Figure 1. PlyC eliminates intracellular Spy in a dose-dependent manner.
(a) Spy/A549 co-cultures, treated with 10 µg/ml penicillin and 200 µg/ml gentamicin for 1 hr to remove extracellular bacteria, were incubated for another hour with antibiotic-free growth medium in dilutions of PlyC beginning at 50 μg/ml (440 nM), or with 50 μg/ml heat-denaturated PlyC (70°C for 30 min.), 50 μg/ml (1890 nM) Ply700, or 50 μg/ml (890 nM) B30. Viable intracellular Spy were enumerated as colonies on agar plates that had been incubated with serial dilutions of cell lysates. (b) A primary tonsillar epithelial cell co-culture was treated with 10 μg/ml penicillin and 200 μg/ml gentamicin for 1 hr after which the antibiotic was removed and the tonsil cells were incubated with 50, 10, or 1 μg/ml PlyC for 1h before lysis and enumeration of Spy. All experiments were performed in triplicate biological replicates, with mean and standard deviations displayed. Statistical analysis using Student's t-test is reported as *p<0.05; **p<0.005; ***p<0.001; ns: not significant.
Figure 1—figure supplement 1. Adherent and internalized colony counts of Spy recovered from co-culture assay.
