Pyruvate Oxidase as a Critical Link between Metabolism and Capsule Biosynthesis in Streptococcus pneumoniae

Abstract

The pneumococcus is one of the most prodigious producers of hydrogen peroxide amongst bacterial pathogens. Hydrogen peroxide production by the pneumococcus has been implicated in antibiotic synergism, competition between other bacterial colonizers of the nasopharynx, and damage to epithelial cells. However, the role during invasive disease has been less clear with mutants defective in hydrogen peroxide production demonstrating both attenuation and heightened invasive disease capacity depending upon strain and serotype background. This work resolves these conflicting observations by demonstrating that the main hydrogen peroxide producing enzyme of the pneumococcus, SpxB, is required for capsule formation in a strain dependent manner. Capsule production by strains harboring capsules with acetylated sugars was dependent upon the presence of spxB while capsule production in serotypes lacking such linkages were not. The spxB mutant had significantly lower steady-state cellular levels of acetyl-CoA, suggesting that loss of capsule arises from dysregulation of this intermediary metabolite. This conclusion is corroborated by deletion of pdhC, which also resulted in lower steady-state acetyl-CoA levels and phenocopied the capsule expression profile of the spxB mutant. Capsule and acetyl-CoA levels were restored in the spxB and lctO (lactate oxidase) double mutant, supporting the connection between central metabolism and capsule formation. Taken together, these data show that the defect in pathogenesis in the spxB mutant is due to a metabolic imbalance that attenuates capsule formation and not to reduced hydrogen peroxide formation.

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

“…In this study, we provide genetic evidence that the pyruvate dehydrogenase complex (PDHC) is a functional pathway for the production of acetyl-CoA under aerobic conditions, consistent with previous findings in the TIGR4 strain (41), and that pyruvate formate lyase (PFL) is the major pathway for acetyl-CoA synthesis under anaerobic conditions. Note that acetate kinase (AckA) and phosphate acetyltransferase (Pta) are reversible enzymes and are represented here with double-headed arrows (8).…”

“…In strong support of this assignment, deletion of S. pneumoniae TIGR4 genes sp1163 and sp1164 (corresponding to spd1127 and spd1128 in the D39 strain) results in significantly reduced (≈50%) acetyl-CoA levels (Fig. 1) (41). We show here that spd1025 to spd1028 are absolutely essential for growth in a Δ spxB strain.…”

“…Transformation of a ΔPDHC (Δ spd1025 – spd1028 ) amplicon into Δ spxB strains or a Δ spxB amplicon into ΔPDHC strains yielded no colonies, while transformation of Δ spxB or ΔPDHC amplicons into wild-type strains and transformation of positive control Δ pbp1b amplicons into ΔPDHC or Δ spxB strains yielded many colonies. These results suggest that under aerobic conditions, either the SpxB or PDHC pathway must be present to produce acetyl-CoA, either directly from pyruvate by this candidate pyruvate dehydrogenase complex (41) or from Ac~P by the phosphotransacetylase (Pta) (Fig. 1).…”

Cell Invasion and Pyruvate Oxidase-Derived H 2 O 2 Are Critical for Streptococcus pneumoniae-Mediated Cardiomyocyte Killing

“…We speculate that these mutations somehow impacted the bacteria at a structural level, modulating the expression of or destabilizing the capsular polysaccharide, thereby allowing stronger host cell interactions due to the unmasking of what is yet the unidentified surface determinant(s) (28). Supporting this possibility, capsule levels varied in our ΔcbpA and ΔlicA mutants (unpublished data) and have been previously described to be altered in ΔspxB backgrounds (29,30), and the localization of pneumolysin in the cell wall (31) may also play a structural role important for the interaction of the bacteria with the cell. These altered interactions may have been missed by other investigators due to the use of nonencapsulated pneumococci in most of the adhesion/invasion studies described in the literature.…”

“…In fact, the corresponding gene has been shown to be important for colonization (36), competence (37), and capsule production (29). Its deletion, therefore, can also attenuate the bacterium in a manner that is substantially greater than inhibition of H 2 O 2 production.…”