause oxidative damage to cell membranes or nucleic acids (Gabbianelli *et al.*, [@bib72]). Highly reactive singlet oxygen reacts with proteins, lipids, DNA and RNA (Glaeser *et al.*, [@bib83]). Hydroxyl radicals are formed during the Fenton reaction in which H~2~O~2~ is converted to OH• in the presence of metal ions \[H~2~O~2~ + Fe(II) → OH• + OH^−^ + Fe(III)\]. The highly reactive OH• targets DNA, attacking deoxyribose residues resulting in nicks in double-stranded DNA which can compromise chromosome fidelity and ultimately lead to cell death (Imlay, Chin and Linn [@bib104]; Rai *et al.*, [@bib169]). Carbonylated proteins are an additional and irreversible byproduct of metal-catalyzed oxidative stress (Amici *et al.*, [@bib4]; Dalle-Donne *et al.*, [@bib51]). Carbonylation often results in loss of protein function and is further reviewed in Dalle-Donne *et al.* ([@bib51]). Heavily carbonylated proteins can form aggregates, are resistant to proteasomal degradation and are thought to be associated with certain neurodegenerative disorders (Dalle-Donne *et al.*, [@bib51]). ROS also plays a role in the activation of the inflammasome, an important arm of the innate immune defense, whereby pattern-recognition receptors on host cells identify danger to the host, via recognition of pathogen-associated molecular patterns from microbes and damage-associated molecular patterns released from injured tissue (Bauernfeind and Hornung [@bib18]). The most studied inflammasome is the Nod-like receptor (NLR) family, pyrin domain-containing 3 (NLRP3). The production of ROS is an important upstream event of NLRP3 activation (Tschopp and Schroder [@bib200]). ROS have diverse roles within the host, participating in biological processes including cellular signaling, chemotaxis, antigen cross-presentation, autophagy, mammalian cell growth, proliferation and differentiation, and the adaptive immune response, which have been reviewed elsewhere (Thannickal and Fanburg [@bib196]; Droge [@bib54]; Lam, Huang and Brumell [@bib121]; Yang *et al.*, [@bib216]).

IMMUNE CELLS THAT GENERATE ROS {#sec3}
==============================

It has been long established that during phagocytosis of microbes, phagocytes such as neutrophils and monocytes exhibit increased O~2~ consumption (Sbarra and Karnovsky [@bib180]; Iyer, Islam and Quastel [@bib106]; Rossi and Zatti [@bib176]). This phenomenon is known as respiratory or oxidative burst and is a very important component of innate immunity and the host defense against microbes. A synergy between myeloperoxidase (MPO) (contained within neutrophil azurophilic granules released into the phagosome during the degranulation process), H~2~O~2~ produced by neutrophils, and a halide, often chloride (Fig. [1](#fig1){ref-type="fig"}), results in intracellular killing of Gram-positive and Gram-negative bacterial species (McRipley and Sbarra [@bib140]; Klebanoff [@bib118]). GAS is naturally deficient in catalase and carotenoid pigment expressed by other Gram-positive bacteria such as *Bacillus* spp. (Zuber [@bib218]) and *S. aureus* (Liu *et al.*, [@bib129]), and may be a susceptible target to oxidative burst killing initiated by host cells (Kwinn and Nizet [@bib120]). Specifically, leukocytes release ROS O~2~•^−^ and H~2~O~2~ into the intracellular milieu through the assembly of membrane-bound nicotinamide adenine dinucleotide phosphate (NADH) oxidase 2 (NOX2) on the phagosomal membrane following phagocytosis (Babior, Kipnes and Curnutte [@bib14]; DeLeo *et al.*, [@bib52]; Nauseef [@bib148]). ROS is also generated by the terminal enzyme of purine catabolism, xanthine oxidase (Harrison [@bib90]), the electron transport chain within mitochondria (Murphy [@bib146]; Santos *et al.*, [@bib179]), peroxisomes (Antonenkov *et al.*, [@bib9]) and the endoplasmic reticulum (Santos *et al.*, [@bib179]). There are seven known members of the NOX family, NOX1--5 and DUOX1--2 (Lam, Huang and Brumell [@bib121]), with NOX2 producing the greatest amount of ROS in human tissues. The O~2~•^−^ and H~2~O~2~ produced by NOX2 are subsequently converted to oxidizing radicals (such as OH•) and oxidizing halogens (e.g. hypochlorite, ClO^−^), both of which are powerful microbicidal agents (Babior [@bib13]). The generation of ROS by NOX mediates the formation of neutrophil extracellular traps (NETs) (Fuchs *et al.*, [@bib71]; Bianchi *et al.*, [@bib20]). NETs are released from activated neutrophils and comprise a mixture of granule proteins, antimicrobial peptides and nuclear constituents such as chromatin and histones that combine to form extracellular fibers capable of trapping and killing bacteria (Brinkmann *et al.*, [@bib28]). Therefore, bacteria including GAS have evolved strategies not only to reduce ROS production, but also to further inhibit the generation of NETs and innate immune clearance. GAS resistance to lethal doses of H~2~O~2~ can be induced *in vitro* by growth in the presence of oxygen (Ricci, Janulczyk and Bjorck [@bib173]), 5% ethanol (King, Horenstein and Caparon [@bib117]) or sub-lethal concentrations of H~2~O~2~ (King, Horenstein and Caparon [@bib117]).

![Generation of reactive oxygen intermediates (light blue) by enzymes in human neutrophils (dark blue) upon phagocytosis, and the enzymatic detoxification mechanisms utilized by GAS bacteria (orange). Oxygen (O~2~) can be converted to superoxide (O~2~•^−^) following the activation of NOX in neutrophils. Superoxide may be converted to H~2~O~2~ by the GAS enzyme SodA. Both superoxide and hydrogen peroxide may be converted to hydroxyl radicals (OH•) during the Fenton reaction. Bacteria express iron chelators and transporters to maintain iron homeostasis and prevent the formation of bactericidal hydroxyl radicals. The neutrophil enzyme MPO catalyzes the production of bactericidal HOCl from H~2~O~2~ and Cl^−^ during the oxidative burst. Hydrogen peroxide can be detoxified to water by the enzyme Gpo, AhpC or NoxA. Following the gain of an electron, hydroxyl radicals can be converted to water. Abbreviations: O~2~, oxygen; O~2~•^−^, superoxide; NOX, NADH oxidase; H~2~O~2~, hydrogen peroxide; SodA, superoxide dismutase; OH•, hydroxyl radicals; MPO, myeloperoxidase; HOCl, hypochlorous acid; Cl^−^, chloride anion; GpoA, glutathione peroxidase; AhpC, alkyl hydroperoxidase; NoxA, NADH oxidase A reductase.](fuu009fig1){#fig1}

GAS PRODUCES ROS {#sec4}
================

Certain, but not all, GAS strains are H~2~O~2~ producers when cultured under anaerobic conditions (Saito *et al.*, [@bib177]). Agar-based techniques, involving the Prussian blue forming reaction, have been developed to differentiate H~2~O~2~ producing from non-producing GAS isolates (Saito *et al.*, [@bib178]). The GAS enzymes lactate oxidase (Seki *et al.*, [@bib182]; Kietzman and Caparon [@bib115]) and NOX (Gibson *et al.*, [@bib80]) are responsible for the secretion of up to millimolar concentrations of H~2~O~2~ into the extracellular milieu (Malke *et al.*, [@bib136]; Cleary and Larkin [@bib44]; Gibson *et al.*, [@bib80]; Saito *et al.*, [@bib177]). The reason why GAS generates peroxide is not fully understood. The H~2~O~2~ produced by H~2~O~2~-producing GAS may function to kill other bacterial species in order to help establish infections, as is the case for *Streptococcus pneumoniae* (Pericone *et al.*, [@bib158]) and commensal *Lactobacillus* spp. (Aroutcheva *et al.*, [@bib10]). In invertebrate models of GAS pathogenesis, GAS production of H~2~O~2~ resulted in the death of *Caenorhabditis elegans* (Jansen *et al.*, [@bib108]; Bolm *et al.*, [@bib21]). However, not all GAS isolates causing disease, be it severe or mild, produce H~2~O~2~, and in a study determining H~2~O~2~ production (25 of the 46 clinical strains examined accumulated H~2~O~2~ in the growth medium) there was no correlation between disease state and a H~2~O~2~-producing phenotype (Saito *et al.*, [@bib177]). In this study, non-H~2~O~2~-producing strains were found to be resistant to *in vitro* killing by phagocytes isolated from patients with chronic granulomatous disease (CGD), and *in vivo* subcutaneous injection of H~2~O~2~ non-producing strains resulted in a greater swelling in CGD mice footpads, accompanied by a higher mortality rate, compared to the H~2~O~2~-producing strains (Saito *et al.*, [@bib177]).

GAS INDUCES ROS {#sec5}
===============

GAS, like other nasopharyngeal pathogens, is exposed to a range of O~2~ concentrations during the infection process, encountering 20% O~2~ on the nasopharynx, 5% O~2~ in the lower respiratory tract and virtually anaerobic conditions in blood (Yesilkaya *et al.*, [@bib217]). During contact with host innate immune cells such as leukocytes and macrophages, GAS induces ROS. In turn, the action of the ROS can result in host cell injury and/or death. For instance, following the production of H~2~O~2~ by host neutrophils, a synergistic interaction between a secreted GAS hemolysin, streptolysin S (SLS) and H~2~O~2~ results in injury to vascular endothelial cells (Ginsburg *et al.*, [@bib81]; Ginsburg and Varani [@bib82]). ROS induced by GAS invasion has also been shown to trigger apoptosis of infected epithelial cells due to mitochondrial dysfunction (Aikawa *et al.*, [@bib2]). Specifically, ROS was produced following activation of the GTPase Rac1 (Aikawa *et al.*, [@bib2]), previously reported to mediate ROS production (Suzukawa *et al.*, [@bib194]).

SURFACE MOLECULES INVOLVED IN THE OXIDATIVE STRESS DEFENSE OF GAS {#sec6}
=================================================================

One factor contributing to the success of GAS as a human pathogen is its ability to evade and/or defend against the host innate immune response. One such defense mechanism is oxidative stress resistance, for which GAS contain an orchestrated repertoire of surface proteins and polysaccharides to counter the ROS produced by host cells (Table [1](#tbl1){ref-type="table"}).

M protein {#sec6-1}
---------

GAS strains are distinguished serologically on the basis of immunovariable surface-anchored M proteins (Facklam *et al.*, [@bib59]). The M protein is a vaccine antigen and major virulence factor of GAS, which affects adherence and invasion to host cells via interaction with multiple host proteins including fibrinogen (Smeesters, McMillan and Sriprakash [@bib184]; Ghosh [@bib77]; Anderson et al., [@bib6]), and inhibits phagocytosis of GAS to overcome innate immunity (Smeesters, McMillan and Sriprakash [@bib184]). There are four known types of granules within neutrophils, the two major granules are the primary (azurophilic) and secondary (specific) granules (Pham [@bib160]). Azurophilic granules contain MPO, and play an active role in the digestion of phagocytosed material, while specific granules are secretory and take part in initiation of the host inflammatory response (Borregaard and Cowland [@bib22]). Live wild-type GAS (expressing M and M-like proteins), after internalization by human neutrophils, increased oxidative burst (specifically H~2~O~2~ production) and membrane traffic responses compared to mutants lacking M protein, specifically via inhibition of the fusion of azurophilic granules with phagosomes (Staali *et al.*, [@bib190]). This is an additional M protein-mediated mechanism of GAS evasion of the host immune system, even once entrapped within phagosomes. Conversely, M1 protein spontaneously released from the surface of GAS or after enzymatic cleavage activates neutrophils as determined by release of heparin-binding protein (Herwald *et al.*, [@bib97]; Macheboeuf *et al.*, [@bib134]). Moreover, M1 protein triggers the release of MPO from neutrophils mediating lung damage (Soehnlein *et al.*, [@bib187]). Additionally, surface-released M1 protein forms a pathological network with fibrinogen, which circulates in high concentrations in human blood (Macheboeuf *et al.*, [@bib134]). This complex contributes to lung damage and inflammation, a condition characterized by high ROS levels (Herwald *et al.*, [@bib97]; Soehnlein *et al.*, [@bib187]; Macheboeuf *et al.*, [@bib134]). Consequently, these findings suggest that the M1 protein released from GAS cell surface may contribute to induction of ROS production; however, further research is required to corroborate the link between M protein and ROS induction (Allen and Stephens [@bib3]).

Hyaluronan capsule {#sec6-2}
------------------

The surface capsule of GAS is composed solely of hyaluronan (HA); a high-molecular-mass polysaccharide comprised of glucuronic acid and *N*-acetylglucosamine (Kendall, Heidelberger and Dawson [@bib114]). The GAS capsule is structurally identical to the HA widely distributed throughout human tissues, allowing GAS to mimic host structures and thwart detection by the host immune system. The capsule promotes GAS survival by obstructing antibody binding to epitopes on the bacterial surface, complement deposition (Dale *et al.*, [@bib50]) and opsonophagocytosis (Foley and Wood [@bib67]; Dale *et al.*, [@bib50]). The capsule of GAS has also been reported to contribute to resistance against H~2~O~2~. Encapsulated strains grow in aggregates, taking up oxygen at a slower rate than non-encapsulated derivative strains (Cleary and Larkin [@bib44]). HA capsule-mediated aggregation is thought to mechanically shield GAS from destruction by oxygen metabolites such as H~2~O~2~ (Cleary and Larkin [@bib44]).

GAS Mac-1-like protein (Mac-1/IdeS) {#sec6-3}
-----------------------------------

Following a proteomic analysis of GAS culture supernatants, a secreted protein with homology to the α-subunit of human Mac-1, designated the GAS Mac-1-like protein (Mac-1, also known as IdeS), was discovered. Human Mac-1, also known as CDIIb or CD18, is a leukocyte adhesion glycoprotein functioning in cell--cell and cell--substrate adhesive interactions including binding complement product iC3b (Corbi *et al.*, [@bib47]). Consequently, human Mac-1 plays a role in regulating leukocyte migration, phagocytosis and oxidative killing. GAS Mac-1 is thought to function through molecular mimicry by binding CD16 on the surface of neutrophils, consequently inhibiting opsonophagocytosis and production of ROS (Lei *et al.*, [@bib124]). Lei and colleagues further concluded that Mac-1 prevents any receptor--antibody interaction, i.e. activation by IgG binding as an immune suppression strategy by GAS (Lei *et al.*, [@bib124]). In addition, recombinant Mac-1-mediated proteolytic cleavage of the hinge region of IgG is hypothesized to prevent the recognition of antibody-opsonized GAS by Fc receptors of immune cells and by the complement system (von Pawel-Rammingen *et al.*, [@bib207]). During studies utilizing recombinant Mac-1 incubated with serum containing anti-GAS antibodies, subsequent proteolytic degradation inhibited ROS production by human neutrophils (Lei *et al.*, [@bib124]; Söderberg and von Pawel-Rammingen [@bib186]). However, a study comparing a Mac-1-deficient mutant GAS strain to the wild-type and complemented strains demonstrated that native Mac-1 expressed by live GAS does not reduce ROS production or other neutrophil functions (Okumura *et al.*, [@bib153]).

Mac-2 {#sec6-4}
-----

A second Mac-1-like protein has been identified, designated Mac-2 (Lei *et al.*, [@bib125]). Previously, Mac-2 has been reported to interfere with opsonophagocytosis by blocking Fcγ receptors on phagocytic cells (Agniswamy *et al.*, [@bib1]). In initial studies, Mac-2 did not inhibit ROS production by neutrophils stimulated with IgG-coated latex beads (Lei *et al.*, [@bib125]). Subsequent studies measuring immunocomplex-induced oxidative burst in whole blood containing recombinant Mac-2 indicated that Mac-2 contributes to the inhibition of opsonophagocytosis-induced ROS production *ex vivo*. However, this inhibition did not enhance streptococcal survival in bactericidal assays (Söderberg, Engström and von Pawel-Rammingen [@bib185]).

ENZYMATIC DETOXIFICATION MECHANISMS {#sec7}
===================================

GAS is a facultative anaerobic organism that generates energy from glycolysis due to a deficiency in heme-containing protein complexes for oxidative phosphorylation. The growth kinetics of this aerotolerant pathogen are substantially enhanced in the presence of oxygen (Gabbianelli *et al.*, [@bib72]). GAS is a member of the lactic acid bacteria family and naturally lacks catalase, a heme-containing peroxidase and highly efficient H~2~O~2~-detoxification enzyme expressed by numerous other bacterial species to survive in aerobic environments. Nevertheless, GAS persists in oxygen-rich anatomical sites of the human host, and has evolved effective strategies to combat bactericidal H~2~O~2~ and other ROS generated by host innate immune cells, such as neutrophils and macrophages, at the site of infection (Gibson *et al.*, [@bib80]). The GAS genome harbors peroxidases, Sod and NADH oxidase to directly decompose ROS (Gibson and Caparon [@bib79]; Gerlach, Reichardt and Vettermann [@bib76]; Gibson *et al.*, [@bib80]; King, Horenstein and Caparon [@bib117]). Indirect ROS resistance mechanisms comprise proteins involved in the repair of ROS-damaged biomolecules (e.g. DNA or proteins) or metal ion transporters involved in maintaining metal homeostasis and oxidative stress resistance (Table [1](#tbl1){ref-type="table"}). Next, we highlight conserved ROS defense mechanisms employed by multiple serotypes of GAS (Fig. [2](#fig2){ref-type="fig"}).

![The GAS proteins involved in oxidative stress resistance. Several surface-associated GAS molecules play a role in ROS suppression: M protein impairs azurophilic granule fusion with the phagolysosome in host neutrophils; HA capsular polysaccharide promotes bacterial clumping and enhances H~2~O~2~ resistance; recombinant Mac-1/IdeS binds CD16 on neutrophils inhibiting phagocytosis and ROS production; and Mac-2 binds neutrophil FCγ receptors to inhibit phagocytosis and ROS production. Enzymes involved in superoxide detoxification include NoxA/NOXase, SodA and AhpC. GpoA plays a role in cellular redox homeostasis and protects cells from the deleterious effects of ROS. Chaperone protein HtrA/DegP and DNA polymerase PolA1 function to repair protein or DNA damaged by ROS, respectively. Cation homeostasis, important for oxidative stress resistance, is regulated by several GAS proteins: PmtA, an iron efflux system important for H~2~O~2~ resistance; MtsABC, involved Fe^3+^, Zn^2+^ and Mn^2+^ transport and required for enhanced resistance to superoxide and H~2~O~2~; Shr, a surface-associated heme receptor involved in iron sequestration and H~2~O~2~ resistance; and Dpr/MrgA, plays a pivotal role in resistance to oxidant stress by functioning as an iron (Fe^2+^) chelator and preventing the production of bactericidal hydroxyl radicals via the Fenton reaction. Abbreviations: ROS, reactive oxygen species; HA, hyaluronan; Mac-1/IdeS, Mac-1-like protein; NoxA/NOXase, NADH oxidase A; SodA, superoxide dismutase; AhpC, alkyl hydroperoxidase reductase C; GpoA, glutothione peroxidase; HtrA/DegP, high-temperature requirement A; PolA1, DNA polymerase I; PmtA, PerR-regulated metal transporter A; MtsABC, metal transporter of *Streptococcus* ABC; Shr, streptococcal hemoprotein receptor; Dpr/MrgA, Dps-like peroxide resistance protein.](fuu009fig2){#fig2}

Superoxide dismutase A (SodA) {#sec7-1}
-----------------------------

Sod represents the first line of bacterial defense against superoxide stress by converting O~2~•^−^ into H~2~O~2~ and O~2~, thereby protecting cells from the toxic effects of superoxide (Fridovich [@bib70]; Liochev and Fridovich [@bib127]). Sods are distinguished by their metal cofactors, which are ferrous (Fe^3+^), manganese (Mn^2+^), nickel (Ni^2+^) or copper/zinc (Cu^2+^/Zn^2+^). *Escherichia coli* has three Sods cofactored with Cu^2+^/Zn^2+^ (periplasm), Mn^2+^ or Fe^3+^ (both cytoplasmic). GAS Sods are highly conserved among different serotypes as well as highly homologous to Sods from other Gram-positive bacteria (Gerlach, Reichardt and Vettermann [@bib76]; Poyart *et al.*, [@bib167]). GAS has a single Mn^2+^-dependent superoxide dismutase, designated SodA, which enhances GAS growth under aerobic conditions (Gibson and Caparon [@bib79]). The H~2~O~2~ generated by SodA is neutralized by endogenous GAS peroxidases (Gerlach, Reichardt and Vettermann [@bib76]). A *sodA* mutant in serotype M14 GAS strain HSC5 (Port, Paluscio and Caparon [@bib164]) is more susceptible to superoxide challenge (Gibson and Caparon [@bib79]). *E. coli* mutants deficient in the periplasmic Cu^2+^/Zn^2+^ Sod are more susceptible to H~2~O~2~, but exhibit equivalent sensitivity towards superoxide challenge (Gort, Ferber and Imlay [@bib84]). Additionally, heterologous expression of SodA from *Streptococcus thermophilus* strain AO45 in four otherwise SodA-negative lactobacilli promoted bacterial survival after challenge with various concentrations of H~2~O~2~ up to 1.6 mM. Treatment of *Lactobacillus gasseri* engineered to express SodA in the presence of an iron chelator, 2′-2′-dipyridyl (DIP) was protective following lethal challenge with 2.5 mM H~2~O~2~. This phenotype was attributed to inhibition of the Fenton reaction, which is downstream of H~2~O~2~ formation (Bruno-Barcena *et al.*, [@bib29]). Extracellular SodA derived from various GAS clinical isolates and reference strains failed to provoke an efficient immune response (McMillan *et al.*, [@bib139]); however, secretion of SodA from serotype M12 GAS strain 12714 into the extracellular milieu protects against the extracellular ROS produced by innate immune cells (Fig. [2](#fig2){ref-type="fig"}) (Gerlach, Reichardt and Vettermann [@bib76]). SodA might be indirectly regulated by the peroxide response regulator (PerR) in GAS as PerR does not bind to the SodA promoter directly, but *perR* mutants in serotype M1 GAS strain AP1 have lower transcript levels of SodA (Ricci, Janulczyk and Bjorck [@bib173]). In response to superoxide, both SodA and the cell surface adhesin fibronectin-binding protein F (PrtF, also known as Sfb) are transcriptionally up-regulated (Gibson *et al.*, [@bib78]; Gibson and Caparon [@bib79]). Furthermore, PrtF is transcriptionally regulated in response to oxygen concentration (VanHeyningen *et al.*, [@bib205]).

Alkyl hydroperoxide reductase (AhpC) {#sec7-2}
------------------------------------

Using a genome-wide screen for peroxidase-related genes in GAS, King and colleagues identified two genes, alkyl hydroperoxide reductase (*ahpC*) and glutathione peroxidase (*gpoA*), which inactivate inorganic and organic peroxides and thereby enhance GAS resistance to ROS (King, Horenstein and Caparon [@bib117]; Brenot, King and Caparon [@bib24]). Alkyl hydroperoxide reductase (AhpC) catalyzes the pyridine nucleotide-dependent reduction of organic hydroperoxides and H~2~O~2~ (Poole and Ellis [@bib162]). AhpC is present in GAS and closely related streptococci, including *S. mutans* and *S. agalactiae* (group B *Streptococcus*); however, AhpC is absent in *S. pneumoniae* (pneumococcus) and Gram-negative bacteria such as *E. coli* (Yesilkaya *et al.*, [@bib217]). GAS AhpC is an NADH-dependent H~2~O~2~-degrading peroxidase that is directly up-regulated in response to oxidative stress, but is not directly regulated by transcription factor PerR (Brenot, King and Caparon [@bib24]), a negative regulator of the inducible peroxide resistance response in GAS. Compared to wild-type serotype M14 GAS strain HSC5, an isogenic in-frame *ahpC* deletion mutant lacking the central 45 amino acids adjacent to the putative active site residue was more susceptible to a 5--10 mM range of paraquat (methyl viologen), a redox-cycling agent that increases intracellular levels of superoxide; however, the mutation had no effect on growth rate kinetics under aerobic conditions, SodA expression levels or bacterial survival following exposure to 1 mM paraquat (Hassan and Fridovich [@bib92]; King, Horenstein and Caparon [@bib117]). GAS *ahpC* mutants were more susceptible than wild-type to 5--20% cumene hydroperoxide as measured by zone of growth inhibition in disk diffusion assays (King, Horenstein and Caparon [@bib117]). Catalase treatment rescued the *ahpC* mutant phenotype, indicating that AhpC enhances GAS resistance to H~2~O~2~; however, following challenge with millimolar concentrations of H~2~O~2~, no significant decrease in H~2~O~2~ resistance was observed between the *ahpC* mutant and the parental strain (King, Horenstein and Caparon [@bib117]). Interestingly, an *ahp* mutant in a catalase-deficient *E. coli* mutant accumulated more H~2~O~2~ intracellularly indicating a primary role for Ahp in scavenging intracellular H~2~O~2~ (Seaver and Imlay [@bib181]). Recent observations indicate that bacteria are exposed to 5--10 μM H~2~O~2~ in neutrophil phagosomes (Mishra and Imlay [@bib141]), concentrations where peroxidases including AhpC are more kinetically efficient scavengers of H~2~O~2~ than catalase. However, the enzymatic activity of AhpC is limited by NADH availability and AhpC becomes saturated at H~2~O~2~ concentrations around 20 μM (Mishra and Imlay [@bib141]; Imlay [@bib103]). These findings indicate that AhpC from serotype M14 GAS strain HSC5 is not critical for survival in *in vitro* studies using super-physiological concentrations of 4 mM H~2~O~2~, where AhpC is saturated (King, Horenstein and Caparon [@bib117]; Brenot, King and Caparon [@bib24]), but is required for full virulence in a mouse model of soft-tissue infection *in vivo* (Brenot, King and Caparon [@bib24]).

Glutathione peroxidase (GpoA) {#sec7-3}
-----------------------------

Glutathione peroxidase (Gpo) is a selenoprotein oxidoreductase important for maintaining cellular redox homeostasis and for protecting cells from the deleterious effects of ROS (Arthur [@bib11]). Initial findings using an in-frame *gopA* mutant indicated that GpoA in serotype M14 GAS strain HSC5 is important for resistance against 5--10 mM paraquat (King, Horenstein and Caparon [@bib117]). Even though GpoA was not involved in resistance against direct challenge with H~2~O~2~, the *gpoA* mutant was rescued by addition of catalase, which detoxifies H~2~O~2~, indicating that GpoA also contributes to resistance against downstream products of superoxide (King, Horenstein and Caparon [@bib117]). Subsequent studies with an in-frame *gpoA* mutant in GAS strain HSC5 showed that GpoA contributes to GAS virulence in *in vivo* models of disease characterized by acute inflammation, a condition marked by high ROS levels (Mittal *et al.*, [@bib142]), including subcutaneous and systemic infection models (Brenot *et al.*, [@bib25]). However, GpoA was not essential for virulence in a zebrafish (*Danio rerio*) model of streptococcal myositis, a disease characterized by the absence of inflammatory cell infiltrate (Brenot *et al.*, [@bib25]). These findings suggest that GpoA is essential for GAS to adapt to oxidative stress and indicates that high, non-physiological concentrations of ROS tested in earlier *in vitro* studies may have neglected the important role played by GpoA *in vivo* (King, Horenstein and Caparon [@bib117]; Brenot *et al.*, [@bib25]).

NADH oxidase A (NoxA) {#sec7-4}
---------------------

The NADH oxidase (NoxA or NOXase) expressed by GAS is an important enzyme involved in H~2~O~2~ decomposition and the regeneration of NAD^+^, which plays a pivotal role in several pathways including DNA repair, post-translational protein modifications and apoptosis (Massudi *et al.*, [@bib137]). Single crossover *noxA* mutants in serotype M6 GAS strain JRS4 and serotype M14 GAS strain HSC5 showed reduced growth under high O~2~ conditions and after challenge with paraquat (Gibson *et al.*, [@bib80]). In addition, NoxA-deficient mutants accumulated almost three times more H~2~O~2~ in culture supernatant compared to parental strains. All *noxA* mutant phenotypes were rescued by the addition of catalase to the growth medium (Gibson *et al.*, [@bib80]). Nox enzymes are generally described as a peroxide-resistance mechanism employed by catalase-negative bacteria including GAS. Accordingly, heterologous expression of Nox from *Enterococcus faecalis* in the *noxA* GAS mutants reduced H~2~O~2~ accumulation in culture supernatant (Gibson *et al.*, [@bib80]). A study using purified recombinant Nox revealed that NoxA from GAS is the most potent enzyme among all tested NADH oxidases from multiple bacteria (Gibson *et al.*, [@bib80]; Gao *et al.*, [@bib74]). This finding indicates that GAS has optimized catalase-independent mechanisms to detoxify H~2~O~2~ and that NoxA is an important pathway for GAS to tolerate high O~2~ environments and promote resistance to ROS (Gibson *et al.*, [@bib80]; Gao *et al.*, [@bib74]).

ENZYMATIC REPAIR MECHANISMS {#sec8}
===========================

A sub-lethal dose of ROS damages proteins, DNA and has multiple adverse effects on the bacterium. Therefore, an immediate repair of the damage is essential for the survival of the bacterium. GAS is equipped with efficient repair mechanisms (Table [1](#tbl1){ref-type="table"}). However, this rather indirect contribution to resistance to oxidative stress is proportionally difficult to determine. In the following section, we focus on several key proteins contributing to the repair of ROS-induced cell damage (Fig. [2](#fig2){ref-type="fig"}).

High-temperature requirement A (HtrA) {#sec8-1}
-------------------------------------

High-temperature requirement A (HtrA, also designated DegP) is a dual-functional serine protease and chaperone protein that either refolds or degrades damaged proteins destined for secretion into the extracellular environment (Spiess, Beil and Ehrmann [@bib189]). Homologs of this protein are present in many Gram-negative bacteria including *E. coli* (Bringer *et al.*, [@bib27]) and Gram-positive bacteria such as *S. aureus* (Rigoulay *et al.*, [@bib174]). HtrA in serotype M14 GAS strain HSC5 is involved in secretion of virulence factors via the ExPortal, a discrete cytoplasmic membrane microdomain involved in the biogenesis of secreted GAS proteins (Rosch and Caparon [@bib175]). HtrA indirectly affects the maturation kinetics of the secreted cysteine protease streptococcal pyrogenic exotoxin B (SpeB), with the absence of a functional HtrA causing a delay in the biogenesis of active SpeB protease (Cole *et al.*, [@bib45]). Studies with an insertional *htrA* mutant in serotype M6 GAS strain S43 (ATCC 12348) showed a growth defect at 37 and 44°C, an inhibition zone around disks with 200 mM paraquat, and HtrA was necessary for full virulence in a mouse model of infection (Jones *et al.*, [@bib111]). A more recent study using an insertional *htrA* mutant in GAS confirmed the earlier report showing a growth defect at 37°C (Jones *et al.*, [@bib111]; Lyon and Caparon [@bib132]); however, an in-frame *htrA* mutant in serotype M14 GAS strain HSC5 had no growth defect at 37°C (Lyon and Caparon [@bib132]). Therefore, it remains unclear if the defect in oxidative stress resistance observed with *htrA* mutant in GAS strain S43 might be due to secondary effects rather than the direct activity of HtrA.

DNA polymerase I (PolA1) {#sec8-2}
------------------------

Recently, a DNA polymerase I (PolA1) was identified in GAS, expressed from the same five-gene operon as the peroxide stress response regulator PerR (Toukoki and Gryllos [@bib199]). An in-frame *polA1* mutant in a serotype M3 GAS strain 003Sm was hypersensitive to challenge with 10 mM H~2~O~2~ compared to the wild-type and complemented strains. In addition, the mutant was rescued after challenge with H~2~O~2~ in the presence of an iron chelator deferoxamine mesylate (desferal, DFM) or the hydroxyl radical scavenger thiourea suggesting that the hypersensitivity of the mutant occurred via the Fenton reaction (Toukoki and Gryllos [@bib199]). Adaptation with sub-lethal H~2~O~2~ prior to H~2~O~2~ lethal challenge decreased the killing of the double *perR polA1* mutant 9-fold indicating that PolA1 is involved in a PerR-dependent oxidative stress defense (Toukoki and Gryllos [@bib199]). Furthermore, the *polA1* mutant exhibited a reduction in the repair of DNA damage initiated by UV light or ciprofloxacin (Toukoki and Gryllos [@bib199]), a fluoroquinolone antibiotic that induces double-strand DNA breaks via entrapment of topoisomerases during DNA cleavage (Chen *et al.*, [@bib40]). PolA1 also contains a 5′--3′ exonuclease domain and increased mutation rates associated with PolA1 function to promote genetic diversity (Toukoki and Gryllos [@bib199]).

ROLE OF CATIONS IN GAS RESISTANCE TO OXIDATIVE STRESS {#sec9}
=====================================================

The acquisition of metal ions such as iron (Fe), manganese (Mn), cobalt (Co) and zinc (Zn) is important for the survival of bacterial pathogens inside the human host. Metal ions enhance oxidative stress resistance by directly detoxifying ROS, serving as cofactors for enzymes such as SodA, and acting as signaling molecules for the transcriptional regulation of genes involved in ROS defense (Yesilkaya *et al.*, [@bib217]). However, high intracellular concentrations of metal ions may be bactericidal or induce oxidative stress (Finney and O\'Halloran [@bib64]); therefore, GAS has evolved effective systems to tightly regulate metal ion homeostasis (Nelson [@bib150]). A recent study in serotype M1T1 GAS strain 5448, a representative of the globally disseminated serotype M1T1 clone (Cole *et al.*, [@bib46]), demonstrated that mutants in a putative Zn^2+^ efflux/activator system, *czcD* (Spy_0653) and *gzcA* (Spy_0654) resulted in increased susceptibility to Zn^2+^ and clearance by innate immune cells *in vitro* and in a mouse model of soft-tissue infection (Ong *et al.*, [@bib155]). These findings indicate that Zn^2+^ contributes to clearance of bacterial pathogens by acting as an antimicrobial factor, and that Zn^2+^ efflux systems are important for full GAS virulence.

Neutrophils are the first line of defense against invading pathogens. Calprotectin from neutrophils binds and reduces the availability of Mn^2+^ and Zn^2+^ at the site of infection, which decreases the activity of the Mn^2+^-dependent Sod in *S. aureus* (Kehl-Fie *et al.*, [@bib113]). Inhibition of Sod results in a higher susceptibility of *S. aureus* by promoting clearance of innate immune cells *in vitro* and *in vivo* (Kehl-Fie *et al.*, [@bib113]). MtsABC from GAS functions as a Mn^2+^ transporter and GAS deficient in MtsABC are more susceptible to superoxide probably due to the decreased activity of SodA (Janulczyk, Ricci and Bjorck [@bib110]), as described in more detail below. In addition, GAS requires low levels of Fe^3+^ for growth and survival in the human host; however, GAS preferably utilizes Mn^2+^ as cofactor for enzymes possibly to minimize iron-induced radical formation via the Fenton reaction as described for *S. pneumoniae* (Ong *et al.*, [@bib156]). GAS has multiple metal transporters and receptors. Herein, we focus on the contribution of metal transport systems to ROS resistance (Fig. [2](#fig2){ref-type="fig"}).

PerR-regulated metal transporter A (PmtA) {#sec9-1}
-----------------------------------------

In a genome-wide transcriptome analysis between serotype M14 GAS strain HSC5 and an in-frame *perR* mutant in the absence of a stress stimulus, six genes were identified with at least 3-fold differential expression (Brenot, Weston and Caparon [@bib26]). Among those genes, five were up-regulated and one was down-regulated. One of the up-regulated genes in the *perR* mutant is the PerR-regulated metal transporter A (PmtA), which has a PerR-binding site and is a putative iron efflux protein in GAS. Overexpression of PmtA in the *perR* mutant is responsible for a higher resistance of up to 1.75 mM zinc, concentrations to which the wild-type and *pmtA* mutant were susceptible. In addition, an in-frame *pmtA* mutant was more sensitive to challenge with 4 mM H~2~O~2~ compared to the wild-type. However, in an *in vivo* mouse model of soft-tissue infection, *pmtA* was not found to be up-regulated compared to mid-log growth phase (Brenot, Weston and Caparon [@bib26]). Therefore, the exact role of PmtA *in vivo* remains unclear. A similar global transcriptional analysis in serotype M3 GAS strain 003Sm confirmed that *pmtA* is highly regulated by PerR. Interestingly, PerR-regulated genes were essential for GAS pharyngeal colonization and ROS-dependent phagocyte resistance indicating an important role for PmtA in physiology and potentially virulence (Gryllos *et al.*, [@bib87]).

Dps-like peroxide resistance protein (Dpr) {#sec9-2}
------------------------------------------

In the presence of iron, H~2~O~2~ is readily converted to the highly reactive OH• through the Fenton reaction. The Dps-like peroxide resistance protein (Dpr, also designated MrgA) plays a pivotal role in resistance to oxidant stress by functioning as a chelator of intracellular iron (Fe^2+^) to prevent the production of bactericidal hydroxyl radicals via the Fenton reaction (Andrews, Robinson and Rodriguez-Quinones [@bib7]; Tsou *et al.*, [@bib201]; Haikarainen *et al.*, [@bib88]; Ge and Sun [@bib75]). Dpr has also been reported to protect GAS from pH-induced stress (Tsou *et al.*, [@bib201]), and is transcriptionally repressed by PerR (Brenot, King and Caparon [@bib24]; Tsou *et al.*, [@bib202]), as discussed below. A study using an in-frame allelic exchange *dpr* mutant in serotype M1 GAS strain A-20 found that the mutant was hypersensitive to killing by 5 mM H~2~O~2~, compared to wild-type and complemented strains (Tsou *et al.*, [@bib201]). Addition of the iron chelator DFM leads to a dose-dependent increase in survival of the mutant strain following *in vitro* challenge with 5 mM H~2~O~2~ (Tsou *et al.*, [@bib201]). Similarly, an in-frame *dpr* mutant in serotype M14 GAS strain HSC5 did not exhibit a defect in growth under aerobic conditions, nor the ability to degrade peroxide, and was hypersensitive to high concentrations of H~2~O~2~, compared to wild-type (Brenot, King and Caparon [@bib24]). These data indicate that Dpr functions as an iron chelator by reducing free iron and thereby increasing the fitness of GAS inside the human host. In *Bacillus subtilis* and *S. aureus* (Chen and Helmann [@bib41]), MrgA plays a central role in protection from a lethal challenge of peroxide. In *S. mutans*, Dpr is important for growth under aerobic conditions (Yamamoto *et al.*, [@bib215]). The transcriptional regulator PerR directly regulates the expression of Dpr in GAS (Brenot, King and Caparon [@bib24]).

Metal transporter of *Streptococcus* ABC (MtsABC/SiaABC) {#sec9-3}
--------------------------------------------------------

In order to establish an infection and survive within the host, many Gram-positive bacterial pathogens utilize ATP-binding cassette (ABC) transporters to acquire essential nutrients and metal ions such as copper, manganese, iron, zinc and cobalt (Claverys [@bib43]; Higgins [@bib98]). Metal homeostasis plays an important role in GAS resistance to oxidative stress and virulence (Ge and Sun [@bib75]), and three metal ABC transporters have been described so far: 1) MtsABC is involved in the uptake and transport of a variety of cations in GAS (Fe^3+^, Zn^2+^ and Mn^2+^) (Janulczyk, Pallon and Bjorck [@bib109]; Janulczyk, Ricci and Bjorck [@bib110]); 2) SiaABC/HtsABC transports heme (Payne [@bib157]); and 3) FtsABC is the principal transporter of Fe^3+^ and heme (Hanks *et al.*, [@bib89]; Ge and Sun [@bib75]). Compared to the wild-type strain, a *perR* mutant exhibited a reduction in *mtsA* transcript levels suggesting that PerR up-regulates *mtsABC* transcription (Ricci, Janulczyk and Bjorck [@bib173]). MtsABC-deficient GAS exhibit reduced growth rates in metal-depleted medium and under aerobic conditions (Janulczyk, Ricci and Bjorck [@bib110]). The *mtsABC* mutant in serotype M1 GAS strain AP1 was hypersensitive to killing by 5 mM H~2~O~2~ and paraquat-induced superoxide radicals following growth in the presence of 2 or 10 mM paraquat, in comparison to wild-type (Janulczyk, Ricci and Bjorck [@bib110]). In GAS, Mn-dependent SodA plays an important role in bacterial resistance to oxidative stress (Gibson and Caparon [@bib79]; Fridovich [@bib70]; Ferretti *et al.*, [@bib62]). The reduced SodA enzymatic activity of the *mtsABC* mutant, most likely due to low intracellular levels of manganese resulting from defective manganese transport, may account for this defect in ROS resistance. Supplementation of the culture medium with 30 μM MnCl~2~ restored SodA enzymatic activity and growth rate to wild-type levels in the presence of paraquat. The *mtsABC* mutant strain was ∼30-fold less virulent compared to wild-type in an *in vivo* mouse air sac model of subcutaneous infection. These data demonstrate that metal cation acquisition by MtsABC plays a key role in GAS growth, oxidative stress resistance and virulence (Janulczyk, Ricci and Bjorck [@bib110]). Additionally, MtsABC shares homology with the pneumococcal surface antigen A, the lipoprotein part of an ABC Mn^2+^ transporter, which also functions as an adhesin and is necessary for full virulence in a mouse model of soft-tissue infection (Berry and Paton [@bib19]).

Streptococcal hemoprotein receptor (Shr) {#sec9-4}
----------------------------------------

Iron availability is essential for bacterial growth, survival and the establishment of infection within the human host (Payne [@bib157]). Streptococcal hemoprotein receptor (Shr) is the first protein of the streptococcal iron acquisition (Sia) operon and is necessary for maintenance of iron homeostasis (Bates *et al.*, [@bib16]). Shr is a surface-associated and secreted receptor for heme, which is the main source of iron in human blood (Bates *et al.*, [@bib16]; Fisher *et al.*, [@bib65]). Insertional inactivation of the *shr* gene in serotype M1 GAS strain SF370 enhanced bacterial survival after challenge with 5 mM H~2~O~2~, compared to wild-type. However, the addition of hemoglobin increased the survival of wild-type to the same level of the *shr* mutant, suggesting that Shr is involved in hemoglobin-dependent resistance to H~2~O~2~ (Bates *et al.*, [@bib16]). A recent study demonstrated that Shr is important for survival of serotype M1T1 GAS strain 5448 in human whole blood and full virulence in two *in vivo* mouse models of infection, underscoring the important role for metal cations in GAS pathogenesis (Dahesh, Nizet and Cole [@bib48]). These studies reveal that during bloodstream infections, hemoglobin sequestration by Shr is an important defense mechanism of GAS to survive inside the human host, presumably by capturing free Fe^2+^ and thereby preventing the production of highly bactericidal hydroxyl radicals.

GAS REGULATION OF THE OXIDATIVE STRESS RESPONSE {#sec10}
===============================================

Stress response plays an important role in the regulation of virulence and gene expression in bacterial pathogens; however, the σ^B^ general stress response pathway involved in the regulation of stress-induced genes in many Gram-positive species (Volker *et al.*, [@bib206]) is absent in GAS. A coordinated response to oxidative stress is necessary for GAS to establish an infection and cause disease. Several two-component systems or stand-alone transcriptional regulators respond to extracellular stimuli and have been linked to the GAS oxidative stress response, including PerR, Rgg/RopB, Ihk-Irr, MtsR and CiaRH.

PerR regulon {#sec10-1}
------------

Regulation of the inducible peroxide resistance response in GAS is primarily coordinated by the peroxide-sensing PerR transcriptional regulator, a 155 amino acid zinc-containing metalloprotein, which is a member of the ferric uptake regulator (Fur) family of metal-binding transcriptional regulators (Herbig and Helmann [@bib96]; Mongkolsuk and Helmann [@bib143]; Brenot, King and Caparon [@bib24]; Moore and Helmann [@bib144]). PerR is a negative transcriptional regulator, or repressor, of the inducible peroxide resistance response in GAS (King, Horenstein and Caparon [@bib117]; Ricci, Janulczyk and Bjorck [@bib173]), and contributes to GAS iron homeostasis, oxidative stress response and virulence (Ricci, Janulczyk and Bjorck [@bib173]; Brenot, King and Caparon [@bib24]; Gryllos *et al.*, [@bib87]) (Table [2](#tbl2){ref-type="table"}). PerR generally acts as a repressor by directly binding the promoter of the target gene (Mongkolsuk and Helmann [@bib143]; Hayashi *et al.*, [@bib93]). Peroxide sensing by PerR requires regulatory metal ions, and under oxidative stress conditions PerR conformational changes induced by the oxidation of metal ions reduce the DNA-binding affinity of PerR, de-repressing target gene expression (Dubbs and Mongkolsuk [@bib56]) (Fig. [3](#fig3){ref-type="fig"}). The PerR of *B. subtilis* has been well characterized and shown to repress the expression of target genes by binding to conserved promoter sequences known as Per boxes (Chen, Keramati and Helmann [@bib42]). However, with the exception of *pmtA* and *ahpCF*, the Per box sequence is not fully conserved in promoters regulated by PerR in GAS (Brenot, King and Caparon [@bib24]; Brenot, Weston and Caparon [@bib26]). In Gram-positive species such as *B. subtilis* and *S. aureus*, PerR coordinately controls the oxidative stress response genes and iron homeostasis (Chen, Keramati and Helmann [@bib42]; Horsburgh *et al.*, [@bib100]; Helmann *et al.*, [@bib94]) in order to prevent the Fenton reaction, whereby intracellular iron reacts with H~2~O~2~ to form highly toxic and oxidizing hydroxyl radicals (OH•).

![Transcriptional regulator PerR regulates the inducible peroxide resistance response. **(A)** Under normal conditions, PerR has a bound zinc ion (Zn^2+^) and generally represses transcription of PerR-regulated genes by directly binding conserved sequences, known as Per boxes, in the promoter of each target gene. **(B)** Under conditions of peroxide stress (H~2~O~2~), a ferrous ion (Fe^2+^) is bound, resulting in the oxidation of histidine residues and conformational changes within PerR. **(C)** Conformational changes reduce the DNA-binding affinity of PerR, resulting in the dissociation of PerR from the PerR boxes, and transcription of the target gene. **(D)** GAS PerR regulates genes involved in virulence, sugar metabolism/transport, metal ion efflux and housekeeping metabolic pathways.](fuu009fig3){#fig3}

###### 

PerR-regulated genes identified by microarray analyses and confirmed by quantitative real-time PCR (qRT-PCR) for different GAS serotypes.

  M type                                       ORF no.^a^   Growth phase       Gene       Protein^b^                                                   Relative expression^c^   Reference^d^   
  -------------------------------------------- ------------ ------------------ ---------- ------------------------------------------------------------ ------------------------ -------------- ---------------------------------------
  *Oxidative stress -- virulence (putative)*                                                                                                                                                   
  M3                                           0466         Mid-exponential    *adcA*     Putative adhesin (zinc-binding)                              −4.6                     −3.2           Gryllos *et al.* ([@bib87])
  M3                                           0815         Mid-exponential    *hylIII*   Putative hemolysin III                                       +3.0                     −1.2           Gryllos *et al.* ([@bib87])
  M3                                           1093         Mid-exponential    *pmtA*     Putative metal transport ATPase                              −11.3                    −15.8          Gryllos *et al.* ([@bib87])
  M14                                          05800        Mid-exponential    *pmtA*     Putative metal transport ATPase                              n/a                      −30.0          Brenot, Weston and Caparon ([@bib26])
  M3                                           1770         Late exponential   *ahpC*     Putative alkyl hydroperoxidase                               −1.7                     −1.9           Gryllos *et al.* ([@bib87])
  M14                                          08790        Mid-exponential    *ahpC*     Putative alkyl hydroperoxidase                               n/a                      −1.3           Brenot, King and Caparon ([@bib24])
  M3                                           1095         Late exponential   *mf4*      Putative mitogenic factor/DNase                              −2.2                     −2.1           Gryllos *et al.* ([@bib87])
  M1                                           1436         Mid-exponential    *mf3*      Secreted DNase virulence factor                              n/a                      +4.3           Wen *et al.* ([@bib213])
  M3                                           0298         Late exponential   *prtS*     Interleukin 8 protease                                       −2.1                     −2.0           Grifantini *et al.* ([@bib85])
  M3                                           0482         Late exponential   *sagC*     SLS-associated ORF                                           −2.1                     −1.6           Grifantini *et al.* ([@bib85])
  M14                                          06285        Mid-exponential    *mrgA*     Peroxide resistance protein                                  n/a                      −2.8           Brenot, King and Caparon ([@bib24])
  M14                                          08560        Mid-exponential    *lsp*      Laminin-binding protein                                      n/a                      −100           Brenot, Weston and Caparon ([@bib26])
  M1 T1                                        1415         Mid-exponential    *sda1*     DNase and virulence factor                                   n/a                      +2.5           Wang *et al.* ([@bib212])
  *Sugar metabolism/transport*                                                                                                                                                                 
  M3                                           1489         Late exponential   *lacR.1*   Putative lactose PTS repressor                               +1.9                     +2.5           Gryllos *et al.* ([@bib87])
  M3                                           1484         Mid-exponential    *lacB.1*   Galactose 6-phosphate isomerase                              −2.3                     +1.1           Gryllos *et al.* ([@bib87])
  M3                                           1658         Late exponential   *lacB.2*   Galactose 6-phosphate isomerase                              +5.0                     +6.6           Gryllos *et al.* ([@bib87])
  M3                                           1658         Late exponential   *lacB.2*   Galactose-6-phosphate isomerase                              −4.9                     −2.0           Grifantini *et al.* ([@bib85])
  M3                                           1654         Late exponential   *lacE*     Putative PTS enzyme IIBC                                     +4.0                     +10.7          Gryllos *et al.* ([@bib87])
  M3                                           1659         Late exponential   *lacA2*    Galactose-6-phosphate isomerase                              −2.4                     −2.5           Grifantini *et al.* ([@bib85])
  M3                                           1660         Late exponential   *lacR.2*   Putative lactose PTS repressor                               +1.9                     +2.2           Gryllos *et al.* ([@bib87])
  M3                                           1487         Late exponential   *pts*      Putative PTS enzyme IIB                                      +2.9                     +5.9           Gryllos *et al.* ([@bib87])
  *Metal -- ABC transport*                                                                                                                                                                     
  M3                                           0069         Mid-exponential    *adcR*     Putative transcriptional repressor                           −1.9                     −1.3           Gryllos *et al.* ([@bib87])
  M3                                           0069         Mid-exponential    *adcR*     Putative transcriptional repressor                           +1.6                     +2.3           Grifantini *et al.* ([@bib85])
  M3                                           0071         Mid-exponential    *adcB*     Zinc/manganese ABC transporter                               +2.0                     +1.7           Grifantini *et al.* ([@bib85])
  M3                                           0319         Mid-exponential    *mtsB*     Iron ABC transporter                                         −1.9                     −1.0           Grifantini *et al.* ([@bib85])
  M3                                           1557         Late exponential   *siaD*     Putative ABC transporter                                     −1.9                     −1.7           Grifantini *et al.* ([@bib85])
  M3                                           1560         Late exponential   *shp*      Heme/ferrichrome-binding protein                             −2.0                     −1.8           Grifantini *et al.* ([@bib85])
  M1                                           0453         Mid-exponential    *mtsA*     Iron ABC transporter                                         n/a                      −2.0           Hanks *et al.* ([@bib89a])
  *General biosynthesis/metabolism*                                                                                                                                                            
  M3                                           0027         Mid-exponential    *purE*     Phosphoribosylaminoimidazole carboxylase catalytic subunit   +2.2                     +1.2           Gryllos *et al.* ([@bib87])
  M3                                           1615         Mid-exponential    *rpsN*.2   30S subunit ribosomal protein S14                            −3.3                     −2.5           Gryllos *et al.* ([@bib87])
  M14                                          07960        Mid-exponential    *rpsN*.2   30S subunit ribosomal protein S14                            n/a                      −4.0           Brenot, Weston and Caparon ([@bib26])
  M3                                           0302         Late exponential   *nrdI.2*   Putative ribonucleotide reductase                            −2.7                     −1.5           Gryllos *et al.* ([@bib87])
  M3                                           0013         Late exponential   n/a        Putative amino acid permease                                 −2.5                     −1.5           Grifantini *et al.* ([@bib85])
  M3                                           0217         Late exponential   *oppC*     Oligopeptide permease                                        −2.0                     −1.9           Grifantini *et al.* ([@bib85])
  M3                                           1363         Late exponential   *cysM*     Putative *O*-acetylserine lyase                              +2.1                     1.0            Grifantini *et al.* ([@bib85])
  M3                                           1794         Late exponential   *nrdD*     Ribonucleoside triphosphate reductase                        −2.9                     −2.5           Grifantini *et al.* ([@bib85])
  *Hypothetical*                                                                                                                                                                               
  M3                                           1208         Mid-exponential    n/a        Conserved hypothetical protein                               +2.0                     +2.0           Gryllos *et al.* ([@bib87])
  M3                                           1724         Late exponential   *phtD*     Hypothetical protein                                         −2.3                     −5.6           Gryllos *et al.* ([@bib87])
  M14                                          08555        Mid-exponential    *phtD*     Hypothetical protein                                         n/a                      −100           Brenot, Weston and Caparon ([@bib26])
  M14                                          05535        Mid-exponential    *phtY*     Hypothetical protein                                         n/a                      −30            Brenot, Weston and Caparon ([@bib26])
  *Miscellaneous*                                                                                                                                                                              
  M3                                           0840         Mid-exponential    n/a        Putative transcriptional repressor                           +3.2                     +4.6           Grifantini *et al.* ([@bib85])
  M14                                          08775        Mid-exponential    *csp*      Putative cold shock protein                                  n/a                      +2.5           Brenot, King and Caparon ([@bib24])

^a^Open reading frame (ORF) number of published GAS genome sequence: M1 strain SF370 (Ferretti *et al.*, [@bib62]), M1T1 strain MGAS5005 (Sumby *et al.*, [@bib193]), M3 strain MGAS315 (Beres *et al.*, [@bib18a]) and M14 strain HSC5 (Port, Paluscio and Caparon [@bib164]).

^b^NCBI annotation.

^c^Fold-change of expression in wild-type GAS compared to expression in the isogenic *perR* mutant.

^d^Gryllos *et al.* ([@bib87]): Transcriptome comparisons performed between wild-type M3 GAS strain 003Sm and isogenic *perR* mutant strain 003Sm*perR*Δ grown to mid-exponential phase (OD~600nm~ = 0.25) or late-exponential phase (OD~600nm~ = 0.6) phase.

Grifantini *et al.* ([@bib85]): Wild-type M3 GAS strain 003Sm and *perR* mutant 003Sm*perR*Δ were grown to mid-exponential phase (OD~600nm~ = 0.25) or late-exponential phase (OD~600nm~ = 0.6) at which point they were challenged with H~2~O~2~ (final concentration 0.5 mM) for 15 min at 37°C or with water as a control.

Brenot, King and Caparon ([@bib24]): Total RNA extracted from mid-exponential phase (OD~600nm~ = 0.3) cultures of wild-type serotype M14 GAS strain HSC5 and the isogenic *perR* mutant.

Brenot, Weston and Caparon ([@bib26]): RNA from HSC5 and the isogenic PerR-deficient mutant (HΔPer) was isolated from mid-exponential phase cultures (OD~600nm~ = 0.3).

Wen *et al.* ([@bib213]): Transcriptome comparisons were performed between wild-type M1 strain AP-20 and isogenic *perR* mutant strain (SW-612) cultured to mid-log phase (OD~600nm~ ∼ 0.5).

Wang *et al.* ([@bib212]): Wild-type serotype M1 GAS strain A-20 and isogenic *perR* mutant (SW-612) were grown for 3 h and treated with 0.5 mM of H~2~O~2~ for another 2 h prior to RNA extraction and real-time PCR analysis.

Hanks *et al.* ([@bib89a]): Serotype M1 GAS strain MGAS5005 and the isogenic *perR* mutant were harvested at an OD~600nm~ of 0.2, 0.4 or 0.7 and immediately processed to isolate total RNA.

n/a; not available.

Structure of PerR {#sec10-2}
-----------------

The unique 11 amino acid residue N-terminal HXH metal-binding motif of PerR is highly conserved among GAS isolates and plays a role in oxidative stress sensing, metal ion binding and GAS virulence (Makthal *et al.*, [@bib135]). Fluorescence polarization assays indicate that metal ion binding enhances the DNA binding affinity of PerR, but has no influence on the sequence-specific DNA binding. Gel mobility shift assays demonstrate that PerR directly senses peroxide stress conditions through iron-dependent metal-catalyzed oxidation, relieving repression of target genes by dissociating from the binding site in the promoter region (Makthal *et al.*, [@bib135]). The PerR crystal is a homodimer containing two metal-binding sites within the dimerization domain (site 1) and interdomain region (site 2) (Makthal *et al.*, [@bib135]). The zinc-binding at site 1 contributes to structural integrity and PerR dimerization, while the metal-binding at site 2 is essential for peroxide sensing, gene regulation and full virulence in a mouse model of systemic GAS infection (Makthal *et al.*, [@bib135]). Under normal conditions in *B. subtilis*, PerR has a bound zinc ion and represses transcription of PerR-regulated genes by directly binding to the PerR boxes in the promoter region. In the presence of oxidative conditions, a ferrous ion (Fe^2+^) is bound, resulting in the oxidation of three histidines, the release of PerR from the PerR boxes and de-repression of the PerR regulon (Herbig and Helmann [@bib96]; Lee and Helmann [@bib123]) (Fig. [3](#fig3){ref-type="fig"}).

Transcriptomic and proteomic studies of PerR-deficient GAS {#sec10-3}
----------------------------------------------------------

The PerR regulon varies considerably among GAS serotypes. For example, the PerR regulon in serotype M5 GAS consists of 6 genes (Brenot, King and Caparon [@bib24]; Brenot, Weston and Caparon [@bib26]), whereas the M3 GAS PerR regulon contains 42 genes (Gryllos *et al.*, [@bib87]). Importantly, not all studies identify the same PerR-regulated genes, perhaps reflecting differences in growth phase (mid-exponential vs. late-exponential) or serotype-dependent variation among GAS isolates (Table [2](#tbl2){ref-type="table"}).

### Serotype M1 GAS {#sec10-3-1}

Precise allelic replacement mutagenesis of *perR* in serotype M1 GAS strain AP1 was performed by replacing the DNA- and metal-binding domains of *perR* (residues 202 to 361) with a kanamycin resistance gene, and complemented with a plasmid containing a functional *perR* gene and promoter region (Ricci, Janulczyk and Bjorck [@bib173]). Northern blot analysis of the AP1 *perR* mutant cultured to exponential growth phase revealed a 48% reduction in iron (^55^Fe) incorporation from the culture medium, and a 2.4-fold reduction in the transcription of metal-binding ABC transporter *mtsA* (Janulczyk, Pallon and Bjorck [@bib109]), compared to wild-type (Ricci, Janulczyk and Bjorck [@bib173]), suggesting that PerR positively activates *mtsABC* transcription (Ricci, Janulczyk and Bjorck [@bib173]). The *in vitro* growth rate of the AP1 *perR* mutant was similar to wild-type following growth in Todd-Hewitt medium supplemented with 0.2% yeast extract (THY), metal-depleted THY and iron-repleted THY (100 μM ferric citrate) under aerobic conditions. The *perR*-deficient mutant was hyperresistant to H~2~O~2~ stress as assessed by growth of mid-log phase cultures exposed to 5 mM peroxide stress for 30 min. The *perR* mutant was more sensitive to the superoxide anion, as indicated by poor growth in the presence of 10 mM paraquat. Compared to wild-type, the PerR-deficient strain had an ∼3-fold transcriptional reduction of *sodA* suggesting that PerR plays a role in the regulation of SodA expression and resistance to superoxide stress in GAS. High intracellular concentrations of H~2~O~2~ may de-repress ROS-responsive regulation, resulting in the partial repression of SodA to minimize H~2~O~2~ production, and the down-regulation of *mtsABC* to reduce intracellular Fe^2+^ and the production of bactericidal hydroxyl radicals by the Fenton reaction. Compared to the parental A-20 strain, the *perR* mutant was attenuated for virulence in a BALB/c mouse skin air sac infection model of GAS infection, consistent with previous reports (Ricci, Janulczyk and Bjorck [@bib173]; Brenot, King and Caparon [@bib24]; Gryllos *et al.*, [@bib87]). It is unclear whether the attenuation was a consequence of *perR* inactivation, the down-regulation of *sodA* expression or the reduced ability of the *perR* mutant to grow under iron-restricted conditions *in vivo*.

Secreted proteins from wild-type serotype M1 GAS strain A-20 and an isogenic *perR* mutant (SW-612) cultured to stationary phase in conditional medium with a protease inhibitor cocktail were compared using two-dimensional (2D) gel electrophoresis to ascertain how PerR regulates the expression of the GAS secretome (Wen *et al.*, [@bib213]). Twenty-five proteins were down-regulated and 13 proteins were up-regulated in the *perR*-deficient mutant, compared to wild-type (Wen *et al.*, [@bib213]). Approximately 50% of the PerR-regulated proteins identified were predicted to play a role in sugar metabolism and stress response. Activation of sugar metabolic pathways may be necessary for GAS to mobilize the additional energy needed to survive and proliferate under oxidative stress conditions (Wen *et al.*, [@bib213]).

### Serotype M3 GAS {#sec10-3-2}

Mutation of *perR* in M3 GAS strain 003Sm enhanced sensitivity to phagocytic clearance by whole human blood and mouse macrophages (Gryllos *et al.*, [@bib87]). Inhibition of phagocyte oxidative burst with diphenyleneiodonium chloride rescued the *perR* mutant phenotype, suggesting that the PerR regulon enhances GAS resistance to phagocyte oxidative burst. Competitive co-infection experiments with wild-type and *perR*-deficient bacteria demonstrated that the *perR* mutant was more rapidly cleared and attenuated for virulence in a baboon model of GAS pharyngitis. Comparative analysis of global gene expression in wild-type M3 GAS and the isogenic *perR* mutant cultured to late-exponential phase identified 42 genes regulated by the PerR regulon (Gryllos *et al.*, [@bib87]). Approximately 50% of the PerR-dependent genes were predicted to encode proteins involved in oxidative stress resistance, virulence, sugar transport and metabolism, which may reflect the increased energy required for GAS to survive the harsh oxidative conditions generated by the host innate immune response (Gryllos *et al.*, [@bib87]). These data indicate that PerR regulates the expression of a diverse set of genes, enhances GAS resistance to phagocytic clearance and contributes to pharyngeal colonization in a non-human primate model of GAS pharyngeal colonization (Gryllos *et al.*, [@bib87]).

Grifantini and coworkers (Grifantini *et al.*, [@bib85]) conducted a transcriptomic analysis of a *perR* mutant in serotype M3 GAS strain 003Sm cultured in the presence of H~2~O~2~. Analysis of wild-type and *perR* mutant transcriptomes revealed that 76 of 237 peroxide-regulated genes were PerR dependent. The PerR-regulated genes, which encode purine and deoxyribonucleotide biosynthesis enzymes, peptide transport and heme uptake, were mostly down-regulated. 53% of the 161 PerR-independent genes were repressed, and encoded for proteins with similar functions to PerR-regulated genes (Grifantini *et al.*, [@bib85]). The 75 up-regulated genes encoded for proteins involved in the detoxification of ROS, repair of damaged DNA, cofactor metabolism and pilus biosynthesis. The strong activation of metabolic enzymes and DNA damage repair mechanisms may play a key role in GAS survival in oxidative environments *in vivo*, similar to *S. aureus* (Chang *et al.*, [@bib34]; Grifantini *et al.*, [@bib85]; Le Breton *et al.*, [@bib122]; Toukoki and Gryllos [@bib199]). PerR-dependent regulation was restored by complementation of the *perR* mutant with the wild-type PerR protein, but not with a modified PerR containing a mutation in one of the two metal-binding sites (Grifantini *et al.*, [@bib85]). Metal content analysis revealed that PerR binds zinc and iron, and that iron oxidation plays a key role in the PerR response (Grifantini *et al.*, [@bib85]). The binding of PerR to the promoter following treatment with EDTA suggests that iron is not required for DNA binding, but is required for optimal PerR-regulated peroxide responses. Reduced iron/heme uptake and increased Mn^2+^ import may augment the substitution of Fe^2+^ with the Fenton-insensitive Mn^2+^ and permit maximal enzymatic activity under oxidative conditions, as previously described for *E. coli* (Anjem, Varghese and Imlay [@bib8]).

### Serotype M5 GAS {#sec10-3-3}

Comparative transcriptomic analysis of wild-type M5 GAS and an isogenic *perR* mutant cultured to mid-log growth phase in the absence of oxidative stress revealed one highly down-regulated gene (*czcD*), a newly identified GAS virulence factor (Ong *et al.*, [@bib155]) and five highly up-regulated genes in the *perR* mutant (Brenot, Weston and Caparon [@bib26]). Of the five up-regulated genes, only *pmtA*, encoding for PmtA involved in metal homeostasis and transport, contained a PerR-binding site in the promoter region and was directly repressed by PerR (Brenot, Weston and Caparon [@bib26]). The ABC-type metal transporter and transcriptional regulator, AdcR, repressed the expression of the remaining genes (*phtY, phtD, lsp* and *rpsN2*). Mutagenesis of all genes in the *perR* mutant background revealed that only *pmtA* contributes to H~2~O~2~ stress resistance *in vitro* (Brenot, Weston and Caparon [@bib26]). The indirect effect on the remaining genes was attributed to metal ion starvation mediated by AdcR (Brenot, Weston and Caparon [@bib26]). The overexpression of *pmtA* also up-regulated the AdcR-regulated genes, suggesting a link between metal ion homeostasis and the PerR and AdcR oxidative stress responses. Up-regulation of PmtA enhanced the resistance of M5 GAS *perR* mutants to H~2~O~2~ *in vitro* (Brenot, Weston and Caparon [@bib26]). However, PmtA overexpression increased metal efflux and may potentiate metal starvation, which could (at least in part) explain the reduced virulence of *perR* mutants *in vivo*.

### Serotype M14 GAS {#sec10-3-4}

The growth kinetics of a *perR* mutant in serotype M14 GAS strain HSC5 containing an in-frame deletion in the N-terminal DNA-binding region of PerR was similar to wild-type under aerobic conditions (King, Horenstein and Caparon [@bib117]). However, compared to wild-type bacteria, *perR* mutant survival was dramatically enhanced in lethal concentrations of H~2~O~2~ (King, Horenstein and Caparon [@bib117]). De-repression of the transcription of *ahpC, gpoA* and *mrgA* was not observed for the HSC5 *perR* mutant (King, Horenstein and Caparon [@bib117]), in contrast to a *perR* mutant reported for *B. subtilis* (Chen and Helmann [@bib41]; Bsat *et al.*, [@bib30]). These data suggest that in M14 GAS, the transcription of *ahpC, gpoA* and *mrgA* is not induced under conditions of peroxide stress, and is not repressed by PerR (King, Horenstein and Caparon [@bib117]). Primer extension analysis and DNase 1 protection assays indicate that the transcriptional regulator PerR binds to a single promoter upstream of *ahpC* (Brenot, King and Caparon [@bib24]). The regulation of *ahpC* is growth phase dependent and independent of PerR (Brenot, King and Caparon [@bib24]). MrgA is regulated by PerR and plays a key role in oxidative stress resistance in *S. mutans* (Yamamoto *et al.*, [@bib215]), *B. subtilis* and *S. aureus* (Chen and Helmann [@bib41]). In contrast to a previous report (King, Horenstein and Caparon [@bib117]), quantitative real-time PCR analysis of mid-log phase wild-type and *perR* mutant cultures for serotype M14 GAS strain HSC5 revealed higher *mrgA* transcripts for the *perR* mutant, indicating that PerR directly represses *mrgA* transcription through the binding of PerR to the *mrgA* promoter region (Brenot, King and Caparon [@bib24]). PerR is required for full virulence in C57Bl/6J mouse models of subcutaneous and systemic intraperitoneal mouse infections, and a zebrafish model of intramuscular infection (Brenot, King and Caparon [@bib24]).

PerR regulation of GAS DNases {#sec10-4}
-----------------------------

GAS DNases Sda1, Spd, MF3 and SpnA are important virulence factors (Iwasaki, Igarashi and Yutsudo [@bib105]; Aziz *et al.*, [@bib12]; Buchanan *et al.*, [@bib31]; Chang *et al.*, [@bib33]). Mitogen factor 3 (MF3) and streptodornase 1 (Sda1) are bacteriophage-encoded, whereas Spd and SpnA are chromosomally encoded (Hasegawa *et al.*, [@bib91]; Sumby *et al.*, [@bib193]). PerR directly binds to the promoter region and positively regulates the expression of MF3, a secreted DNase and virulence factor in serotype M1 GAS (Hasegawa *et al.*, [@bib91]; Wen *et al.*, [@bib213]). The expression of *sda1*, encoding for the bacteriophage-encoding DNase Sda1, is up-regulated under oxidative stress conditions in wild-type M1 GAS bacteria, but not in a *perR*-deficient mutant. Gel mobility shift assays revealed that PerR directly binds to the *sda1* promoter region. Mutation of a PerR metal binding site (histidine-99) reduced *sda1* expression in GAS pretreated with H~2~O~2~ (Wang *et al.*, [@bib212]). The PerR-dependent expression of *sda1* may facilitate GAS evasion of the host innate immune response. Sda1 expression enhances GAS degradation of NETs, promoting neutrophil survival and systemic dissemination of GAS from the infection site to normally sterile sites (Walker *et al.*, [@bib211]; Cole *et al.*, [@bib46]). Sda1 also prevents Toll-like receptor 9 recognition of degraded bacterial DNA to promote GAS immune escape (Uchiyama *et al.*, [@bib203]). In M3 GAS, the expression of the phage-encoded DNase MF4 (spyM3_1095) is PerR dependent and induced under conditions of oxidative stress and DNA damage (Banks, Lei and Musser [@bib15]).

Transcriptional regulator Rgg/RopB {#sec10-5}
----------------------------------

Rgg, also known as RopB, is a DNA-binding global transcriptional regulatory protein that plays a key role in coordinating the expression of cell wall-associated and secreted virulence factors (Chaussee, Ajdic and Ferretti [@bib36], Chaussee *et al.*, [@bib39], [@bib38]), secondary amino acid metabolic enzymes (e.g. arginine and serine catabolism) (Chaussee *et al.*, [@bib37]), and proteins involved in thermal and oxidative stress resistance in GAS. The Rgg of GAS is a 280 amino acid (∼33.2 kDa) polypeptide with an N-terminal helix--turn--helix motif that binds to the promoter regions of Rgg-regulated genes (Neely *et al.*, [@bib149]; Anbalagan *et al.*, [@bib5]). In serotype M49 GAS strain NZ131, Rgg regulates the expression of several secreted GAS virulence factors including cysteine protease SpeB, C5a peptidase, M protein, cytolysin streptolysin O and streptokinase (Lyon, Gibson and Caparon [@bib133]; Chaussee, Ajdic and Ferretti [@bib36]; Chaussee *et al.*, [@bib39], [@bib38]). Inactivation of *rgg* affects the expression of several transcriptional regulatory genes, including *fasBCA, mga, sagA* and *covRS/csrRS* (Chaussee *et al.*, [@bib38]), indicating that Rgg interacts directly or indirectly with other global transcriptional regulators.

Chaussee, Callegari and Chaussee ([@bib35]) used a comparative proteomic approach to identify the Rgg-regulated cytoplasmic proteins from mid-log and stationary phase cultures of wild-type serotype M49 GAS and an isogenic *rgg* mutant strain. Cytoplasmic proteins were identified by 2D gel electrophoresis and tandem mass spectrometry and revealed a growth phase-dependent Rgg regulation of proteins associated with arginine metabolism (ArcABC), histidine (HutI) and serine (SdhA) in the exponential growth phase (Chaussee, Callegari and Chaussee [@bib35]). Thermal and oxidative stress response proteins, including ClpE and ClpL, were expressed in the *rgg* mutant, but not wild-type (Chaussee, Callegari and Chaussee [@bib35]). Compared to wild-type, the Rgg-deficient strain was more resistant to heat-shock and puromycin (Chaussee, Callegari and Chaussee [@bib35]), an aminoacyl-tRNA analog that inhibits protein synthesis and induces a heat-shock-like response in Gram-negative and Gram-positive bacteria (VanBogelen and Neidhardt [@bib204]; Frees and Ingmer [@bib69]; Steiner and Malke [@bib191]). The *rgg* mutant was also hypersensitive to killing by superoxide free radicals induced by 50 mM paraquat exposure (Chaussee, Callegari and Chaussee [@bib35]). The oxidoreductases AhpC (SPy2079) and Nox1 (SPy2080) were more abundant in the *rgg*-deficient strain, indicating the de-repression of enzymes associated with GAS resistance to oxidative stress (Chaussee, Callegari and Chaussee [@bib35]).

More recently, a serotype M49 GAS strain deficient in Rgg decomposed more H~2~O~2~ and was more resistant to 4 mM H~2~O~2~-mediated killing, compared to the wild-type parental strain (Pulliainen *et al.*, [@bib168]). A double mutant deficient in *rgg* and *perR* was resistant to 4 mM H~2~O~2~ and did not exhibit changes in the expression of PerR target gene Dpr/MrgA, compared to the *perR* mutant, suggesting that Rgg H~2~O~2~ regulation may be independent of the PerR regulon (Pulliainen *et al.*, [@bib168]). Transcriptomic analysis revealed that the *ahpCF* operon expression was up-regulated in *rgg* mutant GAS (Pulliainen *et al.*, [@bib168]), suggesting that *ahpCF* is regulated by Rgg. The *ahpCF* gene products are involved in reducing organic peroxides, decomposing H~2~O~2~ and enhancing resistance against oxidative stress (Ellis and Poole [@bib58]; Poole *et al.*, [@bib163]). In addition to *ahpC*, serine protease and chaperone HtrA, also known as DegP, is up-regulated in *rgg* mutant GAS (Chaussee, Callegari and Chaussee [@bib35]; Dmitriev *et al.*, [@bib53]). The H~2~O~2~-resistant *rgg* mutant was more virulent in a mouse model of systemic GAS infection (Pulliainen *et al.*, [@bib168]).

Two-component regulator Ihk-Irr {#sec10-6}
-------------------------------

Voyich and coworkers (Voyich *et al.*, [@bib209]) reported the discovery of Ihk-Irr, a two-component global gene regulatory system that enhances GAS resistance to neutrophil killing and facilitates the lysis of host cells at the site of infection. Upon exposure of GAS to human neutrophils, 276 genes (∼16% of the M1 GAS genome) were differentially transcribed (Voyich *et al.*, [@bib209]). Eleven GAS virulence genes were up-regulated including *sic* (streptococcal inhibitor of complement), *speH* (streptococcal pyrogenic exotoxin H), *ndoS* (endoglycosidase S), *smeZ* (streptococcal mitogenic exotoxin Z) and *speB* (streptococcal pyrogenic exotoxin B), which are known to contribute to neutrophil resistance or modulation of the human innate immune system (Lukomski *et al.*, [@bib130]; Lei *et al.*, [@bib124]; Hoe *et al.*, [@bib99]). GAS genes encoding proteins involved in DNA repair and resistance to ROS-mediated cell damage were up-regulated, including *bsa* (glutathione peroxidase), *ahpC* (alkylhydroperoxidase), *dnaK* (Hsp70) and *nox1* (NADPH oxidase) (Voyich *et al.*, [@bib209]). Phagocytosis of GAS by neutrophils induced the up-regulation of nine genes encoding for proteins that participate in cell wall biogenesis, perhaps in response to the cell wall damage induced by phagocytosis and ROS (Voyich *et al.*, [@bib209]). Metabolic pathway genes were also up-regulated, presumably because an increase in energy metabolism is essential for GAS survival during phagocytosis. Phagocytosis altered the expression of genes encoding for proteins involved in the global transcriptional regulation of GAS. The *mga* regulon, encoding for a transcriptional regulator of virulence factors (Kihlberg *et al.*, [@bib116]), was up-regulated during phagocytosis. The genes encoding the two-component gene regulatory system, *ihk* and *irr*, were up-regulated, suggesting a role for this regulatory system in host innate immunity resistance (Voyich *et al.*, [@bib209]). A serotype M6 GAS strain JRS4 lacking the *irr* gene was hypersensitive to killing by human neutrophils following phagocytosis, indicating that Ihk-Irr plays a key role in the expression of genes necessary for GAS to survive phagocytosis. The *irr* gene is highly expressed in humans with GAS pharyngitis, as assessed by quantitative real-time PCR, suggesting that Ihk-Irr is important for GAS survival in humans (Voyich *et al.*, [@bib209]).

Microarray analysis comparing the global gene expression profiles of a wild-type M6 GAS and a corresponding *irr* mutant demonstrated that Ihk-Irr controls the expression of ∼20% of genes in the GAS genome (Voyich *et al.*, [@bib208]). Several genes involved in oxidative stress resistance were differentially regulated by *ihk-irr*, including *nrdH* (glutaredoxin), *trx* (thioredoxin), *trxR* (thioredoxin reductase), *nox* (NADH peroxidase), *rnr* (ribonucleotide reductase) and *bsaA* (glutathione peroxidase) (Voyich *et al.*, [@bib208]). Multiple virulence-associated genes, including *fbp* (putative fibronectin-binding protein-like protein A), *mf* and *mf3* (DNases), and *sagA* (SLS), and multiple genes involved in cell wall biosynthesis were regulated by the Ihk-Irr regulon (Voyich *et al.*, [@bib208]). The up-regulation of cell wall biosynthesis genes may protect the bacteria from cationic neutrophil antimicrobial peptides such as LL-37 and cathepsin G, which disrupt the bacterial cell membrane (Ganz *et al.*, [@bib73]; Peschel *et al.*, [@bib159], Poyart *et al.*, [@bib166]). During phagocytosis bacteria are exposed to ROS and antibacterial granule components stored within phagocytic vacuoles. Real-time PCR transcript analysis indicated that *ihk* and *irr* expression are induced upon GAS exposure to H~2~O~2~ and neutrophil primary granules (Voyich *et al.*, [@bib208]). In contrast to wild-type GAS, Irr-deficient GAS were more rapidly killed after phagocytosis through enhanced sensitivity to solubilized neutrophil primary granules, which are composed of α-defensins, elastase and cathepsin G (Sorensen *et al.*, [@bib188]). The *irr*-deficient strain was more susceptible than wild-type to killing by H~2~O~2~ across a range of different concentrations, and was hypersensitive to killing by the cell envelope active antimicrobial peptides LL-37 and cathepsin G. Inactivation of *irr* delayed the formation of skin abscesses and resulted in smaller abscesses, indicating that Irr is required for full GAS virulence in a mouse model of subcutaneous infection. In a bacteremia model of infection, *irr* mutant bacteria were more rapidly cleared from the blood 24 h post-infection compared to wild-type, indicating that Ihk-Irr plays an important role in GAS pathogenesis (Voyich *et al.*, [@bib208]).

Transcriptional regulator MtsR {#sec10-7}
------------------------------

Iron is important for many bacterial metabolic functions including the electron transport chain and DNA synthesis repair. However, excess iron is potentially lethal to the bacterial cell through the generation of highly toxic oxygen radicals by the Fenton reaction. In order to maintain iron homeostasis, bacteria express metal-dependent transcription regulators belonging to the Fur or the DtxR family (Andrews, Robinson and Rodriguez-Quinones [@bib7]). GAS acquires iron from heme, hemoglobin, haptoglobin--hemoglobin complexes, ferritin, myoglobin and catalase but not transferrin or lactoferrin (Francis, Booth and Becker [@bib68]; Eichenbaum *et al.*, [@bib57]; Bates *et al.*, [@bib16]). The GAS multimetal transport system (*mts*) is involved in the acquisition of manganese, zinc and iron (Janulczyk, Pallon and Bjorck [@bib109], [@bib110]; Ge and Sun [@bib75]), and is essential for GAS growth in metal-restricted media and full virulence in a mouse model of GAS infection (Janulczyk, Ricci and Bjorck [@bib110]). Transcriptional regulator MtsR is a member of the DtxR family of metal-dependent regulatory proteins involved in the coordination of iron homeostasis, oxidative stress resistance and virulence (Jakubovics, Smith and Jenkinson [@bib107]). Analysis of an *mtsR* mutant in serotype M49 GAS strain NZ131 by western blot and RNA analysis revealed that constitutive transcription of the 10-gene *sia* (streptococcal iron acquisition) operon (Bates *et al.*, [@bib17]), encoding for the hemoprotein receptor Shr, heme-binding protein Shp (Lei *et al.*, [@bib125]) and an ABC transporter, mediates GAS iron uptake from hemoproteins (Bates *et al.*, [@bib16]). Electrophoretic mobility gel shift assays indicated that MtsR directly binds to the *sia* promoter in an iron- and manganese-dependent manner to repress the expression of the *sia* operon (Bates *et al.*, [@bib17]). Excess iron accumulation causes oxidative stress via the Fenton reaction (Ratledge and Dover [@bib170]; Touati [@bib198]; Bates *et al.*, [@bib16]). The *mtsR* mutant accumulated more intracellular iron compared to wild-type in ^55^Fe uptake assays in complete medium, and was hypersensitive to H~2~O~2~, indicating that MtsR plays a role in GAS resistance to oxidative stress (Bates *et al.*, [@bib17]). In zebrafish models of intramuscular and intraperitoneal GAS infection, the *mtsR* mutant was attenuated for virulence (Bates *et al.*, [@bib17]).

Two-component regulator CiaRH {#sec10-8}
-----------------------------

CiaRH is a two-component regulator of GAS gene expression that is up-regulated under conditions of oxidative stress (Riani *et al.*, [@bib172]). Similar to the function of the sensor kinase CiaH in *S. pneumoniae*, the CiaH (Spy_1236) in serotype M1 GAS strain 1529 promotes growth under acidic conditions (pH 6.0) and resistance to oxidative stress following exposure to supraphysiologic concentrations of H~2~O~2~ (61 mM) for 15 min at room temperature (Tatsuno *et al.*, [@bib195]). Pneumococcal CiaH directly up-regulates HtrA, which is involved in resistance to oxidative stress, as described above (Seol *et al.*, [@bib183]; Ibrahim *et al.*, [@bib101]). However, in serotype M1 GAS, the expression of HtrA in the *ciaH*-null mutant was not down-regulated compared to wild-type, suggesting that the contribution of the CiaH sensor kinase to oxidative stress resistance may not be mediated via HtrA (Tatsuno *et al.*, [@bib195]).

CONCLUDING REMARKS {#sec11}
==================

Bacterial pathogens have evolved a plethora of sophisticated defense mechanisms to counter oxidative damage and highly toxic ROS generated from atmospheric oxygen and the oxidative burst from phagocytes. ROS, including H~2~O~2~, hydroxyl radicals and superoxide anions, are capable of damaging proteins, DNA, membrane lipids, and may induce cell death (Nunoshiba *et al.*, [@bib152]; Storz and Imlay [@bib192]; Imlay [@bib102]). Neutrophils are the first line of defense for the host innate immune system and promote bacterial clearance at the infection site through phagocytosis, a process whereby bacteria are killed by ROS and microbicidal granule components stored within phagocytic vacuoles (Mayadas *et al.*, [@bib138]). Bacterial ROS resistance mechanisms include direct detoxification of harmful reactive oxygen molecules by enzymes (catalases, peroxidases and Sods), repair mechanisms and alteration of intracellular metal ion concentrations (Faulkner and Helmann [@bib61]). GAS resistance to ROS generated by the human innate immune response enables this preeminent human pathogen to survive in the human host under the harsh conditions of oxidative stress.

Compared to other bacterial pathogens, GAS is equipped with surface-associated and secreted factors and unique molecular mechanisms to promote aerotolerance and combat ROS-induced stress *in vitro* and *in vivo*. While GAS lacks catalase, it has evolved additional mechanisms to defend against oxidative stress, including 1) novel surface and secreted molecules (M protein, HA capsule, Mac-1/IdeS and Mac-2); 2) enzymes directly involved in peroxide or superoxide detoxification (SodA, AhpC, GpoA and NoxA); 3) enzymes involved in the repair of ROS-damaged protein or DNA (HtrA/DegP and PolA1); 4) transporters involved in the maintenance of metal ion homeostasis (PmtA, Dpr, MtsABC/SiaABC and Shr); and 5) ROS response regulators (PerR, Rgg/RopB, Ihk-Irr, MtsR and CiaRH).

In Gram-positive bacteria, peroxide-sensing transcriptional regulators are responsible for regulating the oxidative stress response (Imlay [@bib102]; Dubbs and Mongkolsuk [@bib56]). The peroxide-sensing transcriptional regulator, PerR, is the chief peroxide responsive regulator in GAS (King, Horenstein and Caparon [@bib117]; Ricci, Janulczyk and Bjorck [@bib173]; Grifantini *et al.*, [@bib85]). Future work is needed to increase our understanding of the mechanisms of ROS resistance, the complex regulatory networks that coordinate GAS--host interactions and the response to ROS, and how these contribute to GAS pathogenesis and human infection. In addition, many of the proteins involved in ROS resistance are virulence factors. Therefore, these proteins are potential targets for the development of novel anti-GAS therapeutics and immune-boosting agents for the prevention and treatment of streptococcal diseases.

This work was supported by the National Health and Medical Research Council of Australia (APP1033258 to JNC) and the National Institutes of Health (AI077780 and AI48176 to VN).

*Conflict of interest statement*. None declared.
