The Group B Streptococcus NADH oxidase Nox‐2 is involved in fatty acid biosynthesis during aerobic growth and contributes to virulence

Abstract

SummaryNumerous Streptococcaceae produce an H2O-forming NADH oxidase, Nox-2, which has been generally implicated in aerobic survival. We examined the roles of Nox-2 in Group B Streptococcus (GBS), a leading agent of neonatal infections. While nox2 inactivation caused an aerobic growth arrest, no improvement was seen by addition of antioxidants to cultures, suggesting that this defect was not due to accumulation of toxic oxygen species. Using several approaches, we show that the observed inability of the nox2 mutant to grow aerobically is mainly due to an underlying defect in fatty acid (FA) biosynthesis: (i) the nox2 aerobic growth defect is fully and rapidly complemented by adding oleic acid to culture medium, and (ii) direct assimilation of this unsaturated FA in both wild type (WT) and nox2 GBS membranes is demonstrated and correlated with mutant growth rescue. We propose that NAD + depletion in the nox2 mutant results in reduced acetyl-CoA production, which perturbs FA biosynthesis and hence blocks growth in aerobiosis. The nox2 aerobic growth defect was also complemented when GBS respiration metabolism was activated by exogenous haem and menaquinone. The membrane NADH oxidase activity generated by the functional respiratory chain thus compensates the cytoplasmic NADH oxidase deficiency. The nox2 mutant was attenuated for virulence, as assessed in lung, intraperitoneal and intravenous murine infection models. As the nox2 defect seems only to affect aerobic growth of GBS, its reduced virulence supports the suggestion that aerobic conditions and NADH oxidase activities are relevant to the GBS infection process.

Identification of a Conserved Sequence in Flavoproteins Essential for the Correct Conformation and Activity of the NADH Oxidase NoxE of Lactococcus lactis

“…In pathogenic streptococci that encounter oxygen upon host colonization, NADH oxidases contribute to oxygen tolerance by decreasing the intracellular NADH/NAD ratio and/or by direct elimination of oxygen (2,10,14,37,38). In L. lactis, which is particularly exposed to oxygen when it is used for milk fermentation in the dairy industry, the role of NADH oxidases in oxygen tolerance has not been investigated.…”

“…Their inactivation in several streptococci strongly reduces the aerobic growth. Inactivation of nox2 in Streptococcus pneumoniae and Streptococcus agalactiae reduces growth in aerated media by 80% (37,38). The growth defect of the S. agalactiae mutant has been attributed to a defect in fatty acid production resulting from nonproduction of the fatty acid precursor acetyl-coenzyme A (CoA) due to strictly homolactic fermentation (37).…”

“…Inactivation of nox2 in Streptococcus pneumoniae and Streptococcus agalactiae reduces growth in aerated media by 80% (37,38). The growth defect of the S. agalactiae mutant has been attributed to a defect in fatty acid production resulting from nonproduction of the fatty acid precursor acetyl-coenzyme A (CoA) due to strictly homolactic fermentation (37). Nox2 inactivation in Streptococcus mutans also leads to strict homolactic fermentation on glucose; however, it does not affect growth on glucose but greatly reduces growth on mannitol (11).…”

Stress Physiology of Lactic Acid Bacteria

“…If not fully reduced by Nox, environmental oxygen can form toxic superoxide and H 2 O 2 . Strains of GBS and pneumococcus lacking Nox were unable to grow under vigorous agitation conditions and showed attenuated virulence in animal models (514,515). An E. faecium Tn917 transposon insertion mutant of nox showed a 98% reduction of NADH oxidase activity (516).…”

Streptococcus mutans NADH Oxidase Lies at the Intersection of Overlapping Regulons Controlled by Oxygen and NAD + Levels

“…Our laboratory has previously shown that Nox is the major oxygen-metabolizing enzyme used by S. mutans, responsible for reduction of approximately 40% of the dissolved oxygen encountered in dental plaque, and that the Nox enzyme shows elevated activity under conditions of both oxidative stress and low pH (21). Mutation of nox in the genomic type strain, S. mutans UA159, has demonstrated that NADH oxidase plays a role in the acid stress response, as inactivation of nox invokes changes in membrane fatty acid composition and metabolic output reminiscent of those seen during exposure to low pH (21,(23)(24)(25). In addition to reducing oxygen, Nox is also critical in the regeneration of NAD ϩ , a key molecule in the production of pyruvate during carbon metabolism in streptococci (26)(27)(28)(29).…”