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

Structural interpretation of the amino acid sequence of a second domain from the Artemia covalent polymer globin

Artemia has a complex extracellular hemoglobin of Mr 260,000 comprising two globin chains (Mr 130,000) each of which is a polymer of eight covalently linked domains of Mr 16,000. The primary structure of this polymeric globin was studied to understand how globin folded domains are ordered within a globin chain and, in turn, how the latter associate into a functional hemoglobin molecule. Here we report the amino acid sequence of a second domain, E7 (Mr 16,081, excluding the heme), and interpretations of sequence data by computer-assisted alignment and modeling. This clearly shows that, as with domain E1 (Moens, L. Van Hauwaert, M.-L. De Smet, K. Geelen, D. Verpooten, G. Van Beeumen, J. Wodak, S. Alard, P. & Trotman, C. (1988) J. Biol. Chem. 263, 4679-4685), domain E7 is compatible with a globin folded structure of the β-type chain. Several specific differences of domains E7 and E1 from the classic globins are identified. They possibly can be interpreted in terms of specific requirements for a double octameric functional molecule.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

ELECTRON-MICROSCOPIC ANALYSIS AND BIOCHEMICAL-CHARACTERIZATION OF A NOVEL METHANOL DEHYDROGENASE FROM THE THERMOTOLERANT BACILLUS-SP C1

Methanol dehydrogenase from the thermotolerant Bacillus sp. C1 was studied by electron microscopy and image processing. Two main projections can be distinguished: one exhibits 5-fold symmetry and has a diameter of 15 nm, the other is rectangular with sides of 15 and 9 nm. Subsequent image processing showed that the 5-fold view possesses mirror symmetry. The rectangular views can be divided into two separate classes, one of which has 2-fold rotational symmetry. It is concluded that methanol dehydrogenase is a decameric molecule, and a tentative model is presented. The estimated molecular weight is 430,000, based on a subunit molecular weight of 43,000. The enzyme contains one zinc and one to two magnesium ions per subunit. N-terminal amino acid sequence analysis revealed substantial similarity with alcohol dehydrogenases from Saccharomyces cerevisiae, Zymomonas mobilis, Clostridium acetobutylicum, and Escherichia coli, which contain iron or zinc but no magnesium. In view of the aberrant structural and kinetic properties, it is proposed to distinguish the enzyme from common alcohol dehydrogenases (EC 1.1.1.1) by using the name NAD-dependent methanol dehydrogenase