Pyruvate oxidase influences the sugar utilization pattern and capsule production in Streptococcus pneumoniae

Abstract

Pyruvate oxidase is a key function in the metabolism and lifestyle of many lactic acid bacteria and its activity depends on the presence of environmental oxygen. In Streptococcus pneumoniae the protein has been suggested to play a major role in metabolism and has been implicated in virulence, oxidative stress survival and death in stationary phase. Under semi-aerobic conditions, transcriptomic and metabolite profiling analysis of a spxB mutant grown on glucose showed minor changes compared to the wild type, apart from the significant induction of two operons involved in carbohydrate uptake and processing. This induction leads to a change in the sugar utilization capabilities of the bacterium, as indicated by the analysis of the growth profiles of the D39 parent and spxB mutant on alternative carbohydrates. Metabolic analysis and growth experiments showed that inactivation of SpxB has no effect on the glucose fermentation pattern, except under aerobic conditions. More importantly, we show that mutation of spxB results in the production of increased amounts of capsule, the major virulence factor of S. pneumoniae. Part of this increase can be attributed to induction of capsule operon (cps) transcription. Therefore, we propose that S. pneumoniae utilizes pyruvate oxidase as an indirect sensor of the oxygenation of the environment, resulting in the adaption of its nutritional capability and the amount of capsule to survive in the host.

Figure 1. Inactivation of spxB led to an increase in capsule production.

(A) Phenotype on Glc-M17 agar plate of D39 (D39), D39 containing the pGh9 T7 plasmid (D39pGh9 T7) and D39 in which the plasmid is excised leaving the ISS1 element in spxB (D39spxB). (B) Phenotype on Glc-M17 agar plate of TIGR4 transparent variant (T4 transparent), TIGR4 opaque variant (T4 opaque) and TIGR4 in which the pGh9 T7 plasmid is inserted into the genome (T4 pGh9 T7). (C–F) TEM pictures of S. pneumoniae grown in broth to exponential phase and stained with LRR. (C) D39, 9700 times magnified; (D) D39spxB 9700 times magnified; (E) D39, 135.000 times magnified; (F) D39spxB 135.000 times magnified.

Figure 2. Effect of spxB mutation on capsule production.

Estimation of capsule was performed based on the determination of its glucuronic acid content in strains D39 (white bars), D39spxB (dark grey bars) and D39spxB ⁺ (complemented strain) (light grey bars) in late-exponential (A) and early-stationary (B) phases of growth. Cultures were grown in CDM containing 1% (wt/vol) Glc at 37^°C, with pH control (6.5), and under semi-aerobic conditions (for details see Materials & Methods). Determinations were done twice in three independent cultures and the values are means ± SD. * = P≤0.01 students t-test. For D39spxB ⁺, the capsule was determined twice for each of two independent cultures.

Figure 3. Effect of spxB mutation and addition of catalase on cps transcription.

Transcription of the cps promoter was estimated by measuring the β-galactosidase activities of strains D39 (bars with white background), D39spxB (bars with grey background) and D39spxB and its parent in the presence of 200 U/ml bovine liver catalase (spotted bars) harbouring pORI Pcps (see Table 1) in mid-exponential (A, left panel and B) and late-exponential (A, right panel) phases of growth. Cultures were grown in CDM containing 1% (wt/vol) Glc (A) or in BHI (B) at 37^°C, and under semi-aerobic conditions. All the determinations were done at least in triplicate and the values are means ± SD. a.u. = arbitrary units.

Figure 4. Growth profiles of strains D39, D39spxB and D39spxB ⁺ under semi-aerobic conditions.

Cultures were grown in A) M17 without pH control (initial pH 6.5) or B) CDM with pH control (pH 6.5), with 1% (wt/vol) Glc at 37^°C, in both cases under semi-aerobic conditions (for details see Materials and Methods). The plotted growth curves are from a representative experiment. For each condition at least three independent experiments were performed, except for the complemented strain (D39spxB ⁺) which was performed twice, and the error was below 15%. Symbols: (triangles), D39spxB; (squares), D39; (diamonds), D39spxB ⁺. Arrows in the CDM grown cultures indicate sampling time for capsule determination and intracellular metabolite analysis (Figures 2 and 5).

Figure 5. Effect of spxB mutation on intracellular phosphorylated metabolites during growth in Glc-CDM under semi-aerobic conditions.

Phosphorylated metabolites were measured by ³¹P-NMR in ethanol extracts of D39 and D39spxB cells grown to late-exponential (LExp) (A, B) and early-stationary (EStat) (C, D) phases of growth in CDM supplemented with 1% (wt/vol) Glc as in Figure 4B. The results are averages of three to four independent growths and the average accuracy was ± 15%. Symbols: (dark grey bars), D39, (light grey bars), D39spxB. FBP, fructose 1,6-bisphosphate; G6P, glucose 6-phosphate; PEP, phosphoenolpyruvate; α-G1P, α-glucose 1-phosphate; Ac–P, acetyl-phosphate; CDP-Cho, CDP-choline; UDP-GlcUA, UDP-glucuronate; UDP-Glc, UDP-glucose; UDP-GlcNAc, UDP-N-acetylglucosamine; UDP-MurNAcp-Pep, UDP-N-acetylmuramoyl-pentapeptide; UDP-GlcN, UDP-glucosamine. LExp, late-exponential phase of growth; EStat, early-stationary phase of growth; A and C, phosphomonoesters; B and D, diphosphodiesters; (*), quantification was impaired by overlapping resonances in NMR spectra.

Figure 6. Model of the spxB deletion effect on glucose metabolism and capsule production in S. pneumoniae.

Glucose is converted by the conventional glycolytic pathway to pyruvate. Fermentation products result from the action of different competing enzymes (lactate dehydrogenase, pyruvate formate-lyase, pyruvate oxidase and hypothetically the pyruvate dehydrogenase complex). Overall, our data show that SpxB represses carbohydrate specific pathways (ketogluconate and hyaluronic acid utilization), capsule production, and stimulates formation of acetyl-P and acetate at the expense of pyruvate and lactate. Pyruvate and end-products are represented by three-dimensional bars and the height of the bar represents the relative amount; grey slashed arrows, function not experimentally verified; boxed metabolites indicate higher metabolite accumulation. Thick pointed-line, repression; Thick black arrow, activation. KDG, ketogluconate; HA, hyaluronic acid; GlcNAc, N-acetylglucosamine, GlcUA, glucuronic acid; Ac–P, acetyl-phosphate; α-G1P, α-glucose 1-phosphate; G6P, glucose 6-phosphate; FBP, fructose 1,6-bisphosphate; PEP, phosphoenolpyruvate.

Figure 7. Effect of spxB mutation on the ability to grow on specified sugars.

Growth profiles of S. pneumoniae D39 (A) and its derivative D39spxB (B) in CDM containing 0.25% (wt/vol) of the specified sugar. Cultures were prepared in 250 µl in 96-well microtiter plates and growth monitored at 595 nm and 37^°C. Symbols: (black diamonds), Glc; (grey diamonds), GlcUA; (grey triangles), Rha; (grey circles), GlcA; (black squares), GlcNAc; (white triangles) 1 GlcUA : 2 Glc: 3 Rha. Zoomed areas: expansions of time-points 5 to 20 h for growths in rhamnose, gluconic acid and glucuronic acid. Each point of the growth curves is an average of triplicate experiments each consisting of two independent cultures, and the error was in all cases below 20%, except for rhamnose which was below 30%.

Figure 8. Growth and fermentation profiles in S. pneumoniae D39spxB and its parent strain D39 under aerobic conditions.

Growth curves, substrate consumption and end-products formed by D39 (A) and D39spxB (B) strains growing on Glc-CDM, at 37^°C, with pH control (6.5), and under aerobic conditions (for details see supplemental Materials and Methods S1). Culture supernatant samples for substrate and end-product analysis by HPLC and/or ¹H-NMR were harvested at denominated time-points along growth (bars in the plots). The plotted growth curves, substrate consumption curves and end-products bars are from a representative experiment. For each condition at least two independent experiments were performed. For the growth and substrate consumption curves the error in each point was below 10%. For the end-products concentrations, the error was below 7% for major products (> 3.5 mM) and 30% for minor products (< 3.5 mM) Symbols: (white circles), substrate consumption; (black squares), growth curve; white bars, lactate; dark grey bars, pyruvate; light grey bars, acetate; stripped bars, hydrogen peroxide. The data on D39 is also presented in [67].