Figure 3
Impact of ClpC on the intracellular survival of S. aureus DSM20231 in keratinocytes and endothelial cells. Cells of the keratinocyte cell line HaCaT (a, b) and the endothelial cell line Ea.hy926 (c, d) were infected with the S. aureus strains DSM20231 (WT; black bars), the clpC mutant PBM001 (clpC; white bars), and the clpC complemented PBM001 derivative (clpC/clpC+, grey bars) at a MoI of 100, respectively, and co-cultured for 90 min. Extracellular and adherent bacteria were subsequently removed by washing and lysostaphin/gentamicin treatment, and the infected cell lines cultured for up to 168 h in cell culture medium supplemented with gentamicin. At 24, 96, and 168 h post lysostaphin/gentamicin treatment, eukaryotic cells were removed, lysed by sonication, and surviving bacteria in lysates determined by plate counting. (a, c) CFU rates recovered from infected HaCaT (a) and Ea.hy926 (c) lysates at the time points indicated. Data are presented as mean + SD (n = 8 biological replicates). (b, d) Relative survival (%) of intracellular bacteria in HaCaT (b) and Ea.hy926 (d) cells in relation to the CFU rates seen immediately after the lysostaphin/gentamycin treatment. Data are presented in box and whisker plots (n = 8 biological replicates). *P < 0.05; **P < 0.01 (Kruskal Wallis test followed by Dunn’s post-hoc test). TpL/G, time post lysostaphin/gentamicin treatment. Horizontal dashed lines indicate the limit of detection.
