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, 48 (12), 4800-7

Reduced Content of Lysyl-Phosphatidylglycerol in the Cytoplasmic Membrane Affects Susceptibility to Moenomycin, as Well as Vancomycin, Gentamicin, and Antimicrobial Peptides, in Staphylococcus Aureus

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Reduced Content of Lysyl-Phosphatidylglycerol in the Cytoplasmic Membrane Affects Susceptibility to Moenomycin, as Well as Vancomycin, Gentamicin, and Antimicrobial Peptides, in Staphylococcus Aureus

Hiromi Nishi et al. Antimicrob Agents Chemother.

Abstract

An association between moenomycin resistance and vancomycin intermediate resistance in Staphylococcus aureus was demonstrated previously. Thus, to elucidate the mechanism of vancomycin intermediate resistance, we searched for factors contributing to moenomycin resistance. Random Tn551 insertional mutagenesis of methicillin-resistant S. aureus strain COL yielded three mutants with decreased susceptibilities to moenomycin. Correspondingly, these mutants also exhibited slightly decreased susceptibilities to vancomycin. Genetic analysis revealed that two of the mutants had Tn551 insertions in the fmtC (mprF) gene, which is associated with the synthesis of lysyl-phosphatidylglycerol. The third Tn551 insertion was located in the lysC gene, which is involved in the biosynthesis of lysine from aspartic acid. Consequently, mutations in both of these loci reduced the lysyl-phosphatidylglycerol content in the cell membrane, giving it a more negative net charge. The positively charged antibiotic gentamicin and cationic antimicrobial peptides such as beta-defensins and CAP18 were more effective against the mutants. The levels of moenomycin and vancomycin binding to intact cells was also greater in the mutants than in the wild type, while the binding affinity was not altered when cells boiled in sodium dodecyl sulfate were used, indicating that both agents had higher affinities for the negatively charged membranes of the mutants. Therefore, the membrane charge of S. aureus appears to influence the efficacies of moenomycin, vancomycin, and other cationic antimicrobial agents.

Figures

FIG. 1.
FIG. 1.
Population analysis of mutants grown in the presence of increasing concentrations of methicillin, moenomycin, and vancomycin. Overnight cultures of S. aureus wild-type strain COL (squares), HN001 (circles), and HN002 (triangles) were diluted and plated on TSA containing various concentrations of antibiotics. Colonies were counted after 48 h of incubation at 37°C.
FIG. 2.
FIG. 2.
Gradient plate analysis of the mutants. Vancomycin gradient plates (containing a vancomycin concentration gradient from 0 to 4 μg/ml) were inoculated with a suspension of 107 bacterial cells, applied with a sterile cotton swab along the gradient, and grown for 24 h at 37°C.
FIG. 3.
FIG. 3.
Restriction map of the fmtC and lysC loci in S. aureus. Arrows indicate the positions of Tn551 insertions.
FIG. 4.
FIG. 4.
Growth curves of S. aureus COL (closed symbols) and lysC mutant HN002 (open symbols) grown in different types of CDM. Growth was determined by measuring the optical density (OD) at 660 nm in CDM (squares), CDM without lysine (circles), CDM without aspartic acid (triangles), and CDM without both lysine and aspartic acid (diamonds).
FIG. 5.
FIG. 5.
Antibacterial activities of hBD3 and CAP18. Different concentrations of the peptides were added to 200 μl of 10 mM sodium phosphate buffer (pH 6.8) containing 105 bacterial cells. After incubation at 37°C for 2 h, serial dilutions were plated onto TSA. The numbers of CFU were counted after 24 h of incubation at 37°C. Bacterial survival was determined from the number of bacteria that survived in the presence of the peptide as a percentage of the total number of CFU growing in the absence of peptide. The values shown correspond to the means of three independent experiments. Symbols: squares, wild-type strain COL; triangles, mutant HN001; circles, mutant HN002.
FIG. 6.
FIG. 6.
Moenomycin and vancomycin binding to S. aureus cells. Different concentrations of moenomycin or vancomycin were mixed with intact S. aureus cells (closed symbols) or S. aureus cells boiled in SDS (open symbols). The amount of antibiotic binding to S. aureus cells was determined as described in Materials and Methods. Symbols: squares, wild-type strain COL; triangles, HN001; circles, HN002.

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