Characterization of the Streptococcus pneumoniae NADH oxidase that is required for infection.
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 (O2(-)) 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 microM, but was unable to oxidize NADPH. Oxidation of NADH was independent of exogenous FAD or FMN.
Yu
Jun
J
Anti-Infectives Research, SmithKline Beecham Pharmaceuticals Research and Development, 1250 S. Collegeville Road, Collegeville, PA 19426, USA1.
Bryant
Alexander P
AP
Anti-Infectives Research, SmithKline Beecham Pharmaceuticals Research and Development, 1250 S. Collegeville Road, Collegeville, PA 19426, USA1.
Marra
Andrea
A
Anti-Infectives Research, SmithKline Beecham Pharmaceuticals Research and Development, 1250 S. Collegeville Road, Collegeville, PA 19426, USA1.
Lonetto
Michael A
MA
Anti-Infectives Research, SmithKline Beecham Pharmaceuticals Research and Development, 1250 S. Collegeville Road, Collegeville, PA 19426, USA1.
Ingraham
Karen A
KA
Anti-Infectives Research, SmithKline Beecham Pharmaceuticals Research and Development, 1250 S. Collegeville Road, Collegeville, PA 19426, USA1.
Chalker
Alison F
AF
Anti-Infectives Research, SmithKline Beecham Pharmaceuticals Research and Development, 1250 S. Collegeville Road, Collegeville, PA 19426, USA1.
Holmes
David J
DJ
Anti-Infectives Research, SmithKline Beecham Pharmaceuticals Research and Development, 1250 S. Collegeville Road, Collegeville, PA 19426, USA1.
Holden
David
D
Department of Infectious Diseases, Imperial College School of Medicine, London W12 0NN, UK2.
Rosenberg
Martin
M
Anti-Infectives Research, SmithKline Beecham Pharmaceuticals Research and Development, 1250 S. Collegeville Road, Collegeville, PA 19426, USA1.
McDevitt
Damien
D
Anti-Infectives Research, SmithKline Beecham Pharmaceuticals Research and Development, 1250 S. Collegeville Road, Collegeville, PA 19426, USA1.
D000483:Alleles; D000818:Animals; D004195:Disease Models, Animal; D006801:Humans; D051379:Mice; D009097:Multienzyme Complexes / Q000235:genetics* / Q000378:metabolism*; D009154:Mutation; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics* / Q000378:metabolism*; D010033:Otitis Media / Q000382:microbiology / Q000503:physiopathology; D010802:Phylogeny; D011008:Pneumococcal Infections / Q000382:microbiology / Q000503:physiopathology*; D012141:Respiratory Tract Infections / Q000382:microbiology / Q000503:physiopathology; D013296:Streptococcus pneumoniae / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development / Q000472:pathogenicity*; D014774:Virulence
D009097:Multienzyme Complexes; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases