Figure 2. Binding of M-C7.1 to multiple peptide resistance factor (MprF) and membrane localization of the M-C7.1-targeted MprF loop 7.

(A) Detection of M-C7.1 binding to MprF. Plasmid-encoded native and green fluorescent protein(GFP)-tagged MprF variants were expressed in S. aureus SA113ΔspaΔmprF and living cells were preincubated with M-C7.1 or the isotype control monoclonal antibody (mAB) L-1 (in order to form MprF–mAB complexes). MprF variants complexed with M-C7.1 or control mAB L-1, respectively, were detected by blue native PAGE followed by Western blotting using two different primary (anti-GFP or M-C7.1) and corresponding secondary antibodies. SA113ΔspaΔmprF expressing the empty vector (pRB) served as negative control. Molecular masses in kDa of marker proteins are given on the left of the blot. Arrows mark both the MprF–M-C7.1 complex at 900 kDa and the MprF oligomers at 250 and 500 kDa, which were previously described (Ernst et al., 2015). (B) Cellular localization of the antigen epitope of M-C7.1 using the substituted cysteine accessibility method (SCAM) for specific loops between the MprF transmembrane segments (TMSs). The substituted cysteine T263C is localized in M-C7.1’s target peptide sequence in MprF. Substitution of A99C served as inside control, substitution of T480C served as outside control (see topology model, part C). S. aureus SA113ΔmprF expressing the empty vector (pRB) and an MprF variant lacking all native cysteines (wild-type [WT] (-Cys)) served as additional negative controls. All MprF variants were plasmid-encoded, FLAG tagged at the C-terminus to allow immunoprecipitation and detection, and were expressed in S. aureus SA113ΔmprF. Substituted extracellular cysteine residues were labeled with Na*-(3-maleimidylpropionyl)-biocytin (MPB) (outer leaflet signal, green in overlay), while labeling of substituted internal cysteine with MPB was performed after the blocking of external cysteines with 4-acetamino-4′-maleimidylstilbene-2,2′-disulfonic acid (AMS) (inner leaflet signal, green in overlay). MprF was detected via antibody staining by an anti-FLAG antibody (red in overlay). (C) MprF topology showing location and amino acid exchanges of artificial cysteine residues for SCAM detection in green.

Figure 2—figure supplement 1. Effects of cysteine replacement and insertion on multiple peptide resistance factor (MprF) function, assessed by measuring daptomycin susceptibility.

Minimal inhibitory concentrations (MICs) of daptomycin against the indicated S. aureus strains are shown. The mprF deletion mutant with empty pRB474 plasmid served as a negative control, whereas cysteine depleted mprF-expressing variants show a fourfold increased daptomycin MIC while wild-type mprF-expressing positive control shows a tenfold increased MIC. The means + standard error of the mean (SEM) of results from three independent experiments are shown. Values that are significantly different from the values determined for S. aureus SA113ΔmprF bearing pRB474 (pRB), calculated by Student’s paired t-test, as indicated (**p < 0.01; ***p < 0.0001).

Figure 2—figure supplement 2. Detection of M-C7.1 binding to multiple peptide resistance factor (MprF).

An overlay of single channels from Figure 2A showing antihuman IgG binding to M-C7.1 in red and an antigoat IgG binding to the primary anti-GFP IgG in green. SA113ΔspaΔmprF expressing the empty vector (pRB) served as negative control. Molecular masses in kDa of marker proteins are given on the left of the blot. Arrows mark the MprF–M-C7.1 complex at 900 kDa and the MprF oligomers at 250 and 500 kDa as previously described (Ernst et al., 2015). Further explanations are found in figure legend of Figure 2A.