EndoS confers resistance to phagocytic killing. (A) GAS strains AP1, 5448, and their respective ndoS mutants were grown in human saliva supplemented with 5% serum, and the culture supernatants were analyzed by SDS-PAGE under nonreducing conditions. Note the lack of fully intact IgGs in the AP1 culture supernatants. (B) GAS 5448 and an isogenic ndoS mutant were grown in human saliva supplemented with 5% human serum. IgGs were purified by Protein G and analyzed by SDS-PAGE (top) and LCA blot (bottom). Addition of recombinant EndoS (rEndoS) was used to complement the mutation. (C and D) Saliva-grown GAS 5448 and an isogenic ndoS mutant were challenged with human MDMs (C) and human PMNs (D) in the presence of serum with a high (dark gray) or low (light gray) anti-M1 IgG response. Survival rates were determined by enumerating bacteria both in the initial inoculum and after incubation with the phagocytic cells. Data from three independent experiments with different cell donors (each performed in triplicate, total n = 9) were combined and analyzed using a Kruskal–Wallis test followed by Dunn's multiple comparison test (not significant [ns], P > 0.05; **, P < 0.01). The bar represents the mean, with the standard error depicted as error bars. Each individual data point is represented with a cross, showing the variability between the individual experiments and the replicates within the same experiment.