title: Biological and Chemical Adaptation to Endogenous Hydrogen Peroxide Production inStreptococcus pneumoniaeD39.
abstract: The catalase-negative, facultative anaerobeStreptococcus pneumoniaeD39 is naturally resistant to hydrogen peroxide (H2O2) produced endogenously by pyruvate oxidase (SpxB). Here, we investigate the adaptive response to endogenously produced H2O2. We show that lactate oxidase, which converts lactate to pyruvate, positively impacts pyruvate flux through SpxB and thatΔlctOmutants produce significantly lower H2O2. In addition, both the SpxB pathway and a candidate pyruvate dehydrogenase complex (PDHC) pathway contribute to acetyl coenzyme A (acetyl-CoA) production during aerobic growth, and the pyruvate format lyase (PFL) pathway is the major acetyl-CoA pathway during anaerobic growth. Microarray analysis of the D39 strain cultured under aerobic versus strict anaerobic conditions shows upregulation ofspxB, a gene encoding a rhodanese-like protein (locus tagspd0091),tpxD,sodA,piuB,piuD, and an Fe-S protein biogenesis operon under H2O2-producing conditions. Proteome profiling of H2O2-induced sulfenylation reveals that sulfenylation levels correlate with cellular H2O2production, with endogenous sulfenylation of ≈50 proteins. Deletionof tpxDincreases cellular sulfenylation 5-fold and has an inhibitory effect on ATP generation. Two major targets of protein sulfenylation are glyceraldehyde-3-phosphate dehydrogenase (GapA) and SpxB itself, but targets also include pyruvate kinase, LctO, AdhE, and acetate kinase (AckA). Sulfenylation of GapA is inhibitory, while the effect on SpxB activity is negligible. Strikingly, four enzymes of capsular polysaccharide biosynthesis are sulfenylated, as are enzymes associated with nucleotide biosynthesis via ribulose-5-phosphate. We propose that LctO/SpxB-generated H2O2functions as a signaling molecule to downregulate capsule production and drive altered flux through sugar utilization pathways.IMPORTANCEAdaptation to endogenous oxidative stress is an integral aspect ofStreptococcus pneumoniaecolonization and virulence. In this work, we identify key transcriptomic and proteomic features of the pneumococcal endogenous oxidative stress response. The thiol peroxidase TpxD plays a critical role in adaptation to endogenous H2O2and serves to limit protein sulfenylation of glycolytic, capsule, and nucleotide biosynthesis enzymes inS. pneumoniae.
authors.affiliation: Department of Chemistry, Indiana University, Bloomington, Indiana, USA; Graduate Program in Biochemistry, Indiana University, Bloomington, Indiana, USA.
authors.forename: John P
authors.initials: JP
authors.lastname: Lisher
authors.affiliation: Department of Biology, Indiana University, Bloomington, Indiana, USA.
authors.forename: Ho-Ching Tiffany
authors.initials: HT
authors.lastname: Tsui
authors.affiliation: Department of Biology, Indiana University, Bloomington, Indiana, USA.
authors.forename: Smirla
authors.initials: S
authors.lastname: Ramos-Montañez
authors.affiliation: Department of Biology, Indiana University, Bloomington, Indiana, USA.
authors.forename: Kristy L
authors.initials: KL
authors.lastname: Hentchel
authors.affiliation: Department of Chemistry, Indiana University, Bloomington, Indiana, USA.
authors.forename: Julia E
authors.initials: JE
authors.lastname: Martin
authors.affiliation: Department of Chemistry, Indiana University, Bloomington, Indiana, USA; Department of Chemistry, Laboratory for Biological Mass Spectrometry, Indiana University, Bloomington, Indiana, USA.
authors.forename: Jonathan C
authors.initials: JC
authors.lastname: Trinidad
authors.affiliation: Department of Biology, Indiana University, Bloomington, Indiana, USA; Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana, USA.
authors.forename: Malcolm E
authors.initials: ME
authors.lastname: Winkler
authors.affiliation: Department of Chemistry, Indiana University, Bloomington, Indiana, USA; Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana, USA.
authors.forename: David P
authors.initials: DP
authors.lastname: Giedroc
keywords: Streptococcus pneumoniae; hydrogen peroxide stress; pyruvate oxidase; sulfenylation