1887

Abstract

is an important human pathogen capable of causing serious infections. NADH oxidase, a factor necessary for infection, was previously identified as part of a signature-tagged mutagenesis screen of a clinical isolate, 0100993. The mutant, with a plasmid insertion disrupting the gene, was attenuated for virulence in a murine respiratory tract infection model. A complete refined deletion mutant was generated by allelic-replacement mutagenesis and found to be attenuated for virulence 10-fold in the murine respiratory tract infection model and at least 10-fold in a Mongolian gerbil otitis media infection model, confirming the importance of the NADH oxidase for both types of infection. NADH oxidase converts O to HO. If O is not fully reduced, it can form superoxide anion () and hydrogen peroxide (HO), both of which can be toxic to cells. Bacterial cell extracts from the allelic-replacement mutant were found to lack NADH oxidase activity and the mutant was unable to grow exponentially under conditions of vigorous aeration. In contrast, the mutant displayed normal growth characteristics under conditions of limited aeration. The gene was cloned and expressed in . The purified His-tagged NADH oxidase was shown to oxidize NADH with a of 32 μM, but was unable to oxidize NADPH. Oxidation of NADH was independent of exogenous FAD or FMN.

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2001-02-01
2020-05-29
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