Figure 4. M-C7.1 inhibits the multiple peptide resistance factor (MprF) lysyl-phosphatidylglycerol (LysPG) flippase but not the LysPG synthase.

(A) Detection of phospholipids from S. aureus SA113ΔmprF and wild-type (WT) treated or not treated with 100 µg/ml M-C7.1 or the isotype control monoclonal antibody (mAB) L-1. Polar lipids were separated by thin layer chromatography (TLC) and stained with the phosphate group-specific dye molybdenum blue to detect the well-documented phosphatidylglycerol (PG) and LysPG pattern of S. aureus WT and mprF deletion mutant (Slavetinsky et al., 2012). Percentages of LysPG in relation to total phospholipid content are given below LysPG spots. (B) The repulsion of positively charged cytochrome C corresponds to MprF LysPG synthase plus flippase activity while the synthase activity alone does not affect repulsion. To assess MprF flippase efficiency, unbound cytochrome C in the supernatant was quantified photometrically after incubation with the S. aureus SA113 WT without pretreatment, or with pretreated with M-C7.1 or the isotype control mAB L-1 (WT set to 100%). SA113ΔmprF with or without mAB pretreatment served as positive control. The means + standard error of the mean (SEM) of results from three biological replicates are shown. (C) Annexin V binding to S. aureus SA113 WT compared to incubation with M-C7.1 or the isotype control mAB L-1 was quantified by measuring cell-bound annexin V by fluorescence-activated cell sorting (FACS) and untreated WT samples were set to 100%. SA113ΔmprF with and without mAB incubation served as positive control. Data are expressed as % of untreated WT cells. The means + SEM of results from three biological replicates are shown in (B) and (C). Significant differences compared to WT samples were calculated by Student’s paired t-test (*p < 0.05; **p < 0.01; ***p < 0.0001).