Fig 1.
S. aureus induces a metal-independent phosphoglycerate mutase in response to calprotectin.
(A) The chromosomal location of gpmI and gpmA is depicted. gpmI is part of the glycolytic operon, while gpmA is not part of an operon and is in a separate location. Not to scale. (B) Wild type S. aureus was grown in rich medium in the presence and absence of 240 μg/ml of CP and transcript levels of gpmA and gpmI were assessed by qRT-PCR. Expression was compared to wild type bacteria grown in the absence of CP. * = p≤0.05 relative to media alone by two-way ANOVA on log-transformed values with Tukey’s multiple comparisons test. (C-D) Wild type S. aureus and ΔmntCΔmntH were grown in rich medium in the presence and absence of 240 μg/ml of CP and transcript levels of gpmA (C) and gpmI (D) were assessed by qRT-PCR. Expression was compared to wild type bacteria grown in the absence of CP. * = p≤0.05 relative to media alone by one-way ANOVA on log-transformed values with Tukey’s multiple comparisons test. Error bars indicate SEM. n≥3.
Fig 2.
Expression of a metal-independent phosphoglycerate mutase promotes resistance to manganese starvation.
(A) Wild type S. aureus Newman, ΔgpmA and ΔgpmI and (B) wild type S. aureus Newman and ΔgpmA containing either pOS1 plgt (plgt) or pOS1 plgt:gpmA (plgt:gpmA) were grown in rich medium in the presence of increasing concentrations of CP (t = 7). (C) Wild type USA300 JE2, ΔgpmA and ΔgpmI and (D) wild type USA300 JE2 and ΔgpmA containing either pOS1 plgt (plgt) or pOS1 plgt:gpmA (plgt:gpmA) were grown in rich medium in the presence of increasing concentrations of CP (t = 7). (E) Growth of wild type S. aureus Newman, ΔgpmA and ΔgpmI in the presence of 240 μg/ml of wild type CP as well as the ΔS1 and ΔS2 site mutants (t = 7). (F) Growth of wild type S. aureus Newman, ΔgpmA and ΔgpmI derivatives in NRPMI in the presence and absence of 1 μM MnCl2 or 1 μM ZnSO4 (t = 8). * = p≤0.05 relative to wild type and ΔgpmI by two-way ANOVA with Tukey’s multiple comparisons test. n≥3. Error bars indicate SEM. Also see S1 Fig.
Fig 3.
Expression of a metal-independent isozyme facilitates retention of glycolysis when manganese-starved.
(A-D) S. aureus wild type, ΔgpmA and ΔgpmI were grown in defined medium supplemented with (A) glucose (DM + glucose), (B) Casamino acids (DM + AA), (C) glucose and sodium pyruvate (DM + glucose + pyruvate) or (D) glycerol (DM + glycerol) as a carbon source in the presence of increasing concentrations of CP (t = 7). * = p≤0.05 relative to wild type and ΔgpmI by two-way ANOVA with Tukey’s multiple comparisons test. n≥3. Error bars indicate SEM. Also see S2 Fig.
Fig 4.
Loss of GpmA sensitizes S. aureus to manganese limitation in the absence of manganese transporters.
The growth of (A) wild type, ΔgpmA, ΔmntC ΔmntH and ΔmntC ΔmntH ΔgpmA and (B) wild type, ΔgpmI, ΔmntC ΔmntH and ΔmntC ΔmntH ΔgpmI were assessed in rich medium supplemented with 1 μM MnCl2 and 1 μM ZnSO4 in the presence of increasing concentrations of CP (t = 8). For these assays, the bacteria were precultured overnight in TSB. * = p≤0.05 by two-way ANOVA with Tukey’s multiple comparisons test. n≥3. Error bars indicate SEM. Strains are normalized to growth of parental strains, i.e., ΔgpmA and ΔgpmI are normalized to wild type and ΔmntC ΔmntH ΔgpmA and ΔmntC ΔmntH ΔgpmI are normalized to ΔmntC ΔmntH. C) Wild type S. aureus, ΔgpmA, ΔmntC ΔmntH, ΔmntC ΔmntH ΔgpmA containing either pOS1 plgt (plgt) or pOS1 plgt: gpmA (plgt: gpmA) were grown in rich medium supplemented with 1 μM MnCl2 and 1 μM ZnSO4 in the presence of increasing concentrations of CP (t = 8). For these assays, the bacteria were precultured overnight in TSB. * = p≤0.05 by two-way ANOVA with Tukey’s multiple comparisons test. NS = not significant. n≥3. Error bars indicate SEM. Also see S3 Fig.
Fig 5.
GpmA is necessary for invasive S. aureus disease and resisting manganese starvation.
Wild type C57BL/6 (C57) and CP-deficient C57BL/6 S100A9-/- (CP-/-) mice were infected with either S. aureus wild type, ΔgpmA or ΔgpmI and (A) mean weight loss and (B-C) bacterial burdens in the (B) liver and (C) heart and kidneys were assessed after four days of infection. (A) *,# = p≤0.05 as determined by two-way ANOVA with Tukey’s multiple comparisons test, with * compared to wild type bacteria and # compared to ΔgpmI. (B-C) * = p≤0.05 as determined by Mann-Whitney test for bacterial burdens. The lines indicate the mean. The data are the results from two independent experiments.
Fig 6.
A metal-independent phosphoglycerate mutase enables glycolysis when S. enterica serovar Typhimurium is manganese-starved.
(A & B) Wild type Salmonella and ΔgpmA were grown in rich medium in the presence of (A) increasing concentrations of CP or (B) 240 μg/ml of WT CP or the ΔS1 and ΔS2 CP mutants (t = 8). (C-F) Wild type Salmonella and ΔgpmA were grown in defined medium (DM) supplemented with (C) glucose (DM + glucose), (D) Casamino acids (DM + AA), (E) glycerol (DM + glycerol) or (F) sodium pyruvate (DM + pyruvate) as a carbon source in the presence of increasing concentrations of CP (t = 8). * = p≤0.05 relative to wild type by two-way ANOVA with Sidak’s multiple comparisons test. n≥3. Error bars indicate SEM. Also see S4 Fig.
Table 1.
PCR primers used in this study.