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

Secretion by Overexpression and Purification of the Water-Soluble Streptomyces K15 Dd-Transpeptidase/Penicillin-Binding Protein

Though synthesized with a cleavable signal peptide and devoid of membrane anchors, the 262-amino-acid-residue Streptomyces K15 DD-transpeptidase/penicillin-binding protein is membrane-bound. Overexpression in Streptomyces lividans resulted in the export of an appreciable amount of the synthesized protein (4 mg/litre of culture supernatant). The water-soluble enzyme was purified close to protein homogeneity with a yield of 75%. It requires the presence of 0.5 M-NaCl to remain soluble. It is indistinguishable from the detergent-extract wild-type enzyme with respect to molecular mass, thermostability, transpeptidase activity and penicillin-binding capacity

Glutathione and Catalase Provide Overlapping Defenses for Protection against Respiration-Generated Hydrogen Peroxide in Haemophilus influenzae

Glutathione is an abundant and ubiquitous low-molecular-weight thiol that may play a role in many cellular processes, including protection against the deleterious effects of reactive oxygen species. We address here the role of glutathione in protection against hydrogen peroxide (H(2)O(2)) in Haemophilus influenzae and show that glutathione and catalase provide overlapping defense systems. H. influenzae is naturally glutathione deficient and imports glutathione from the growth medium. Mutant H. influenzae lacking catalase and cultured in glutathione-deficient minimal medium is completely devoid of H(2)O(2) scavenging activity and, accordingly, substantial amounts of H(2)O(2) accumulate in the growth medium. H. influenzae generates H(2)O(2) at rates similar to those reported for Escherichia coli, but the toxicity of this harmful metabolite is averted by glutathione-based H(2)O(2) removal, which appears to be the primary system for protection against H(2)O(2) endogenously generated during aerobic respiration. When H(2)O(2) concentrations exceed low micromolar levels, the hktE gene-encoded catalase becomes the predominant scavenger. The requirement for glutathione in protection against oxidative stress is analogous to that in higher and lower eukaryotes but is unlike the situation in other bacteria in which glutathione is dispensable for aerobic growth during both normal and oxidative stress conditions