The generation of superoxide radicals in an organism is an unavoidable by-product of aerobic respiration. Superoxide radicals can be generated by the univalent reduction of oxygen as the terminal acceptor of the electron transport chain (McCord and Fridovich, 1968; Fridovich, 1970; Imlay and Fridovich, 1991; Messner and Imlay, 1999). Superoxide radicals can also be generated enzymatically through the action of the NOX family of NADPH oxidases (Bedard and Krause, 2007). Superoxide oxidizes [4Fe-4S] clusters of dehydratases, which leads to the loss of iron from said clusters. Superoxide also inhibits transketolase which has a downstream effect on aromatic amino acid synthesis (Imlay, 2003). Due to the near ubiquity of superoxide generation, both eukaryotic and prokaryotic organisms possess SODs, metalloenzymes with roles in the detoxification of superoxide (McCord and Fridovich, 1969; Fridovich, 1995). Four categories of SOD have been identified, categorized by the metal cofactor at their active site (Grace, 1990; Abreu and Cabelli, 2010). Iron-containing SOD ([Fe]-SOD, encoded by sodB) and manganese containing SOD ([Mn]-SOD, encoded by sodA) are similar in both protein sequence and structure, differing primarily in the nature of their metal cofactor. Both [Fe]-SOD and [Mn]-SOD are found in bacteria, while the former are also found in plant cells and the latter are found in mitochondria and peroxisomes (Fridovich, 1995; Abreu and Cabelli, 2010). The third class of SOD is co-factored by nickel and appears to be restricted to Streptomyces spp. (Kim et al., 1996; Youn et al., 1996; Choudhury et al., 1999; Barondeau et al., 2004). The final class of SOD, encoded by sodC, is co-factored by both copper and zinc. This [Cu, Zn]-SOD is structurally unrelated to the single metal co-factored SODs. [Cu, Zn]-SOD was originally thought to be present only in eukaryotic and plant cells but was subsequently identified in E. coli (Fridovich, 1995; Imlay and Imlay, 1996; Abreu and Cabelli, 2010). [Cu, Zn]-SOD homologues have also been identified in Photobacter spp., Pseudomonas spp., Caulobacter crescentus, and Brucella abortus, among others (Puget and Michelson, 1974; Martin and Fridovich, 1981; Bannister and Parker, 1985; Steinman, 1985; Beck et al., 1990; Steinman and Ely, 1990; Benov and Fridovich, 1994). [Cu, Zn]-SODs are transported to the periplasm. As superoxide generated within the bacterial cytosol cannot cross the inner membrane of the cell (Hassan and Fridovich, 1979), [Fe]-SOD and [Mn]-SOD must detoxify endogenously generated superoxide, whereas secreted [Cu, Zn]-SOD must have a role in detoxifying exogenous superoxide generated by the host (Schnell and Steinman, 1995; San Mateo et al., 1998).

The damage to the bacterial cell resulting from superoxide radicals is exacerbated by their involvement in the generation of H₂O₂ (Gonzalez-Flecha and Demple, 1995, 1997). The dismutation of superoxide leads to the generation of dioxygen and H₂O₂. Thus E. coli, when grown exponentially in glucose-containing medium, produces approximately 14 μM H₂O₂ per second in the cytosol (Seaver and Imlay, 2001b). As cell membranes are semi-permeable to H₂O₂, extracellular H₂O₂ generated by host macrophages can also lead to an internal increase in H₂O₂ in both the periplasm and cytosol of a bacterial cell (Seaver and Imlay, 2001b; Imlay, 2008). An increase in hydrogen peroxide in the periplasm could disrupt redox reactions critical for protein function (Kadokura et al., 2003; Heras et al., 2009). An increase in intracellular H₂O₂ is detrimental to the cell through both the oxidation of [4Fe-4S] clusters of dehydratases as well as by protein carbonylation, which can lead to the inactivation of enzymes (Stadtman, 1993; Tamarit et al., 1998; Imlay, 2003). Critically, H₂O₂ can also interact with intracellular free iron to generate toxic hydroxyl radicals, via the Fenton reaction (Imlay, 2003). If this iron is bound to DNA, then hydroxyl radicals generated via the Fenton reaction can damage the DNA (Imlay, 2003). Indeed, experiments that involved repeated passage of an E. coli strain that was unable to scavenge H₂O₂, under aerobic conditions, demonstrated DNA damage mediated by H₂O₂ (Storz and Imlay, 1999; Park et al., 2005).

As opposed to SOD, which combats superoxide derived oxidative stress, bacteria have evolved multiple methods of combating H₂O₂-induced oxidative stress. These methods are discussed in detail below.

The first gene identified in H. influenzae that encodes a homologue of SOD was found fortuitously by Kroll et al. (1991). Characterization of capsulation genes in H. influenzae type b led to the identification of an adjacent ORF, sodC, which was predicted to encode a protein homologous to the [Cu, Zn]-SOD that was first identified in Photobacterium leiognathi (Steinman, 1987; Kroll et al., 1991). However, no detectable [Cu, Zn]-SOD activity could be demonstrated in encapsulated H. influenzae strains. Analyses of the derived amino acid sequence of [Cu, Zn]-SOD from H. influenzae type b showed that the protein possessed only five of the six histidine residues that coordinate the metal ions in other [Cu, Zn]-SODs (Kroll et al., 1991). In contrast both a sodC homolog and functional [Cu, Zn]-SOD were found in 100% of Haemophilus parainfluenzae strains tested (Kroll et al., 1991) as well as other members of the Haemophilus, Actinobacillus and Pasteurellaceae (HAP) group (Langford et al., 1992; Kroll et al., 1995). Analyses of the derived [Cu, Zn]-SOD protein sequences from the HAP group showed they may share a critical property of prokaryotic [Cu, Zn]-SODs; they are exported to the periplasm where they are implicated in protection against exogenous, host-derived superoxide (Kroll et al., 1995; San Mateo et al., 1998). Finally, analysis of [Cu, Zn]-SOD proteins expressed by H. parainfluenzae and Haemophilus ducreyi provided insight into how [Cu, Zn]-SOD functions (Kroll et al., 1995; Forest et al., 2000). Specifically, [Cu, Zn]-SOD has an exposed, histidine-rich N-terminus that binds copper. Kinetically favorable binding of copper to the N-terminal region relative to the active site, but stronger binding of copper to the active site, relative to the N-terminal region suggests the bound copper binds the N-terminal region prior to transport to the protein's active site (Battistoni et al., 2001). The role of the histidine-rich N-terminal was demonstrated by expressing N-terminal truncated [Cu, Zn]-SOD from H. parainfluenzae in E. coli. Loss of the N-terminus, and presumably the ability to scavenge copper for the active site, had a significant impact on the mutated [Cu, Zn]-SOD's ability to protect E. coli from being killed in a phagocytosis assay (Battistoni et al., 2001).

It is now clear that H. influenzae lacks either a gene encoding a [Cu, Zn]-SOD, or the ability to synthesize functional [Cu, Zn]-SOD (Kroll et al., 1991). Further, Norskov-Lauritsen and co-workers showed that sodC was absent in all nontypeable H. influenzae (NTHi) strains investigated (Norskov-Lauritsen, 2009; Norskov-Lauritsen et al., 2009). Recently, it has been recognized that some strains previously thought to be NTHi are non-hemolytic Haemophilus haemolyticus. All NTHi isolates studied lacked both an identifiable homologue of sodC and [Cu, Zn]-SOD activity while the H. haemolyticus isolates posses a sodC gene (Fung et al., 2006). Finally, all of the genome sequences of NTHi currently deposited in Genbank lack an identifiable homologue of sodC.

The lack of SodC activity in H. influenzae raises an interesting question about how H. influenzae deals with exogenously generated superoxide as the immune response of the host likely subjects H. influenzae to superoxide generated by phagocytes. [Cu, Zn]-SODs are localized in the periplasm to detoxify exogenously generated superoxide (Hassan and Fridovich, 1979; Schnell and Steinman, 1995; San Mateo et al., 1998). However, H. influenzae can persist within the host yet lacks [Cu, Zn]-SOD. Neisseria gonorrhoeae similarly lacks an identifiable sodC homologue and detectable [Cu, Zn]-SOD activity (Tseng et al., 2001; Seib et al., 2004). N. gonorrhoeae however, can persist in purulent exudates, rich in polymorphonuclear leukocytes. When the sole SOD-encoding gene, sodB, was inactivated, N. gonorrhoeae exhibited resistance to both endogenously and exogenously generated superoxide in a manganese-dependent manner. When the periplasmic-binding protein of an ABC transport system that takes up manganese was mutated, N. gonorrhoeae was sensitive to endogenously generated, superoxide-mediated oxidative stress. It was thus proposed that manganese scavenges superoxide non-enzymatically and thus protects cells from oxidative stress (Tseng et al., 2001; Seib et al., 2004). In contrast, work by the Imlay group suggested that, in E. coli, manganese has a more complex role as a substitute for iron in mononuclear metalloenzymes (Anjem et al., 2009). The gene that encodes the E. coli manganese importer, MntH, is regulated by OxyR and mntH expression is responsive to H₂O₂ (Kehres et al., 2000). No homologue of E. colimntH can be identified in Haemophilus spp. However, the manganese transporter, MntC, from Synechocystis is homologous to HfeA from NTHi strain 86-028NP. HfeA, annotated as a putative periplasmic chelated iron-binding protein (Harrison et al., 2005), is OxyR-regulated and responsive to H₂O₂-induced oxidative stress (Harrison et al., 2007). Although there are no experimental data to suggest that HfeA has a role in manganese transport, it is possible that Haemophilus imports manganese via an oxidative stress-responsive transport system. This would suggest that NTHi, strain 86-028NP has a system akin to that in E. coli; imported manganese substituting for iron in metalloenzymes and thus protecting the proteins from damage induced by H₂O₂, one of the dismutation products of superoxide.

The role of [Mn]-SOD in H. influenzae is more clear. After the identification of a [Cu, Zn]-SOD, a [Mn]-SOD homolog was subsequently identified and characterized in H. influenzae type b. In contrast to [Cu, Zn]-SOD, this protein was active. Moreover, when expressed in E. coli, [Mn]-SOD from H. influenzae type b was regulated by the fumarate/nitrate reduction transcriptional regulator, Fnr and the ferric uptake regulator, Fur (Kroll et al., 1993). Using degenerate primers based on the sequences of sodA from H. influenzae strain Rd KW20 and NTHi strain 86-028NP, PCR analyses subsequently showed that homologues of sodA were present in all Haemophilus type strains tested (Cattoir et al., 2006).

To date, homologues of neither [Fe]-SOD or [Ni]-SOD have been found in any Haemophilus strain.

Catalase is one of the best-understood enzymes with a role in protection against oxidative stress. A number of subgroups of catalases have been identified, based on heterogeneities in sequence, heterogeneities in function or differences in metal cofactors (Zamocky and Koller, 1999). This review will, unless stated otherwise, refer to classic, heme co-factored catalase. Catalases are predominantly found expressed by species that utilize aerobic respiration and catalyzes the decomposition of H₂O₂ to oxygen and water (Chelikani et al., 2004). There are two homologues of catalase in E. coli, K12; hydroperoxidase I and hydroperoxidase II, encoded by katG and katE respectively (Loewen et al., 1985; Von Ossowski et al., 1991; Imlay, 2008). katG and katE are differentially regulated in E. coli. katE is expressed constitutively during exponential growth but, through regulation by RpoS, is increased in expression during stationary phase. katE expression is not inducible by H₂O₂ treatment (Loewen et al., 1985; Vijayakumar et al., 2004). Conversely, katG is normally expressed at low levels and is induced upon peroxide stress. OxyR mediates this regulation of katG expression in E. coli and S. typhimurium (Christman et al., 1985, 1989).

Although catalase activity in H. influenzae was characterized in 1961 (Biberstein and Gills, 1961), an H. influenzae homologue of catalase was not identified until 1994. Bishai and co-workers screened an H. influenzae library for genes that complemented an E. coli strain that could not express katE in stationary phase (Bishai et al., 1994b) and identified hktE, which encodes a protein homologous to hydroperoxidase II from E. coli. HktE expression was, however, maximal during exponential growth and reduced during stationary phase, unlike hydroperoxidase II in E. coli. An hktE mutant of H. influenzae strain Rd grew similarly to the parent when cultured aerobically in rich medium. However, when the hktE mutant was cultured anaerobically in minimal medium, then shifted to an aerobic growth condition, it exhibited a growth defect, concomitant with a rise in H₂O₂ in the medium. The parent exhibited no such growth defect. These growth defects (generated by the loss of catalase) could be prevented by the addition of a peroxide scavenger, sodium pyruvate, to the growth medium (Vergauwen et al., 2003a). The medium-specific killing of the H. influenzaehktE mutant was due to the absence of glutathione in the minimal medium. In fact, when H. influenzae strain Rd cells were starved for glutathione, the shift to aerobic respiration produced a ∼7-fold increase in catalase activity. Moreover, when the hktE mutant was starved for glutathione and shifted to aerobic metabolism, there was a measurable accumulation of H₂O₂ in the growth medium (Vergauwen et al., 2003a).

HktE's expression was inducible with H₂O₂, a characteristic of E. coli hydroperoxidase II (Bishai et al., 1994b; Harrison et al., 2007). When hktE mutations were generated in both Rd and type b H. influenzae strains, no catalase activity could be detected, even after peroxide-induced stress. Thus, HktE is the sole catalase in H. influenzae. Also, the hktE mutant strains were more sensitive to H₂O₂, as compared to their parent strains (Bishai et al., 1994a). H. influenzae type b hktE mutants were tested in an infant rat intraperitoneal model of virulence, as well as an infant rat intranasal model of colonization. In the intraperitoneal virulence model, when inoculated i.p. at lethal doses, the hktE mutant was impaired in its ability to cause sepsis, relative to the parent. However, when inoculated at sub-lethal doses, the hktE mutant was not impaired in its ability to cause persistent bacteremia, relative to the parent. Finally, in the colonization model, the hktE mutants exhibited similar levels of colonization as the parent (Bishai et al., 1994a; Vergauwen et al., 2006).

The regulation of expression of hktE in H. influenzae must be considered with respect to the niche H. influenzae occupies. As a commensal of the human nasopharynx and a bacterium capable of forming a biofilm, H. influenzae persists in stationary phase. Only after transit to other niches and growth does the organism cause disease. As already discussed, H. influenzae has one catalase homologue, HktE, whose expression is down regulated during stationary phase. Also, hktE expression is induced when subject to peroxide stress. H. influenzae must be able to withstand the relatively high concentrations of oxygen in the nasopharynx. It must also be able to withstand peroxide stress likely encountered when H. influenzae has transitioned to a pathogen and is under assault from the host cells, particularly from the respiratory burst. However, an hktE mutant of H. influenzae type b, although slightly impaired in its ability to kill infant rats when administered at high doses, was not impaired in its ability to colonize or cause bacteremia in a nasal challenge infant rat model (Bishai et al., 1994a). This apparent absence of a role for catalase in virulence agrees with data from Listeria monocytogenes, S. typhimurium, Shigella flexneri and Brucella abortus (Fields et al., 1986; Gaillard et al., 1986; Franzon et al., 1990; Steele et al., 2010), but disagrees with data from Xanthomonas campestris, P. aeruginosa and Staphylococcus aureus, among others (Mandell, 1975; Lee et al., 2005; Jittawuttipoka et al., 2009). Also, a catalase mutant of H. influenzae was not significantly affected by the H₂O₂ generated by S. pneumoniae (Pericone et al., 2000). With the caveat that the infant rat model of H. influenzae may not be ideal for showing host killing by oxidative stress (Bishai et al., 1994a), experimental data suggests that catalase appears to be the enzyme that is utilized primarily in the scavenging of high concentrations of endogenously generated, H₂O₂ (Vergauwen et al., 2003a, 2006) while only being one part of a broader defense against H₂O₂-induced oxidative stress.

Glutathione is a cysteine-containing thiol found in all kingdoms of life. Glutathione has multiple roles within the bacterial cytosol that include control of redox potential, cell signaling and protection against multiple stresses such as oxidative stress. Critically, glutaredoxins can reduce disulfide bonds, utilizing the reducing power of glutathione (Fernandes and Holmgren, 2004; Dalle-Donne et al., 2009). This role for glutaredoxin and glutathione in the regulation of a protein's function was found in E. coli, where OxyR is regulated by glutaredoxin (Zheng et al., 1998; Aslund et al., 1999; Pomposiello and Demple, 2001). The role of glutathione in resistance to oxidative stress was also observed in yeast. When production of glutathione was prevented by a mutation in its synthesis pathway, yeast became more sensitive to oxidative stress induced by H₂O₂ (Grant et al., 1996). However in E. coli, a mutation in glutathione synthetase failed to produce increased sensitivity to both H₂O₂ and the organic hydroperoxide cumene hydroperoxide (CMH) (Greenberg and Demple, 1986). Greenberg and Demple speculated that E. coli exhibits an adaptive response to oxidative stress, in the absence of glutathione import. Other, overlapping means of protection must be present.

H. influenzae can import glutathione from the environment employing the ABC-like dipeptide transporter DppBCDF (Vergauwen et al., 2010). A periplasmic glutathione-binding protein was identified as a previously characterized heme-binding lipoprotein, HbpA, which was subsequently renamed GbpA. GbpA binds both oxidized and reduced glutathione. GbpA also has a reduced affinity for conjugated or modified glutathione, preferentially importing useful forms of glutathione (Vergauwen et al., 2010). H. influenzae does not need glutathione for growth but imported glutathione has been shown to protect an NTHi strain against oxidative stress induced by H₂O₂ or tert-butyl hydroperoxide (t-BOOH) (Pauwels et al., 2003, 2004; Vergauwen et al., 2003a).

This glutathione-based peroxidase activity was assayed in cell-free extracts from an NTHi strain and the activity was inhibited by iodoacetamide, an inhibitor of disulfide bond formation (Vergauwen et al., 2003b). As noted above, a catalase mutant of H. influenzae was not impaired in growth in rich medium, colonized infant rats at parental levels and caused similar levels of bacteremia (Bishai et al., 1994a). Based on these data, it was suggested that H. influenzae must have another method of scavenging peroxide, with glutathione being the electron donor for this peroxidase (Vergauwen et al., 2003a). This glutathione-mediated, disulfide bond-requiring protection against t-BOOH was subsequently found to be dependent on the peroxidase Prx/Grx (subsequently named PgdX). PgdX, a member of the thiol peroxidase family that can reduce H₂O₂ and alkylhydroperoxides via glutathione redox cycling, is a chimeric protein with a peroxiredoxin domain and a glutaredoxin domain (Vergauwen et al., 2001). The peroxiredoxin domain is involved in detoxification of hydroperoxides while the glutaredoxin domain reduces and thus reactivates the peroxiredoxin domain (Pauwels et al., 2003). Subsequent BLAST analysis identified homologues of H. influenzae PgdX in a number of bacteria that are predominantly human pathogens. The homology was strongest over the peroxiredoxin domains (Pauwels et al., 2003). When the crystal structure of PgdX from H. influenzae was solved, it was found to exist as a homotetramer. Within the homotetramer, the peroxiredoxin domain of one monomer interacts with the glutaredoxin domain of the adjacent tetramer. Such a peroxiredoxin-glutaredoxin interaction would allow inter-monomer transfer of electrons between domains (Kim et al., 2003). A pgdX mutant generated in H. influenzae strain Rd exhibited a growth rate similar to the parent when cultured both aerobically and anaerobically in broth (Vergauwen et al., 2006). However, pgdX mutant cells were also more susceptible to t-BOOH and CMH, when compared to the parent (Pauwels et al., 2004). Of great interest was the observation that a pgdX mutant of H. influenzae strain Rd showed increased resistance to H₂O₂. The loss of PgdX was compensated by an increase in catalase expression (Pauwels et al., 2004). Also, if PgdX activity was reduced by decreasing the amount of glutathione available to the cell, catalase activity increased (Vergauwen et al., 2003a,b). PgdX has a kinetic preference for H₂O₂, as compared to alkylhydroperoxides. However, PgdX is optimized to scavenge lesser concentrations of H₂O₂ than catalase. Thus, in the absence of PgdX, low levels of H₂O₂ cannot be efficiently scavenged by catalase. The activity of OxyR is then induced and catalase activity is upregulated. Not unexpectedly, in the absence of both catalase and PgdX, H. influenzae strain Rd cannot decompose H₂O₂ (Vergauwen et al., 2006). These data suggest that both catalase and PgdX scavenge metabolically generated H₂O₂, with PgdX being protective during stationary phase (Vergauwen et al., 2003b, 2006; Pauwels et al., 2004). Thus, due to the lower efficiency of PgdX in scavenging H₂O₂, stationary phase cultures of H. influenzae are more susceptible to oxidative stress (Pauwels et al., 2004).

A mutant of H. influenzae strain Rd that lacks pgdX and hktE is unaffected during either aerobic growth, or after a shift to aerobic growth in rich medium (Vergauwen et al., 2006). This stands in contrast to a mutant strain of E. coli that lacks the ability to scavenge H₂O₂ and exhibits decreased aerobic growth due to presumed DNA damage (Seaver and Imlay, 2001a). Conversely, when the same strain Rd mutant is grown in a chemically defined medium under aerobic conditions, all growth ceases due to H₂O₂ accumulation leading to possible inactivation of amino acid or vitamin synthesis (Vergauwen et al., 2006). Aerobic respiration is required for some strains of H. influenzae to become invasive (Herbert et al., 2003). However, the ability of an H. influenzae, type b strain that lacks both catalase and PgdX to cause bacteremia in infant rats was unaffected. The authors speculate that the extent of the host response to H. influenzae invasion and/or the oxygen level in infant rat blood are not sufficient for the infant rat to prevent H. influenzae bacteremia, even in those strains which lacked catalase and PgdX (Vergauwen et al., 2006). A potentially similar situation occurs in S. typhimurium, wherein loss of catalase, a second peroxidase AhpC or OxyR has little effect on virulence (Buchmeier et al., 1995; Taylor et al., 1998). A recA mutant of S. typhimurium did, however, exhibit a large decrease in virulence in the mouse model (Buchmeier et al., 1995). The ability to repair damaged DNA thus appears to be more critical than the ability to detoxify H₂O₂ and hydroxyl radicals. This is apparent in NTHi where both recA and recN expression were moderately induced by H₂O₂ in NTHi strain 86-028NP (Harrison et al., 2007). This raises an important question. Catalase and PgdX are not essential for the virulence of H. influenzae, type b. Thus, what scavenging enzymes with roles in mitigating oxidative stress are critical for cell survival? Possibly, H. influenzae is more sensitive to superoxide in vivo, although a sodA mutation in H. influenzae type b only affects nasopharyngeal colonization, not invasive disease in an infant rat model of infection (D'Mello et al., 1997). Alternatively, the experimental conditions used to assess H. influenzae's sensitivity to oxidative stress inducing compounds do not accurately recapitulate the conditions encountered in vivo.

Despite the apparent dispensability of PgdX for virulence in H. influenzae, PgdX is important in the ability of H. influenzae to form biofilms (Murphy et al., 2005). PgdX is expressed at a greater level in H. influenzae cells that form biofilms in vitro. In concert, when pgdX mutations were introduced into four H. influenzae strains isolated from patients with COPD, the strains' abilities to form biofilms were impaired, although the level of impairment varied between strains. COPD patients demonstrate elevated levels of H₂O₂, possibly derived from alveolar macrophages and systemic oxidative stress (Boots et al., 2003). The peroxide scavenging ability of PgdX may, therefore, be critical to the survival of H. influenzae in such environments.

The alkylhydroperoxidase AhpC was first identified in S. typhimurium during a search for enzymes that protected the cell against organic hydroperoxides, such as t-BOOH and CMH (Jacobson et al., 1989). As well as this role in S. typhimurium, AhpC was shown to decompose both CMH and H₂O₂ in S. enterica (Jacobson et al., 1989; Niimura et al., 1995; Seaver and Imlay, 2001a). AhpC is also the primary scavenger of low levels of endogenous H₂O₂ in E. coli, while catalase scavenges high levels of H₂O₂ (Seaver and Imlay, 2001a,b). After oxidation, AhpC is reduced using electrons from NADPH that are transferred via the flavoprotein AhpF (Jacobson et al., 1989). In E. coli AhpC and AhpF are encoded in an operon that is regulated by OxyR (Tartaglia et al., 1989). In discussions of the role of catalase in decomposition of H₂O_2, it was assumed, based on the genome sequence of H. influenzae strain Rd, that H. influenzae lacks AhpC (Vergauwen et al., 2006). Homologues of ahpC are present in the Pasteurellaceae, for example tsaA from P. multocida. A microarray-based genomic screen of genes present in NTHi strain 1885MEE subsequently identified an H. influenzae homologue of tsaA. A second H. influenzae homologue of tsaA was identified in NTHi strain 86-028NP (Munson et al., 2004; Harrison et al., 2005). A homologue of AhpF is not present in the NTHi strain 86-028NP genome (Harrison et al., 2005). Critically, expression of tsaA in NTHi strain 86-028NP was not inducible by H₂O₂. If functional, the unresponsiveness to H₂O₂ suggests that TsaA in NTHi strain 86-028NP may have a role in detoxification of other reactive oxygen species (Harrison et al., 2007).

In E. coli, there are two peroxidases, catalase and alkylhydroperoxide (Seaver and Imlay, 2001a). Further, in E. coli, catalase, and AhpC have overlapping functions in the decomposition of H₂O₂. Catalase is the primary scavenger at high levels of H₂O₂, while AhpC is the primary scavenger at low levels of H₂O₂ (Storz et al., 1989; Seaver and Imlay, 2001a,b). Loss of katG, katE, and ahpCF in E. coli results in a strain that can grow under anaerobic conditions but grows poorly under aerobic conditions. This katG, katE, ahpCF mutant strain exhibits increased endogenous hydroxyl radical formation and DNA damage (Park et al., 2005). The work of Vergauwen et al. (2003a) suggested that H. influenzae strain Rd uses two peroxidases, catalase and a glutathione–dependent peroxidase, possibly PgdX, to decompose H₂O₂. Non-enzymatic glutathione-dependent protection also occurs and loss of available glutathione leads to an increase in catalase activity. The AhpCF system is not present in H. influenzae strain Rd, although AhpC is present in some NTHi strains.

E. coli lacks a homologue of PgdX, but there are parallels between PgdX and AhpC that suggest they are functional analogues. In multiple species of bacteria, including H. influenzae, both PgdX and AhpC scavenge H₂O₂ and t-BOOH with similar kinetics; they are both critical during exponential phase growth but not for virulence and they are both highly expressed (O'Toole et al., 1991; Seaver and Imlay, 2001a,b; Pauwels et al., 2003; Vergauwen et al., 2003a, 2006; Yoon et al., 2003; Murphy et al., 2005). Why then does NTHi strain 86-028NP possess both a homologue of AhpC (TsaA) and PgdX? Possibly NTHi strain 86-028NP has both enzymes as an important redundancy in the bacterium's ability to scavenge low concentrations of H₂O₂. Alternatively, the non-responsiveness of TsaA to H₂O₂ treatment in NTHi strain 86-028NP suggests that, in this bacterium, TsaA has an alternative function to that exhibited by its homologue, AhpC, in E. coli.

The ferritin-like protein, Dps, was first identified in E. coli as important for protection against conditions that arise during stationary phase, conditions that include protection from H₂O₂ (Almiron et al., 1992). Expression of dps in E. coli is increased by exposure to H₂O₂, in an OxyR-dependent manner (Altuvia et al., 1994). Dps homologues with roles in protection against oxidative stress were subsequently identified in many gram negative and positive bacteria, including S. enterica serovar Typhimurium, Campylobacter jejuni, B. subtilis, S. mutans and Listeria innocua (Chen and Helmann, 1995; Yamamoto et al., 2000; Ishikawa et al., 2003; Halsey et al., 2004; Su et al., 2005). Dps binds iron, which is then oxidized to Fe³⁺ by H₂O₂. Fe³⁺ then forms a stable ferric oxide mineral core within a cavity in Dps. Thus iron is no longer available to contribute to the Fenton reaction and generate hydroxyl radicals (Park et al., 2005; Chiancone and Ceci, 2010). Critically Dps also binds DNA, which both protects the DNA from damage and slows initiation of chromosome replication. Slowing of replication will allow time for the DNA damage to be repaired (Martinez and Kolter, 1997; Grant et al., 1998; Chodavarapu et al., 2008).

Expression of dps in NTHi strain 86-028NP is induced by the presence of H₂O₂, in an OxyR-dependent manner (Harrison et al., 2007). Moreover, expression of dps in H. influenzae strain Rd is also induced by ArcA (Wong et al., 2007). A dps mutant of H. influenzae strain Rd, when grown anaerobically, is more sensitive to H₂O₂, as compared to the parent strain. Dps is thus protecting the cell preemptively against transitions from growth in low oxygen to aerobic stress. This H₂O₂-specific effect was mitigated when cells were grown aerobically (Wong et al., 2007). The reduced role of Dps in protection against H₂O₂ in H. influenzae strain Rd cells grown aerobically suggests that the other major detoxification systems (catalase, SOD and PgdX) are more critical in aerobically growing cells (Wong et al., 2007).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.