Fig. 3: Pre-incubation with lipid II-binding antibiotics prevents DAP binding to S. aureus and delays DAP-induced killing.

a Binding of DAP-FL to S. aureus cells (OD₆₀₀ = 0.5) pre-incubated with cell wall targeting antibiotics (oxacillin (OXA), teixobactin (TEIX) and oritavancin (ORI) (4-fold MIC)). After pre-incubation for 2 min, cells were washed and incubated with DAP-FL in the presence of Ca²⁺ for 10 min followed by washing and the addition of nile red membrane stain. Cells were washed again and subjected to fluorescence microscopy. In a control (CTRL) pre-incubuation with antibiotics was omitted. Scale bar 1 µm. Representative images are shown. b Quantification of DAP-FL binding measured in the experiment as described in a. Binding is expressed as mean grey value per cell. Box plots represent the interquartile range of the data. The black bar represents the mean and whiskers represent minimal and maximal values, respectively. At least n = 350 cells were evaluated for each condition from three biologically independent experiments. Significance was determined by unpaired Student’s t-test with a 95% confidence interval. ****p < 0.0001, n.s., not significant (p = 0.0835). c Survival of S. aureus challenged with DAP (10 µg ml⁻¹) without (red line) or with (blue line) pre-incubation with TEIX as described in a. Bactericidal effects were not observed in cells only pre-treated with TEIX (black line). Data presented are mean values from n = 3 biologically independent experiments. Error bars represent the standard deviation (SD). Source data are provided as a Source Data file.