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 pre viously identified as part of a signature-tagged mutagenesis screen of a S. pneumoniae clinical isolate, 0100993. The mutant, with a plasmid insertion disrupting the nor gene, was attenuated for virulence in a murine respirat ory tract infection model. A complete refined nor deletion mutant was gener ated by allelic-replacement mutagenesis and found to be attenuated for viru lence 10(5)-fold in the murine respiratory tract infection model and at lea st 10(4)-fold in a Mongolian gerbil otitis media infection model, confirmin g the importance of the NADH oxidase for both types of S. pneumoniae infect ion. NADH oxidase converts O-2 to H2O, If O-2 is not fully reduced, it can form superoxide anion (O-2(-)) 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, t he mutant displayed normal growth characteristics under conditions of limit ed aeration, The S. pneumoniae nor 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 muM, but was unable to oxidize NADPH, Oxidation of NADH was independ ent of exogenous FAD or FMN.