Figure 3. GAS M1 protein protects against histones.

(A) Screening of virulence-associated genes with GAS M1 background of isogenic mutants Δsic, ΔdltA, ΔgacI, ΔhasA, Δscl1, Δpil and Δemm1 mutants compared to GAS M1 WT. (B) Histone H2A MIC assays with GAS WT, Δemm1 mutant and complemented mutant (Δemm1+pM1) as well as LL and heterologous expression of M1 protein in LL (LL+pM1) are shown. (C) Effect of histone treatment on GAS WT and Δemm1 mutant bacteria were visualized using the cell-impermeable DNA dye Sytox Green (green), the cell-permeable DNA dye DAPI (blue) and the membrane dye FM4-64 (red) by confocal microscopy after 3 h of incubation in the presence or absence of histone at 62.5 μg/mL or 150 μg/mL histone H2A and (D) the Sytox Green-positive population in random view fields was quantified. (E) Time-dependency of permeability was assessed using the cell-impermeable dye Sytox Green, which yielded into fluorescent signal upon interaction of dye with bacterial DNA, and was monitored with 1000 μg/mL histone for GAS WT and Δemm1 mutant and bacterial survival was simultaneously calculated via CFU enumeration relative to initial inoculum over a 3 h time course. (F) Bacterial permeability was investigated by treatment with increasing concentrations of histones ranging from 7.8 μg/mL to 1000 μg/mL histones and simultaneous CFU enumeration of bacterial survival relative to initial inoculum after 3 h incubation. Results shown represent average ± SEM values and were analyzed by Student’s t-test in (D–H) or Mann-Whitney test in (A,B) (**P < 0.01, ***P < 0.001). Each dot represents one sample. All experiments have been carried out a minimum of three times run in triplicates, except for (C) which was repeated three times in monoplicates.