Fig. 5: TslA causes membrane damage to S. aureus cells in the absence of Til1 immunity proteins, in a T7SS-dependent manner.

a–c the indicated strain and plasmid combinations were cultured for 2 h, after which 500 ng ml⁻¹ ATc was added to induce plasmid-encoded gene expression. OD₆₀₀ readings, in triplicate, were taken manually every hour. The experiment was repeated three times and each point is an average of 3 biological and 3 technical replicates. Data are presented as mean values ± SD and n = 3 independent biological experiments. d. USA300 and USA300 Δtil1 harbouring pRAB11 or pRAB11 encoding TslA-TlaA1-TlaA2 were cultured with ATc for 1 h 50 min, after which an aliquot of USA300/pRAB11 and USA300 Δtil1/pRAB11 were treated with 10 μM melittin for 5 min. Subsequently, all samples were stained with 200 nM Sytox green and 2 μM DiSC₃(5), spotted onto an agarose pad and imaged by fluorescence microscopy. Representative fields of view, avoiding large clusters of cells are displayed. This experiment was repeated three times, each with similar results. Source data are provided on Figshare (https://doi.org/10.6084/m9.figshare.24648513). e. Fluorescence intensity of Sytox green for individual cells from each group plotted on a log₁₀ axis. At least 100 cells were analysed for each condition, pooled across three biological replicates. The exact number of cells analysed is provided in the Source Data file. Note that due to signal saturation in some fields, quantitative analysis could not be performed. Cells are therefore placed into high and low groups based on negative control values, depicted by the dashed line. The percentage of cells in each group with membrane damage is given in Supplementary Table 2.