Lactate dehydrogenase is the key enzyme for pneumococcal pyruvate metabolism and pneumococcal survival in blood

Abstract

Streptococcus pneumoniae is a fermentative microorganism and causes serious diseases in humans, including otitis media, bacteremia, meningitis, and pneumonia. However, the mechanisms enabling pneumococcal survival in the host and causing disease in different tissues are incompletely understood. The available evidence indicates a strong link between the central metabolism and pneumococcal virulence. To further our knowledge on pneumococcal virulence, we investigated the role of lactate dehydrogenase (LDH), which converts pyruvate to lactate and is an essential enzyme for redox balance, in the pneumococcal central metabolism and virulence using an isogenic ldh mutant. Loss of LDH led to a dramatic reduction of the growth rate, pinpointing the key role of this enzyme in fermentative metabolism. The pattern of end products was altered, and lactate production was totally blocked. The fermentation profile was confirmed by in vivo nuclear magnetic resonance (NMR) measurements of glucose metabolism in nongrowing cell suspensions of the ldh mutant. In this strain, a bottleneck in the fermentative steps is evident from the accumulation of pyruvate, revealing LDH as the most efficient enzyme in pyruvate conversion. An increase in ethanol production was also observed, indicating that in the absence of LDH the redox balance is maintained through alcohol dehydrogenase activity. We also found that the absence of LDH renders the pneumococci avirulent after intravenous infection and leads to a significant reduction in virulence in a model of pneumonia that develops after intranasal infection, likely due to a decrease in energy generation and virulence gene expression.

FIG 1

Schematic representation of sugar metabolism in S. pneumoniae. Sugars are internalized and phosphorylated, and the resulting sugar-phosphates are converted through the Embden-Meyerhof-Parnas pathway to pyruvate. This metabolite is further converted to fermentation end products. Abbreviations: ldh, lactate dehydrogenase; pfl, pyruvate formate lyase; spxB, pyruvate oxidase; adh, alcohol dehydrogenase; ack, acetate kinase; pta, phosphotransacetylase; G6P, glucose 6-phosphate; FBP, fructose 1,6-bisphosphate; DHAP, dihydroxyacetone phosphate; GAP, glyceraldehyde 3-phosphate; 3-PGA, 3-phosphoglycerate; PEP, phosphoenolpyruvate.

FIG 2

Analysis of transformants (A) and schematic representation of the genomic region containing putative ldh (SPD_1078) (B). (A) The presence of the spectinomycin cassette is analyzed in transformants 1 to 7 using the primers SPD1078F and MP127, which has recognition sites in the spectinomycin resistance cassette. D39 is used as a negative control (lane 8). Lane M shows 1-kb DNA markers (New England Biolab), and the size of each band is given in base pairs. (B) The chromosome is represented with a thin solid line, and genes are shown with a block arrow. The genomic locations of SPD1078F and SPD1078R primers, which amplified the genetic locus for mutation, are shown, and the position of the first nucleotide recognized by each primer and the coordinates of ldh have been indicated. The vertical arrows show the positions of mutations in transformants SPD1078-3 and SPD1078-4, and the chevrons represent the orientation of the spectinomycin cassette. The diagram is not drawn to scale.

FIG 3

Growth profiles and end products from glucose and galactose metabolism. D39 and its LDH-deficient mutant were grown in CDM supplemented with 1% glucose (A) or galactose (B) under anaerobic or aerobic conditions at 37°C and initial pH 6.5. Concentrations of substrate in the medium and fermentation end products after growth arrest of strains D39 (◆), SPD1078M (●), and SPD1078comp (▲) on glucose (C) or galactose (D).

FIG 4

Real-time measurements of metabolites during the metabolism of glucose in S. pneumoniae. Kinetics of [1-¹³C]glucose (20 mM) consumption, end product formation, and intracellular metabolites in strain R6, an avirulent derivative of D39 (A) and its ldh mutant SPR1078M (B) at 37°C under anaerobic conditions and pH 6.5. Symbols: ◆, glucose; ▲, FBP; ◼, 3-PGA; –, lactate; ×, acetate; ○, ethanol; △, pyruvate. GCR, glucose consumption rate (μmol min⁻¹ mg⁻¹ of protein).

FIG 5

Impaired virulence of pneumococcal strains defective in galactose catabolic pathways following intranasal infection. (A) Survival time of mice after infection with approximately 1 × 10⁶ CFU pneumococci. Symbols show the times at which mice became severely lethargic. The horizontal bars mark the median times to the severely lethargic state. (B) Growth of bacteria in the blood is shown for D39 (●), SPD1078M (●), and SPD1078comp (◆). Each point is the mean of data from 10 mice. Error bars show the standard errors of the means. *, P < 0.05; **, P < 0.01; ****, P < 0.0001 relative to D39.

FIG 6

(A) Survival of mice after intravenous infection with pneumococcal strains. Each point represents one animal. The median survival time is given with a horizontal bar. ****, P < 0.0001 relative to D39. (B) Numbers of D39 (●), SPD1078M (●), and SPD1078comp (◆) in the blood after intravenous infection. Each point is the mean of data from 10 mice. Error bars show the standard errors of the means.