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Original Article |
andreas.peschel{at}uni-tuebingen.de
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Key Words: host defense peptides • oxygen-independent killing • Staphylococcus aureus virulence • phospholipids • innate immunity
S. aureus has evolved the means to resist antimicrobial host components such as lysozyme, the
Defensins form pores in the bacterial cytoplasmic membrane 5; peptides with similar properties and activity have been found in several vertebrates and invertebrates as well as in plants 10 and bacteria 11. They include molecules with β-sheet structure such as porcine protegrins 12,
Construction of Plasmids and Homologous Recombination.
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The human pathogen Staphylococcus aureus is a major cause of community- and hospital-acquired skin, respiratory, endovascular, soft tissue, bone, and joint infections 1. The increasing prevalence of multidrug resistant strains and the recent appearance of strains with reduced susceptibility to vancomycin, the antibiotic of last resort, raises the specter of untreatable staphylococcal infections and adds urgency to the search for new antiinfective strategies 2. 
-defensins human neutrophil peptide (HNP)1–3 3, and the β-defensin hBD2 4. Defensin peptides constitute a shield against microbial infections on skin, on epithelia of the respiratory, gastrointestinal, and genitourinary tracts (β-defensins), and are found in large amounts in the granules of phagocytes and intestinal Paneth cells (-defensins; reference 5). Although defensins account for 50% of the neutrophil granule proteins, they fail to kill S. aureus effectively. Accordingly, patients with inherited oxidative burst deficiency (chronic granulomatous disease [CGD]) are particularly susceptible to S. aureus infections 6. When keratinocytes from human skin come into contact with bacterial pathogens, they upregulate the expression of the defensin hBD-2 gene 4 and there is evidence that airway epithelial cells respond in a similar manner 7. Diminished defensin activity, caused by increased salt concentration in airway fluids, is thought to contribute to the susceptibility of cystic fibrosis patients to life-threatening S. aureus and Pseudomonas aeruginosa infections 8. The ability of bacterial pathogens to resist host defense peptides has a profound influence on their virulence as demonstrated for Salmonella typhimurium 9. However, the molecular basis for defensin resistance has remained elusive. -helical peptides such as the amphibian magainins 13, and the bacterial lantibiotics bearing thioether bridges 11. Both S. aureus and the coagulase-negative Staphylococcus xylosus show high level innate tolerances to host defense peptides and lantibiotics.
Materials and Methods
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Transposon Mutagenesis and DNA Sequence Analyses.
All bacterial strains were grown in BM broth (1% tryptone, 0.5% yeast extract, 0.5% NaCl, 0.1% K2HPO4, and 0.1% glucose) unless otherwise noted. A mutant library of S. xylosus C2a was constructed using transposon Tn917 and the delivery vector pTV1ts as described previously 3. Mutant clones (4,000) were transferred to BM agar plates containing 3 µg gallidermin/ml and monitored for impaired growth. Tn917-specific primers were used to sequence genomic DNA upstream and downstream from the transposon insertion site by cycle sequencing 14 and the sequence was completed by primer walking. With one of the primers used for sequencing of the S. xylosus mprF, part of the corresponding gene in S. aureus Sa113 was obtained and the sequence was completed by primer walking. The program tblastn with the Microbial Genomes Blast Databases at http://www.ncbi.nlm. nih.gov was used to perform sequence similarity searches. Prediction of the MprF transmembrane topology was accomplished using the program TMAP at http://www.mbb.ki.se/cgi-bin/tmap1.pl. It is based on a multiple alignment of the closely related MprF homologues from S. aureus, S. xylosus, Bacillus subtilis, and Enterococcus faecalis.
For in vitro recombination of DNA, standard methods and vectors were used 14. To delete the mprF gene of S. aureus Sa113, DNA fragments of 885 bp and 1,042 bp flanking mprF were amplified by PCR and cloned together with the ermB gene from Tn551 into the polylinker of the temperature-sensitive shuttle plasmid pBT2 using the restriction sites indicated in Fig. 1 A. After construction in Escherichia coli DH5a 14, the resulting plasmid pBT
mprF was transferred to S. aureus Sa113 by electroporation 15 and mprF deletion mutants were enriched by incubation at 42°C in the presence of 2.5 µg/ml erythromycin; the recombination procedure has recently been described in detail 16. The proper integration of ermB was verified by sequencing of the genomic DNA at the borders of the PCR-derived regions. A 2,345-bp fragment comprising 93% of the mprF gene was deleted. Plasmid pRBmprF was constructed in E. coli DH5 by ligation of a 3,281-bp PCR fragment bearing the mprF gene of S. xylosus C2a, together with 359 bp upstream containing the putative promoter region and 400 bp downstream containing the terminator structure into the SmaI site of the shuttle vector pRB473 17. To construct plasmid pTXmprF, a 2,927-bp PCR fragment encoding the S. xylosus mprF gene was cloned in the expression vector pTX15 via BamHI and MluI to produce a transcriptional fusion with the xylA promoter 18. PCR primers were modified to introduce a BamHI site 34 bp upstream of the mprF start codon and a MluI site 370 bp downstream of the stop codon. The cloning host Staphylococcus carnosus TM300 was transformed with the pTXmprF ligation mixture by protoplast transformation 19. Plasmids pRBmprF and pTXmprF were subsequently transferred to S. aureus Sa113 or S. xylosus C2a by electroporation 15. The xylA promoter of pTXmprF is repressed by the plasmid-encoded repressor XylR and derepression was achieved by the addition of 0.5% xylose to the culture medium 18.
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Dns)-tachyplesin 1 was synthesized, folded, and purified as above with the exception that the NH2 terminus was extended by a further lysine residue, the -amino group of which was dansylated. The purity and quality of the peptide preparations was confirmed by HPLC and electrospray ionization mass spectrometry (ESI-MS). Gallidermin was provided by Dr. K. Thomae (GmbH, Biberach, Germany). Nisin, synthetic (A8,13,18)-magainin II amide, melittin, gramicidin S, and gramicidin D were purchased from Sigma-Aldrich. Minimal inhibitory concentration (MIC) values of the various peptides were determined in serial dilution tests using Luria-Bertani (LB) broth (1% tryptone, 0.5% yeast extract, 0.5% NaCl) as described recently 21. As the activity of human defensins is very sensitive to the salt concentration 20, half-strength LB broth without NaCl was used for them; the tested strains all grew well in this medium.
Phagocytosis and Killing of S. aureus by Human Neutrophils.
Blood was drawn from healthy human volunteers and heparinized. Neutrophils were isolated from peripheral blood as described previously 22 and resuspended in HBSS-HSA (HBSS containing 0.05% human serum albumin). To prepare bacteria for killing experiments, Mueller Hinton Broth was inoculated with 1/100 volumes of overnight cultures and vigorously shaken at 37°C until mid-logarithmic phase was reached. The bacteria were washed twice in HBSS-HSA and adjusted to a density of 8.5 x 107 CFU/ml. Normal human serum was added to a final concentration of 4% and bacteria were opsonized for 10 min at 37°C. Prewarmed bacterial and neutrophil suspensions were mixed to final concentrations of 8.5 x 104 CFU/ml and 5 x 106 neutrophils/ml. 50-µl samples were shaken at 37°C and incubation was stopped by the addition of 2 ml ice-cold distilled water to disrupt the neutrophils. Appropriate sample volumes were spread on LB agar plates and colonies were counted after 24 h incubation at 37°C. When bacteria were incubated for 4 h under the same conditions without neutrophils, no significant changes in the colony numbers compared with the initial counts were observed.
In phagocytosis studies, bacteria were grown as described above, washed, resuspended in PBS, and inactivated by heating for 25 min at 70°C. Subsequently, 0.1 mg/ml FITC was added and the bacteria were labeled at 37°C for 5 h. After washing with PBS, the bacteria were resuspended in HBSS-HSA, adjusted to the same density, and opsonized as described above. 50-µl aliquots of the prewarmed bacterial and neutrophil suspensions were mixed and shaken at 37°C. The final concentrations of bacteria and neutrophils were 8.5 x 107/ml and 2.5 x 106/ml, respectively. Incubation was stopped by addition of 100 µl ice-cold 1% paraformaldehyde. The percentage of neutrophils bearing FITC-labeled bacteria was determined by flow cytometric analysis of 5,000 cells using a FACScanTM (Becton Dickinson).
Killing by Myeloperoxidase.
Bacteria were grown as described for neutrophil-mediated killing and washed twice with PBS containing 0.0005% HSA. 3 x 105 CFU/ml were incubated with 0.05 U myeloperoxidase (MPO)/ml (Calbiochem) and 10 µM H2O2 in the same buffer after preheating all solutions to 37°C. 154 mM NaCl from the PBS buffer was present in the samples to permit generation of toxic chlorinating compounds 23. Samples of 10 µl each were shaken at 37°C and killing was stopped after 45 min by 25-fold dilution in ice-cold PBS. CFUs were counted 24 h after plating appropriate amounts on LB agar.
Animal Studies.
Female NMRI mice aged 5–7 wk were injected in the tail vein with a bacterial suspension containing 1.8 x 107 CFU of either S. aureus Newman wild type or the isogenic mprF mutant and evaluated for weight loss, arthritis, and sepsis over a 7-d period as described previously 24. Mice were subsequently killed and kidneys were removed in order to assay bacterial loads. The differences between the means of the values in all groups were tested for significance with the two-tailed Student's t test. The between-group differences in the mortality rate, frequency of arthritis, body weight losses, and bacterial counts in kidneys were analyzed using the chi-square test. A P value 
0.05 was considered statistically significant. All mice were treated in accordance with institutional guidelines.
Isolation and Detection of Membrane Lipids.
Bacteria were grown overnight in BM broth containing 0.25% glucose, washed once in sodium acetate buffer (20 mM, pH 4.5), and disrupted in the same buffer using glass beads and a Disintegrator S (Biomatic GmbH) as described elsewhere 18. Polar lipids were extracted by the Bligh-Dyer procedure 25, vacuum dried, and dissolved in chloroform/methanol (2:1, by volume). Two-dimensional TLC (2d-TLC) was carried out as described previously 26. In brief, equal amounts of lipid extracts were spotted onto silica 60 F254 HPTLC plates (Merck) and developed with chloroform/methanol/water (65:25:4, by volume) in the first direction and chloroform/acetic acid/methanol/water (80:15:12:4, by volume) in the second direction. All lipids were visualized with molybdatophosporic acid spray (Merck) followed by charring at 120°C and treatment with ammonia vapor to improve the contrast. Phospholipids or amino group–containing lipids were selectively stained with Molybdenum Blue (Sigma-Aldrich) or ninhydrin spray (Merck), respectively. For further analyses, lipid spots were stained with iodine vapor, scraped from the glass plates, and extracted with dichloromethane/methanol (2:1, by volume). Phosphatidylglycerol (PG) and diphosphatidylglycerol (DPG) were purchased from Sigma-Aldrich and used as standards to determine the positions of the PG and DPG spots in 2d-TLC.
Lipid Analysis by Mass Spectrometry and Gas Chromatography.
The masses of lipids from the TLC spot were determined by Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) on an Apex II FT-ICR mass spectrometer (Bruker) in both the positive and negative ESI modes. To analyze the composition of the lipid, the samples were spiked with n-tridecanoic acid and D-lysine as internal standards and incubated at 110°C for 30 min in 200 µl 1.5 M ethanolic HCl. After addition of 200 µl distilled water, the hydrophobic compounds were extracted into pentane and subsequently analyzed by gas chromatography (GC) and GC-MS on an Agilent 6890/5973 (Agilent Technologies) and an HP-5 MS capillary (30 m x 0.25 mm). The ethanolic phase was evaporated to dryness, reesterified with ethanolic HCl to ensure complete esterification, and then trifluoroacetylated (trifluoroacetic anhydride/dichloromethane 4:1 at 110°C for 10 min). The derivatized amino acids were detected by chiral GC-MS on a Lipodex E capillary and determined by enantiomer labeling 27. Finally, the ethanolic layer was trimethylsilylated (bis[trimethylsilyl]trifluoroacetamide in pyridine [1:1]) at 60°C for 30 min and derivatives were analyzed by GC-MS on the HP-5 MS capillary. A sector from a TLC plate without sample but identically developed and worked up (blank) was used to correct the fatty acid amounts determined in corresponding spots from sample-containing plates.
Interaction of Tachyplesin 1 and Gallidermin with Bacterial Cells.
Strains were grown for 5 h in BM medium, harvested, washed three times, and resuspended in ice-cold 50 mM sodium phosphate buffer, pH 7.0. Bacteria (2 x 107/ml) were incubated with 2 µM Lys(Dns)-tachyplesin 1 for 20 min on ice, and pelleted by centrifugation. The relative fluorescence of the supernatant was determined spectrofluorometrically using excitation and emission wavelengths of 340 and 522 nm, respectively. Binding of gallidermin was analyzed in a similar way with the following modifications: bacteria were grown in LB broth with 0.5% xylose, incubated with 2.3 µM (S. aureus) or 11.5 µM (S. xylosus) gallidermin for 20 min at 37°C, and the bacterial density was 1.6 x 108/ml. The amount of gallidermin in the supernatant was determined by RP-HPLC as described previously 3.
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The S. aureus mprF gene was replaced by an erythromycin resistance gene (ermB) via homologous recombination (Fig. 1 A). In the absence of an intact mprF gene, the susceptibility of S. aureus Sa113 to defensin HNP1-3 from human neutrophils, protegrins 3 and 5 from porcine leukocytes, and tachyplesin 1 from horseshoe crab haemocytes was 8- to at least 30-fold higher (Fig. 2). The sensitivity to the lanthionine-containing peptides (lantibiotics) gallidermin from Staphylococcus gallinarum and to nisin from Lactobacillus lactis 11 was increased by factors of 8 to 38. The linear peptides magainin II from clawed frog skin and melittin from honeybee venom 13 were only slightly more active against the mprF mutant (three- to fourfold). The increased sensitivity of the mutant seemed to be restricted to extended cationic peptides, as the small, circular gramicidin S and the linear, neutral gramicidin D (both peptides from Bacillus brevis; reference 30) were almost equally active against wild-type and mutant strains. Interestingly, even the wild-type strain was particularly susceptible to these peptides. Complementation of the mprF mutant with plasmid pRBmprF resulted in normal or considerably higher tolerances to cationic antimicrobial peptides (Fig. 2).
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Disruption of mprF Results in Attenuated Virulence.
The virulence of the mprF mutant was analyzed in a mouse model of sepsis and septic arthritis 24 using S. aureus Newman, which is more virulent than the laboratory strain Sa113. The mprF mutation caused a similar sensitivity to antimicrobial peptides in strain Newman as in strain Sa113 (data not shown). Significant differences in the mortality of NMRI mice injected intravenously with 1.8 x 107 CFU bacteria were noted, with 33% mortality in the wild-type infected group and no deaths in the mutant infected group at day 7 after infection (Table ). The incidence of arthritis in the two groups of animals was also different. At day 7 after infection, the frequencies of joint swelling were 21% for the mutant and 94% for the wild type. In addition, the wild-type infected animals lost significantly more weight during the course of the experiment than the mutant group, and the numbers of bacteria recovered from the kidneys of mice infected with the mprF mutant were significantly lower than those from wild-type infected animals (Table ), suggesting that the mprF mutant causes less systemic effects of disease.
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Dns)-tachyplesin 1, a synthetic fluorescent-labeled derivative of tachyplesin 1, was determined. The modification of tachyplesin 1 caused a 2.5-fold increase in the MIC, but the mprF mutant was still 33-fold more sensitive to the peptide than the wild type (Fig. 2). Whole cells were incubated with Lys(Dns)-tachyplesin 1 and the amount remaining in the supernatant after centrifugation was determined spectrofluorometrically. In the supernatants of wild-type cells, a considerably higher amount of tachyplesin 1 was detected compared with that found in supernatants of the mutant strain (Fig. 5 A), indicating that in the presence of L-PG, antimicrobial peptides bind less efficiently to the cells. The wild-type and mutant bacteria were not killed by Lys(Dns)-tachyplesin 1 at the concentration used. Similar results were obtained when bacteria were incubated with gallidermin and the concentration of unbound lantibiotic was determined by RP-HPLC. Moreover, complementation of the mprF-deficient mutant with plasmid pTXmprF restored bacterial repulsion of gallidermin (Fig. 5 A).
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The mprF mutant no longer produces the unusual L-PG lipid that arises from esterification of PG with L-lysine 36. It remains to be determined whether mprF encodes the as yet unidentified L-PG synthase or whether it is involved in some other essential aspect of L-PG biosynthesis. The putative membrane location of MprF is consistent with a role in lipid metabolism. Positively charged L-PG accounts for up to 38% of the S. aureus membrane lipids, whereas the other phospholipids (PG and DPG) are negatively charged. Therefore, L-PG synthesis most probably has a significant impact on membrane surface charge and interactions with cationic antimicrobial peptides, which have to bind to the charged head groups of the membrane lipids before integration into the hydrophobic core of the cytoplasmic membrane (Fig. 5 B). Accordingly, the mprF mutant was much more susceptible to a broad variety of cationic antimicrobial peptides whereas the neutral gramicidin D was equally active toward wild-type and mprF mutant. The increased binding capacity of mutant cells for a tachyplesin 1 derivative and gallidermin demonstrates that the presence of L-PG leads to reduced attraction and binding of cationic antimicrobial peptides by the bacteria. We have recently demonstrated that reducing the negative charge of the staphylococcal cell wall by modification of the teichoic acid polymers with D-alanine affects the binding of cationic peptides 3, supporting the notion that S. aureus cells protect themselves against host defense peptides by modulating the electrostatic properties of their cell envelope. The location of mprF proximal to an S. aureus homologue of msrA, a locus involved in repair of bacterial proteins damaged by reactive oxygen species 28, indicates a clustering of genes involved in the escape from phagocyte functions.
Up to 60% of healthy individuals are permanently or intermittently colonized by S. aureus, and carriage is an important risk factor for life-threatening infections in patients undergoing surgery, bearing intravascular devices, or those with HIV infection and AIDS 37. The main ecological niches for S. aureus are the nasal epithelia, although these sites are protected by defensins produced by epithelial cells and submucosal glands 38. Defensin resistance thus may play a key role in the capacity of S. aureus to infect host tissues, in particular those of cystic fibrosis or chronic granulomatous disease patients 68. It should be noted that S. aureus produces L-PG in comparatively high amounts, whereas coagulase-negative staphylococci contain only traces (Staphylococcus epidermidis, S. xylosus) or no detectable amounts (Staphylococcus haemolyticus, Staphylococcus saprophyticus) of this unusual lipid 26. It is tempting to speculate that high L-PG content is a prerequisite for S. aureus survival on nasal epithelia and invasiveness.
Lysinylation of phospholipids may play a similar role in other bacteria. L-PG has been described in several bacterial species bearing mprF-related genes including E. faecalis, B. subtilis 39, and P. aeruginosa 40. In the remaining bacteria with mprF homologues, the membrane lipids seem to be only incompletely characterized 41. Defensin-like peptides also play a role in the defense of plants against microbial infections 10, perhaps explaining the presence of a mprF homologue adjacent to other virulence genes in the plant pathogen A. tumefaciens 42. MprF thus represents an interesting new target for novel antimicrobial drugs which block L-PG synthesis and thereby render the bacteria susceptible to antimicrobial host defenses.
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Acknowledgments |
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This work was supported by a fellowship from the European Molecular Biology Organization to A. Peschel (ASTF 9521) and by grants from the German Research Council to A. Peschel (Priority Program No. 1047), H. Kalbacher (Ka 767/4-2), and G. Jung (SFB 323). L.V. Collins acknowledges the support of the EU Biotechnology program (contract no. BIO4CT975130).
Submitted: 12 June 2000
Revised: 25 January 2001
Accepted: 14 March 2001
R.W. Jack's present address is Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Ave., Kowloon Tong, Kowloon, Hong Kong.
M. Otto's present address is Laboratory of Human Bacterial Pathogenesis, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 903 South 4th St., Hamilton, MT 59840.
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References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Lowy F.D.. Staphylococcus aureus infections, N. Engl. J. Med., 339, 1998, 520–532.
Sieradzki K., Roberts R.B., Haber S.W. & Tomasz A.. The development of vancomycin resistance in a patient with methicillin-resistant Staphylococcus aureus infection, N. Engl. J. Med., 340, 1999, 517–523.
Peschel A., Otto M., Jack R.W., Kalbacher H., Jung G. & Götz F.. Inactivation of the dlt operon in Staphylococcus aureus confers sensitivity to defensins, protegrins and other antimicrobial peptides, J. Biol. Chem., 274, 1999, 8405–8410.
Harder J., Bartels J., Christophers E. & Schröder J.-M.. A peptide antibiotic from human skin, Nature., 387, 1997, 861, .[Medline]
Lehrer R.I. & Ganz T.. Antimicrobial peptides in mammalian and insect host defence, Curr. Opin. Immunol., 11, 1999, 23–27.[Medline]
Liese J.G., Jendrossek V., Jansson A., Petropoulou T., Kloos S., Gahr M. & Belohradsky B.H.. Chronic granulomatous disease in adults, Lancet., 347, 1996, 220–223.[Medline]
Diamond G., Kaiser V., Rhodes J., Russel J.P. & Bevins C.L.. Transcriptional regulation of beta-defensin gene expression in tracheal epithelial cells, Infect. Immun., 68, 2000, 113–119.
Goldman M., Anderson G., Stolzenberg E.D., Kari U.P., Zasloff M. & Wilson J.M.. Human beta-defensin-1 is a salt-sensitive antibiotic in lung that is inactivated in cystic fibrosis, Cell., 88, 1997, 553–560.[Medline]
Groisman E.A.. How bacteria resist killing by host defense peptides, Trends Microbiol. Sci., 2, 1994, 444–448.
Hoffmann J.A., Kafatos F.C., Janeway C.A. & Ezekowitz R.A.. Phylogenetic perspectives in innate immunity, Science., 284, 1999, 1313–1318.
Jack R.W., Bierbaum G. & Sahl H.-G., Lantibiotics and Related Peptides, 1998, Springer-Verlag, Berlin, Germanypp. 224 pp.
Kokryakov V.N., Harwig S.S.L., Panyutich E.A., Shevchenko A.A., Aleshina G.M., Shamova O.V., Korneva H.A. & Lehrer R.I.. Protegrinsleukocyte antimicrobial peptides that combine features of corticostatic defensins and tachyplesins, FEBS Lett., 327, 1993, 231–236.[Medline]
Bechinger B.. Structure and functions of channel-forming peptidesmagainins, cecropins, melittin and alamethicin, J. Membr. Biol., 156, 1997, 197–211.[Medline]
Ausubel F.M., Brent R., Kingston R.E., Moore D.D., Seidman J.G., Smith J.A. & Struhl K., Current Protocols in Molecular Biology, 1990, John Wiley and Sons, Inc, New York.
Augustin J. & Götz F.. Transformation of Staphylococcus epidermidis and other staphylococcal species with plasmid DNA by electroporation, FEMS (Fed. Eur. Microbiol. Soc.) Microbiol. Lett., 66, 1990, 203–208.
Brückner R.. Gene replacement in Staphylococcus carnosus and Staphylococcus xylosus, FEMS (Fed. Eur. Microbiol. Soc.) Microbiol. Lett., 151, 1997, 1–8.
Brückner R.. A series of shuttle vectors for Bacillus subtilis and Escherichia coli, Gene., 122, 1992, 187–192.[Medline]
Peschel A., Ottenwälder B. & Götz F.. Inducible production and cellular location of the epidermin biosynthetic enzyme EpiB using an improved staphylococcal expression system, FEMS (Fed. Eur. Microbiol. Soc.) Microbiol. Lett., 137, 1996, 279–284.[Medline]
Götz F. & Schumacher B.. Improvements of protoplast transformation in Staphylococcus carnosus, FEMS (Fed. Eur. Microbiol. Soc.) Microbiol. Lett., 40, 1987, 285–288.
Harwig S.S.L., Ganz L. & Lehrer R.I.. Neutrophil defensinspurification, characterization, and antimicrobial testing, Methods Enzymol., 236, 1994, 160–172.[Medline]
Peschel A., Vuong C., Otto M. & Götz F.. The D-alanine residues of Staphylococcus aureus teichoic acids alter the susceptibility to vancomycin and the activity of autolysins, Antimicrob. Agents Chemother., 44, 2000, 2845–2847.
Schmitz F.J., Veldkamp K.E., Van Kessel K.P., Verhoef J. & Van Strijp J.A.. Delta-toxin from Staphylococcus aureus as a costimulator of human neutrophil oxidative burst, J. Infect. Dis., 176, 1997, 1531–1537.[Medline]
Klebanoff S.J., Waltersdorph A.M. & Rosen H.. Antimicrobial activity of myeloperoxidase, Methods Enzymol., 105, 1984, 399–403.[Medline]
Bremell T., Lange S., Yacoub A., Ryden C. & Tarkowski A.. Experimental Staphylococcus aureus arthritis in mice, Infect. Immun., 59, 1991, 2615–2623.
Bligh E.G. & Dyer W.J.. A rapid method of total lipid extraction and purification, Can. J. Biochem. Physiol., 37, 1959, 911–917.[Medline]
Nahaie M.R., Goodfellow M., Minnikin D.E. & Hajek V.. Polar lipid and isoprenoid quinone composition in the classification of Staphylococcus, J. Gen. Microbiol., 130, 1984, 2427–2437.
Frank H., Nicholson G. & Bayer E.. Enantiomer labelling, a method for the quantitative analysis of amino acids, J. Chromatogr., 167, 1978, 187–196.[Medline]
Wizemann T.M., Moskovitz J., Pearce B.J., Cundell D., Arvidson C.G., So M., Weissbach H., Brot N. & Masure H.R.. Peptide methionine sulfoxide reductase contributes to the maintenance of adhesins in three major pathogens, Proc. Natl. Acad. Sci. USA., 93, 1996, 7985–7990.
Massidda O., Dardenne O., Wahlen M.B., Zorzi W., Coyette J., Shockman G.D. & Daneo-Moore L.. The PBP 5 synthesis repressor (psr) gene in Enterococcus hirae ATCC 9790 is substantially longer than previously reported, FEMS (Fed. Eur. Microbiol. Soc.) Microbiol. Lett., 166, 1998, 355–360.[Medline]
Zuber P., Nakano M.M. & Marahiel M.A.. Peptide antibiotics, Sonenshein A.L.. Bacillus subtilis and Other Gram-positive Bacteria: Physiology and Molecular Genetics, 1993, 897–916, American Society for Microbiology, Washington, DC.
Haest C.W.M., de Gier J., op den Kamp J.A.F., Bartels P. & van Deenen L.L.M.. Changes in permeability of Staphylococcus aureus and derived liposomes with varying lipid composition, Biochim. Biophys. Acta., 255, 1972, 720–733.[Medline]
Short S.A. & White D.C.. Metabolism of phosphatidylglycerol, lysylphosphatidylglycerol, and cardiolipin of Staphylococcus aureus, J. Bacteriol., 108, 1971, 219–226.
Gutberlet T., Dietrich U., Bradaczek H., Pohlentz G., Leopold K. & Fischer W.. Cardiolipin, alpha-D-glucopyranosyl, and lysylcardiolipin from Gram-positive bacteriaFAB MS, monofilm and X-ray powder diffraction studies, Biochim. Biophys. Acta., 1463, 2000, 307–322.[Medline]
Tocanne J.F., Verheij H.M., op de Kamp J.A.F. & van Deenen L.L.M.. Chemical and physicochemical studies of lysylphosphatidylglycerol derivatives. Occurrence of a 2'-3' lysyl migration, Chem. Phys. Lipids., 13, 1974, 389–403.[Medline]
Fischer W. & Arneth-Seifert D.. D-Alanylcardiolipin, a major component of the unique lipid pattern of Vagococcus fluvialis, J. Bacteriol., 180, 1998, 2950–2957.
Lennarz W.J., Bonsen P.P.M. & van Deenen L.L.M.. Substrate specificity of O-L-lysylphosphatidylglycerol synthase. Enzymatic studies on the structure of O-L-lysylphosphatidylglycerol, Biochemistry, 6, 1967, 2307–2312.[Medline]
Kluytmans J., van Belkum A. & Verbrugh H.. Nasal carriage of Staphylococcus aureusepidemiology, underlying mechanism, and associated risk, Clin. Microbiol. Rev., 10, 1997, 505–520.[Abstract]
Lee S.H., Lim H.H., Lee H.M. & Choi J.O.. Expression of human beta-defensin 1 mRNA in human nasal mucosa, Acta Otolaryngol., 120, 2000, 58–61.[Medline]
Fischer W., Nakano M., Laine R.A. & Bohrer W.. On the relationship between glycerophosphoglycolipids and lipoteichoic acids in Gram-positive bacteria. I. The occurrence of phosphoglycolipids, Biochim. Biophys. Acta., 528, 1978, 288–297.[Medline]
Kenward M.A., Brown M.R. & Fryer J.J.. The influence of calcium or manganese on the resistance to EDTA, polymyxin B or cold shock, and the composition of Pseudomonas aeruginosa grown in glucose- or magnesium-depleted batch cultures, J. Appl. Bacteriol., 47, 1979, 489–503.[Medline]
Ratledge C. & Wilkinson S.G., Microbial Lipids, 1988, Academic Press, Londonpp. 963 pp.
Wirawan I.G., Kang H.W. & Kojima M.. Isolation and characterization of a new chromosomal virulence gene of Agrobacterium tumefaciens, J. Bacteriol., 175, 1993, 3208–3212.
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| TABLE OF CONTENTS |
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We used and show the synthetic variant A8,13,18-magainin II amide. 
MICs of the fluorescent derivative Lys(
) 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).
