Fig. 6. S. aureus isolates showing high intracellular replication and delayed host cell death are deficient for the cysteine protease staphopain A.

a, b Comparison between the percentage of cells with high S. aureus intracellular replication (maximum) and the viability of epithelial cells (HeLa) infected with the 191 S. aureus isolates, at 3 (a) and 6 hpi (b). Results are shown as mean of three biologically independent experiments. Group III and V bacterial isolates are highlighted in orange and purple, respectively; six isolates selected from clusters IVa, IVb, and IVc are highlighted in black; all the other isolates are shown in light gray. c Staphopain A protein levels, determined by western blot, in the supernatant of bacterial liquid cultures of the 13 S. aureus isolates belonging to group V. S. aureus USA300; scpA mutant, and isolates BJI001, IE059, IE060 (belonging to clusters IVa and IVb) were used as controls. Western blots are representative of three independent experiments. Ponceau staining of the membranes is shown. d–f Representative fluorescence microscopy images (d), and time course quantification of the percentage of host cells with high bacterial intracellular replication (e), and host cell viability (f), of HeLa cells infected with S. aureus isolates belonging to cluster V (WT, light purple in panels e, f) or with isolates modified to ectopically express both staphopain A and staphostatin A from a plasmid (WT + pScpAB, dark purple in panels e, f). S. aureus scpA mutant and scpA + pScpAB are shown for comparison (dotted lines). Results shown in panels e and f are the mean of three biologically independent experiments; rightmost plots indicate the maximum of replication (e) or minimum of host cell viability (f) for WT or modified bacteria; ***P < 0.001 (statistical analysis is detailed in Supplementary Data 3). Scale bar, 50 µm. g–i Representative fluorescence microscopy images (g), and time course quantification of the percentage of host cells with high bacterial intracellular replication (h) and host cell viability (i), of HeLa cells infected with S. aureus isolates that natively express staphopain A (selected from clusters IVa, IVb, IVc). Infection was performed without (dark gray) or with treatment with the inhibitor of cysteine proteases E-64d (dark red). S. aureus USA300 and scpA mutant are shown for comparison in panels h, i (dotted lines). Results shown in panels h and i are the mean of three biologically independent experiments; rightmost plots indicate the maximum of replication (h) or a minimum of host cell viability (i) in the presence/absence of the inhibitor; **P < 0.01 and ***P < 0.001 (statistical analysis is detailed in Supplementary Data 3). Scale bar, 50 µm. Source data are provided as a Source Data file.