Biological and Chemical Adaptation to Endogenous Hydrogen Peroxide Production in Streptococcus pneumoniae D39

ABSTRACT The catalase-negative, facultative anaerobe Streptococcus pneumoniae D39 is naturally resistant to hydrogen peroxide (H 2 O 2 ) produced endogenously by pyruvate oxidase (SpxB). Here, we investigate the adaptive response to endogenously produced H 2 O 2 . We show that lactate oxidase, which converts lactate to pyruvate, positively impacts pyruvate flux through SpxB and that ΔlctO mutants produce significantly lower H 2 O 2 . 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 of spxB , a gene encoding a rhodanese-like protein (locus tag spd0091 ), tpxD , sodA , piuB , piuD , and an Fe-S protein biogenesis operon under H 2 O 2 -producing conditions. Proteome profiling of H 2 O 2 -induced sulfenylation reveals that sulfenylation levels correlate with cellular H 2 O 2 production, with endogenous sulfenylation of ≈50 proteins. Deletion of tpxD increases 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 H 2 O 2 functions as a signaling molecule to downregulate capsule production and drive altered flux through sugar utilization pathways. IMPORTANCE Adaptation to endogenous oxidative stress is an integral aspect of Streptococcus pneumoniae colonization 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 H 2 O 2 and serves to limit protein sulfenylation of glycolytic, capsule, and nucleotide biosynthesis enzymes in S. pneumoniae .