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Characterization of the Streptococcus pneumoniae NADH oxidase that is required for infection

Anti-Infectives Research, SmithKline Beecham Pharmaceuticals Research and Development, 1250 S. Collegeville Road, Collegeville, PA 19426, USA¹
Department of Infectious Diseases, Imperial College School of Medicine, London W12 0NN, UK²

Author for correspondence: Jun Yu. Tel: +1 510 291 6220. Fax: +1 510 291 6196. e-mail: jyu{at}xenogen.com

Streptococcus pneumoniae 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 S. pneumoniae clinical isolate, 0100993. The mutant, with a plasmid insertion disrupting the nox gene, was attenuated for virulence in a murine respiratory tract infection model. A complete refined nox deletion mutant was generated by allelic-replacement mutagenesis and found to be attenuated for virulence 10⁵-fold in the murine respiratory tract infection model and at least 10⁴-fold in a Mongolian gerbil otitis media infection model, confirming the importance of the NADH oxidase for both types of S. pneumoniae infection. NADH oxidase converts O₂ to H₂O. If O₂ is not fully reduced, it can form superoxide anion () and hydrogen peroxide (H₂O₂), 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 S. pneumoniae nox gene was cloned and expressed in E. coli. The purified His-tagged NADH oxidase was shown to oxidize NADH with a K_m of 32 µM, but was unable to oxidize NADPH. Oxidation of NADH was independent of exogenous FAD or FMN.

Keywords: nox gene, reactive oxygen species, virulence

Abbreviations: STM, signature-tagged mutagenesis

^a Present address: Xenogen Corporation, 860 Atlantic Avenue, Alameda, CA 94501, USA.

^b Present address: Protein Design Labs, 34801 Campus Drive, Fremont, CA 94555, USA.

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