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. 2001 May 7;193(9):1067-76.
doi: 10.1084/jem.193.9.1067.

Staphylococcus aureus resistance to human defensins and evasion of neutrophil killing via the novel virulence factor MprF is based on modification of membrane lipids with l-lysine

A Peschel  1 , R W Jack, M Otto, L V Collins, P Staubitz, G Nicholson, H Kalbacher, W F Nieuwenhuizen, G Jung, A Tarkowski, K P van Kessel, J A van Strijp
Affiliations

Staphylococcus aureus resistance to human defensins and evasion of neutrophil killing via the novel virulence factor MprF is based on modification of membrane lipids with l-lysine

A Peschel et al. J Exp Med. .

"VSports在线直播" Abstract

Defensins, antimicrobial peptides of the innate immune system, protect human mucosal epithelia and skin against microbial infections and are produced in large amounts by neutrophils. The bacterial pathogen Staphylococcus aureus is insensitive to defensins by virtue of an unknown resistance mechanism. We describe a novel staphylococcal gene, mprF, which determines resistance to several host defense peptides such as defensins and protegrins. An mprF mutant strain was killed considerably faster by human neutrophils and exhibited attenuated virulence in mice, indicating a key role for defensin resistance in the pathogenicity of S. aureus. Analysis of membrane lipids demonstrated that the mprF mutant no longer modifies phosphatidylglycerol with l-lysine VSports手机版. As this unusual modification leads to a reduced negative charge of the membrane surface, MprF-mediated peptide resistance is most likely based on repulsion of the cationic peptides. Accordingly, inactivation of mprF led to increased binding of antimicrobial peptides by the bacteria. MprF has no similarity with genes of known function, but related genes were identified in the genomes of several pathogens including Mycobacterium tuberculosis, Pseudomonas aeruginosa, and Enterococcus faecalis. MprF thus constitutes a novel virulence factor, which may be of general relevance for bacterial pathogens and represents a new target for attacking multidrug resistant bacteria. .

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Figure 1
Figure 1
Inactivation of mprF (A), Kyte and Doolittle hydrophobicity profile (B), and putative transmembrane topology (C) of the S. aureus MprF. (A) The deletion caused by Tn917 integration in the S. xylosus mutant XG2 is indicated by the triangle. The mprF gene of S. aureus was disrupted by replacing the mprF gene with the erythromycin resistance gene ermB. T, transcriptional terminator. (C) The positions in the MprF sequence of putative transmembrane segments and the numbers of positive residues in the putative loops are indicated.
Figure 3
Figure 3
Kinetics of killing and phagocytosis of wild-type (•) and mprF mutant bacteria (○) by neutrophils. (A) The numbers of viable bacteria (CFU) after incubation with neutrophils are expressed as percentage of the initial counts (means and SD of three counts from a representative experiment). (B) The percentage of neutrophils bearing FITC-labeled S. aureus cells are given (means and SD of four independent experiments).
Figure 2
Figure 2
MIC values of the indicated antimicrobial peptides against S. aureus wild type and mprF mutant bearing the empty control plasmid pRB473 or the complementation vector pRBmprF. Positively or negatively charged peptide positions are highlighted in black or gray, respectively. The charges of amino acid side chains and the terminal amino and carboxyl groups were considered. *Although human defensin HNP-1 is shown, a mixture of HNP1, HNP2, and HNP3 which differ in the first amino acid was used in the antibacterial assay. ‡We used and show the synthetic variant A8,13,18-magainin II amide. §MICs of the fluorescent derivative Lys(εDns)-tachyplesin 1. Unusual amino acids: Lan, lanthionine; Mla, methyllanthionine; Dhb, dehydrobutyrine; Avc, S-aminovinyl-d-cysteine; Dha, dehydroalanine; O, ornithine.
Figure 4
Figure 4
Detection of membrane lipids (A), structure (B), and mass spectrometry analysis (C) of L-PG. (A) The polar lipids from S. aureus Sa113 wild type, mprF mutant, and mutant complemented with pRBmprF were analyzed by 2d-TLC. The first panel shows the position of the lipids DPG, PG, L-PG, and the putative 2′ L-PG isomer (L-PG2). Two unidentified lipids were negative (P−N−) or positive (P+N+) with both phosphate- or amino group–specific reagents, respectively. (B) Structure of L-PG. (C) Part of the FT-ICR mass spectrogram of the TLC spot lacking in the mprF mutant. The molecular masses of singly protonated ions (m/z) are given above the peaks. The major peaks represent L-PG species with two saturated fatty acids (indicated with a dot; compare Table ). The minor peaks represent species with one unsaturated fatty acid (mass difference of −2) or molecules containing one or two 13C atoms (mass differences of +1 or +2).
Figure 4
Figure 4
Detection of membrane lipids (A), structure (B), and mass spectrometry analysis (C) of L-PG. (A) The polar lipids from S. aureus Sa113 wild type, mprF mutant, and mutant complemented with pRBmprF were analyzed by 2d-TLC. The first panel shows the position of the lipids DPG, PG, L-PG, and the putative 2′ L-PG isomer (L-PG2). Two unidentified lipids were negative (P−N−) or positive (P+N+) with both phosphate- or amino group–specific reagents, respectively. (B) Structure of L-PG. (C) Part of the FT-ICR mass spectrogram of the TLC spot lacking in the mprF mutant. The molecular masses of singly protonated ions (m/z) are given above the peaks. The major peaks represent L-PG species with two saturated fatty acids (indicated with a dot; compare Table ). The minor peaks represent species with one unsaturated fatty acid (mass difference of −2) or molecules containing one or two 13C atoms (mass differences of +1 or +2).
Figure 4
Figure 4
Detection of membrane lipids (A), structure (B), and mass spectrometry analysis (C) of L-PG. (A) The polar lipids from S. aureus Sa113 wild type, mprF mutant, and mutant complemented with pRBmprF were analyzed by 2d-TLC. The first panel shows the position of the lipids DPG, PG, L-PG, and the putative 2′ L-PG isomer (L-PG2). Two unidentified lipids were negative (P−N−) or positive (P+N+) with both phosphate- or amino group–specific reagents, respectively. (B) Structure of L-PG. (C) Part of the FT-ICR mass spectrogram of the TLC spot lacking in the mprF mutant. The molecular masses of singly protonated ions (m/z) are given above the peaks. The major peaks represent L-PG species with two saturated fatty acids (indicated with a dot; compare Table ). The minor peaks represent species with one unsaturated fatty acid (mass difference of −2) or molecules containing one or two 13C atoms (mass differences of +1 or +2).
Figure 6
Figure 6
Alignment of the COOH-terminal 310 amino acids of the S. aureus MprF protein (Sa) with the corresponding parts of hypothetical proteins from S. xylosus (Sx), B. subtilis (Bs), E. faecalis (Ef), P. aeruginosa (Pa), A. tumefaciens (At), S. coelicolor (Sc), M. tuberculosis (Mt), and M. leprae (Ml). Identical or similar amino acids are highlighted in black or gray, respectively. The COOH-terminal ends are indicated by asterisks. The amino acid positions, calculated from the first possible start points, are given, except for the E. faecalis and A. tumefaciens proteins whose entire sequences are not yet available. The mprF sequences from S. aureus and S. xylosus are available from GenBank/EMBL/DDBJ under accession nos. AF145699 and AF145698, respectively.
Figure 5
Figure 5
Increased binding of antimicrobial peptides in the absence of L-PG (A) and putative mode of action of the resistance system (B). (A) S. aureus or S. xylosus wild-type strains (black bars) and mprF mutants (white bars) were incubated with a fluorescent variant of tachyplesin 1 or gallidermin. The amount of unbound tachyplesin 1 or gallidermin in the supernatants was determined. In binding studies with gallidermin, mutant strains bearing the mprF gene on the plasmid pTXmprF (shaded bars) were also tested, whereas the wild-type and mutant strains contained the empty control plasmid pTX16. The means and SD of three independent experiments are shown. (B) The staphylococcal cell wall and membrane are shown. The hydrophobic and positively charged portions of antimicrobial peptides are indicated in gray and white, respectively. A major amount of membrane lipids is esterified with l-lysine residues in wild-type bacteria. The positively charged l-lysyl groups cause a reduced binding capacity of the cell envelope for cationic antimicrobial peptides, whereas mutant cells lacking L-PG accumulate the harmful molecules in the membrane composed mainly of negatively charged lipids.
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