Fig. 1.

Calprotectin is present at the GAS colonization surface in the host. a) Lesions from mice infected intramuscularly at days 1 and 4 post-infection with either saline (mock) (n = 4) or wild type GAS (n = 4) were probed for S100A8, S100A9, and loading control GAPDH by immunoblotting. b) Quantification of calprotectin in the infected muscular tissue lesions by ELISA. Data graphed are mean ± standard deviation. Statistical significance between the mock and GAS-infected samples is indicated by * (P < 0.05). P values were derived from two-sample t-test. c) and d) Calprotectin levels in the subcutaneous abscesses of mice infected with either saline (mock) (n = 4) or wild type GAS (n = 4) were analyzed by immunoblotting (c) and quantified by ELISA (d) at day 3 post infection. Data graphed are mean ± standard deviation. Statistical significance between the mock and GAS-infected samples is indicated by * (P < 0.05). P values were derived from two-sample t-test. e) GAS were grown in triplicate in THY broth supplemented with the indicated concentrations of CP. Growth was monitored by absorption at A₆₀₀ in a microplate reader at the indicated time points. f) Comparison of the number of CFUs of GAS grown in the indicated concentrations of CP. Triplicate cultures were grown at the indicated conditions. Using the optical density of the untreated GAS growth, cells were collected at the start point (t = 0), early exponential (EE, A₆₀₀ – 0.125), mid-exponential (ME, A₆₀₀ – 0.25), late-exponential (LE, A₆₀₀ – 0.375), and stationary (STAT, A₆₀₀ – 0.5). Statistical significance between the untreated and calprotectin treated samples is indicated by ** (P < 0.0001). Data graphed are mean ± standard deviation. P values were derived from two-sample t-test. g) Transcript levels of lmb, adcA, and adcC in CP-treated GAS growth compared to untreated sample measured by qRT-PCR. Three biological replicates were grown and analyzed in triplicate. Data graphed are mean ± standard deviation. h) Metal content analysis of THY broth treated with the indicated concentrations of CP. Metal concentration (M) is denoted in the y-axis (in μM). Samples were analyzed in triplicate and data graphed are mean ± standard deviation. Statistical significance between the untreated and calprotectin treated samples is indicated by ** (P < 0.0001). P values were derived from two-sample t-test.