Article Figures & Data
Figures
- Fig 1
Structures of S. aureus and C. perfringens MprF proteins. The lengths and predicted transmembrane segments of MprF (S. aureus), CpMprF1, and CpMprF2 (C. perfringens) are shown. Extensions of truncated MprF variants MprF-N and CpMprF2-N and of the previously characterized S. aureus synthase domain (Syn) are indicated.
- Fig 2
Aminoacyl phospholipid production by C. perfringens MprF proteins in S. aureus. (A) Detection of aminoacyl phospholipids from wild-type S. aureus and an S. aureus mprF deletion mutant strain expressing empty plasmid pTX16, S. aureus MprF (pTX15mprF), or C. perfringens MprF protein (pTX15cpmprF2 or pTX15cpmprF1). Polar lipids were separated by TLC and stained with the amino group-specific dye ninhydrin. (B) Quantification of aminoacyl phospholipid content. Polar lipids were separated by TLC, stained with the phosphate group-specific dye molybdenum blue, and quantified densitometrically. (C) Detection of phospholipids from S. aureus mprF deletion mutants expressing S. aureus MprF together with empty plasmid pRB474 or S. aureus MprF together with C. perfringens CpMprF1 on separate plasmids. Polar lipids were separated and detected as described for panel B. (D) Quantification of aminoacyl phospholipids as described for panel B. The Ala-PG and Lys-PG contents of an S. aureus mprF deletion mutant expressing mprF and cpmprF1 or mprF alone are indicated. Means and standard errors of the means of at least three independent experiments are shown. *, P < 0.05; **, P < 0.005; ns, not significantly different from S. aureus ΔmprF containing plasmid pTX15mprF (B) or plasmids pTX15mprF and pRB474 (D).
- Fig 3
Impact of C. perfringens MprF proteins on antimicrobial peptide resistance. (A to C) MICs of daptomycin (A), gallidermin (B), and nisin (C) were determined with the S. aureus wild-type strain and mprF deletion mutants expressing empty plasmid (pTX16) and S. aureus MprF (pTX15mprF) and C. perfringens MprF proteins (pTX15cpmprF2 and pTX15cpmprF1). (D) MIC of daptomycin determined with mprF deletion mutants expressing S. aureus MprF together with empty plasmid pRB474, as well as S. aureus MprF together with C. perfringens CpMprF1 on separate plasmids. Means and standard errors of the means of at least three independent experiments are shown. *, P < 0.05; **, P < 0.01; ***, P < 0.0001; ns, not significantly different from S. aureus ΔmprF containing plasmid pTX16 (A to C) or plasmids pTX15mprF and pRB474 (D).
- Fig 4
Impact on daptomycin resistance of Lys-PG flippases expressed in trans with Ala-PG or Lys-PG synthase in S. aureus. MICs of daptomycin were determined for a set of mprF deletion mutants expressing the Lys-PG synthase domain of S. aureus MprF [pRB474mprF(syn)] together with the empty plasmid (pTX16), the Lys-PG flippase domain of S. aureus (pTX15mprF-N), and the Lys-PG flippase domain of C. perfringens (pTX15cpmprF2-N). Another strain set consisted of the mprF deletion mutant expressing the Ala-PG synthase of C. perfringens (CpMprF1) together with the empty plasmid (pTX16) and the Lys-PG flippase domain of S. aureus (pTX15mprF-N) or C. perfringens (pTX15cpmprF2-N). Wild-type S. aureus and the complemented mprF deletion mutant (pRB474mprF/pTX16) served as positive controls, while the mprF deletion mutant expressing empty plasmids pTX16 and pRB474 served as a negative control. Means and standard errors of the means of at least three independent experiments are shown. Statistically significant differences from S. aureus ΔmprF expressing respective aminoacyl phospholipid synthases lacking Lys-PG flippase domains [pRB474mprF(syn)/pTX16; pRB474cpmprF1/pTX16] are indicated as follows: ***, P < 0.0001; **, P < 0.01.
