Fig. 2.

Zinc limitation by calprotectin inhibits GAS growth. Growth curves of indicated strains in THY broth (a), chemically-defined medium devoid of zinc (b), and THY broth supplemented with 10 μM TPEN (c). d) The ∆ adcC mutant was grown in triplicate in nutrient rich THY broth supplemented with the indicated CP concentrations. Growth was monitored by absorption at A₆₀₀ in a microplate reader at the indicated time points. e) The WT, ∆ adcC, and the trans-complemented strains were grown in THY broth and the intracellular metal content was measured by ICP-MS. Statistical significance between the WT, and ∆ adcC mutant is indicated by * (P < 0.05). Data graphed are mean ± standard deviation. P values were derived from two-sample t-test. f) Ten outbred CD-1 mice per strain were injected intramuscularly with 1 × 10⁷ CFU of each strain. Mouse survival depicted using a Kaplan-Meier curve with P values derived by log rank test. * indicates P < 0.05. g) Histopathological analysis of hindlimb lesions from mice infected with each indicated strain. Areas of host tissue damage are boxed, whereas confined, less destructive lesions are circled. h) Twenty mice were infected intramuscularly and mean CFUs recovered from the infected muscle tissue are shown with P value as determined by t-test. * indicates P < 0.05. Data graphed are mean ± standard deviation. i) Lesions from mice infected intramuscularly with the indicated strains were probed for S100A8, S100A9, and loading control GAPDH at days 1 and 3 post-infection by immunoblotting. j) Quantification of calprotectin concentration in the infected muscular tissue lesions by ELISA. Data graphed are mean ± standard deviation. Statistical significance between the WT, and ∆ adcC mutant is indicated by * (P < 0.05). P values were derived from two-sample t-test. k) Ten immunocompetent hairless mice were infected with each indicated strain, and the lesion area produced by each strain was determined. Lesion area was measured day 3 p.i and graphed (mean ± standard error of the mean). P value was derived by log rank test. Statistical significance between the WT, and ∆ adcC mutant is indicated by * (P < 0.05). l) Calprotectin levels in the subcutaneous abscesses of mice infected with the indicated strains were analyzed by immunoblotting (l) and quantified by ELISA (m). Statistical significance of P < 0.05 between the WT and ∆ adcC mutant strain is indicated by *. Data graphed are mean ± standard deviation. P values were derived from two-sample t-test.