Fig 1.
S. pyogenes Central Carbon Metabolism and Mutant Construction.
A. Fermentation pathways. Homo-lactic and mixed-acid fermentation pathways are shown. Major short chain fatty acid end-products are shown in red, other end-products are shown in blue and the enzymes acting on pyruvate are shown in green. Other enzymes listed: ADH, alcohol dehydrogenase; PTA, phosphate acetyltransferase; AckA, acetate kinase. B. Enzyme loci. The chromosomal regions encoding the indicated enzymes are shown, with the annotation and genetic loci listed corresponding the genome of S. pyogenes HSC5 [27]. Enzyme open-reading frames are shown in blue with the location of the constructed in-frame deletions shown in white. Deletion junctions are indicated by “x” and “y” relative to the first base-pair of the open-reading frame. C. Mutant strain nomenclature. The indicated nomenclature was adopted for clarity. Each cell shown represents a mutant strain. For single mutants, the name corresponds to the genotype. For double mutants, the name corresponds to the enzyme that remains active. Mutant genotypes are in parentheses. Specific strain names and genotypes are provided in S1 Table.
Fig 2.
Growth characteristics under ideal conditions.
Shown is the ability of the various mutants listed at the top of the Figure to grow in a standard nutrient rich medium (THY medium) cultured under oxygenated (Shaken, aerobic) or oxygen-limited (Static, anaerobic) conditions, as listed. The top panel (Broth Grown) presents images of culture tubes follow overnight culture in liquid THY medium. The degree of turbidity corresponds to the amount of growth, which was quantitated by spot plating on solid media (THY plates) as shown in the bottom panel (Plated) under the atmospheric conditions indicated. The spot plate data presented are a montage assembled from individual images collected from the same experiment, that are delineated by the boxes numbered at the bottom of the Figure. Culture conditions are described in detail in the Materials and Methods. Strains are as described in Fig 1.
Fig 3.
Growth characteristics under conditions reproducing the soft tissue environment.
Comparison of the growth and changes in culture pH for the mutant panel was analyzed in a medium that reproduces conditions encountered by the bacterium during soft tissue infection (C medium + 0.1% glucose) under (A) oxygen-limited (Static) and (B) oxygenated (Shaken) conditions, with (C) the concentrations of the 3 major SCFA end-products determined following oxygen-limited growth. An exception is Only Pdh, whose SCFA profile was determined following culture in Shaken conditions since this mutant cannot grow under oxygen limited conditions. N.D. indicates levels were below the limit of detection. Data presented represents the mean and standard error of the mean derived from three experiments.
Fig 4.
Non-overlapping roles for mixed acid fermentation in soft-tissue infection.
A subcutaneous ulcer model of soft-tissue infection of SKH1-E mice was used to compare virulence properties between mutant and WT, which allows the determination of (A) tissue damage, measured as the area of the draining ulcer and (B) growth in tissue, measured as the number of recoverable CFUs. Data presented were collected at Day 3, with (C) showing images from representative Day 3 ulcers. For A and B, each symbol represents an individual mouse, pooled from two individual experiments, with differences compared to WT. ***, P < 0.05; ****, P < 0.001; n.s., not significant. N.D. indicates a visible ulcer was not detected.
Table 1.
Growth/Damage Phenotypes.
Fig 5.
Comparison of macrophage infection by WT and ΔPdh.
Cultured Raw264.7 macrophages were infected by the indicated strains as detailed in the Materials and Methods. A. Net change in CFUs recovered between time 0 and 4 hrs of infection. B. Transmission electron microscopy of infected Raw264.7 cells (top), with immunogold staining to detect Lamp1 (middle) Arrows indicate several representative gold particles. The bottom panel shows the regions outlined by the boxed areas in the middle panels at 7.5X magnification. mb, vacuolar membrane. C. Fluorescent microscopy of infected Raw264.7 cells to detect DNA (DAPI), S. pyogenes (Cell Tracker Green) and Lysosomes (LysoTracker Red). Merge overlays all three stains. Images of the 4 panels at the right are magnified from the area indicated by the boxes shown in the merged images at the left. D. Quantitation of the number of bacteria which co-localized with lysosomes. The top and bottom of each box delineates the 75th and 25th percentiles, respectively; the horizontal bar indicates the median. E. Net change in recoverable CFUs between untreated Raw264.7 cells, and cells treated with Bafilomycin A1 (Baf A1). Where indicated, data presented represents the mean and standard error of the mean derived from at least 3 independent experiments and examination of at least 1,000 cells. **, P < 0.01; ***, P < 0.05; ****, P < 0.001.
Fig 6.
Differential expression of IL-10 between WT- and ΔPdh-infected macrophages.
Cultured Raw264.7 cells were infected by WT, ΔPdh or PBS (mock) for 4 hrs. Overall differences in gene expression are shown in Volcano plots of statistically significantly differentially expressed genes for the indicated comparisons (A, B, C). Genes regulated up or down regulated with a log2 fold-change > 1.0 are shown in red and blue, respectively. Supernatants from cells infected and untreated or treated with Bafilomycin A1 (Baf A1) were analyzed to determine concentrations of IL-10 (D) and TNFα (E) and the IL-10/TNFα ratio determined for untreated infected cells (F). Subcutaneous infection of the indicated strains in IL-10+/+ and IL-10-/- C57BL/6 mice is shown, comparing Growth (G), Damage (H) and the Growth/Damage ratio calculated for ΔPdh vs WT (I). For G and H, each symbol represents an individual mouse, pooled from two individual experiments, with differences compared to WT. Where indicated, *, P < 0.05; **, P < 0.05; ****, P < 0.001; n.s., not significant.
Fig 7.
Model for PDH regulation of growth/damage balance.
Shown is a comparison of WT and ΔPdh mutant phenotypes observed in macrophages in vitro and for murine subcutaneous infection. The steps of infection in macrophages are shown in temporal order from left to right by the closed arrows and are labeled as indicated (featuring: L, lysosome; grey oval, nucleus), resulting in the ratio of the cytokines IL-10 and TNFα represented by frequency of the symbols indicated, which correlates (as shown by the open arrows) to the mouse infection phenotypes shown at the right of the Figure. Relative areas of the resulting lesions are represented as small or large in mice whose IL-10 genotypes are noted. Growth/Damage balance (G/D Balance) for IL-10+/+ or IL-10-/- mice are shown by the paired sliders at the very right of the Figure, with the relative degree of Growth (G, green) and Damage (D, red) indicated by a box on a scale from Low to High, as shown.