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

• Published in: Microbiology (Society for General Microbiology) Vol. 147; no. 2; pp. 431 - 438
• Main Authors: Yu, Jun, Bryant, Alexander P, Marra, Andrea, Lonetto, Michael A, Ingraham, Karen A, Chalker, Alison F, Holmes, David J, Holden, David, Rosenberg, Martin, McDevitt, Damien
• Format: Journal Article
• Language: English
• Published: Reading Soc General Microbiol 01.02.2001; Society for General Microbiology
• Subjects: Alleles; Animals; Bacteriology; Biological and medical sciences; Disease Models, Animal; Fundamental and applied biological sciences. Psychology; Humans; Mice; Microbiology; Multienzyme Complexes - genetics; Multienzyme Complexes - metabolism; Mutation; NADH, NADPH Oxidoreductases - genetics; NADH, NADPH Oxidoreductases - metabolism; Otitis Media - microbiology; Otitis Media - physiopathology; Pathogenicity, virulence, toxins, bacteriocins, pyrogens, host-bacteria relations, miscellaneous strains; Phylogeny; Pneumococcal Infections - microbiology; Pneumococcal Infections - physiopathology; Respiratory Tract Infections - microbiology; Respiratory Tract Infections - physiopathology; Streptococcus pneumoniae - enzymology; Streptococcus pneumoniae - genetics; Streptococcus pneumoniae - growth & development; Streptococcus pneumoniae - pathogenicity; Virulence; Enzyme; Rodentia; Streptococcus pneumoniae; Infection; Pathogenicity; Streptococcaceae; Vertebrata; Experimental disease; Mammalia; Enzymatic activity; Mouse; Streptococcal infection; Bacteriosis; Bacteria; Micrococcales; Attenuation; Oxidoreductases; Mutation; Physicochemical properties
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/00221287-147-2-431

Anti-Infectives Research, SmithKline Beecham Pharmaceuticals Research and Development, 1250 S. Collegeville Road, Collegeville, PA 19426, USA 1 Department of Infectious Diseases, Imperial College School of Medicine, London W12 0NN, UK 2 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 10 5 -fold in the murine respiratory tract infection model and at least 10 4 -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 O 2 to H 2 O. If O 2 is not fully reduced, it can form superoxide anion ( ) and hydrogen peroxide (H 2 O 2 ), 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.