Hydrogen peroxide scavenging is not a virulence determinant in the pathogenesis of Haemophilus influenzae type b strain Eagan

BACKGROUND: A potentially lethal flux of hydrogen peroxide (H(2)O(2)) is continuously generated during aerobic metabolism. It follows that aerobic organisms have equipped themselves with specific H(2)O(2 )dismutases and H(2)O(2 )reductases, of which catalase and the alkyl hydroperoxide reductase (AhpR) are the best-studied prokaryotic members. The sequenced Haemophilus influenzae Rd genome reveals one catalase, designated HktE, and no AhpR. However, Haemophilus influenzae type b strain Eagan (Hib), a causative agent of bacterial sepsis and meningitis in young children, disrupted in its hktE gene is not attenuated in virulence, and retains the ability to rapidly scavenge H(2)O(2). This redundancy in H(2)O(2)-scavenging is accounted for by peroxidatic activity which specifically uses glutathione as the reducing substrate. RESULTS: We show here that inside acatalasaemic H. influenzae all of the residual peroxidatic activity is catalyzed by PGdx, a hybrid peroxiredoxin-glutaredoxin glutathione-dependent peroxidase. In vitro kinetic assays on crude hktE(- )pgdx(- )H. influenzae Rd extracts revealed the presence of NAD(P)H:peroxide oxidoreductase activity, which, however, appears to be physiologically insignificant because of its low affinity for H(2)O(2 )(K(m )= 1.1 mM). Hydroperoxidase-deficient hktE(- )pgdx(- )H. influenzae Rd showed a slightly affected aerobic growth phenotype in rich broth, while, in chemically defined medium, growth was completely inhibited by aerobic conditions, unless the medium contained an amino acid/vitamin supplement. To study the role of PGdx in virulence and to assess the requirement of H(2)O(2)-scavenging during the course of infection, both a pgdx single mutant and a pgdx/hktE double mutant of Hib were assayed for virulence in an infant rat model. The ability of both mutant strains to cause bacteremia was unaffected. CONCLUSION: Catalase (HktE) and a sole peroxidase (PGdx) account for the majority of scavenging of metabolically generated H(2)O(2 )in the H. influenzae cytoplasm. Growth experiments with hydroperoxidase-deficient hktE(- )pgdx(- )H. influenzae Rd suggest that the cytotoxicity inflicted by the continuous accumulation of H(2)O(2 )during aerobic growth brings about bacteriostasis rather than bacterial killing. Finally, H(2)O(2)-scavenging is not a determinant of Hib virulence in the infant rat model of infection.