Inactivation of the dlt Operon inStaphylococcus aureus Confers Sensitivity to Defensins, Protegrins, and Other Antimicrobial Peptides

Andreas Peschel,Michael Otto,Ralph W Jack,Hubert Kalbacher,Günther Jung,Friedrich Götz

doi:10.1074/jbc.274.13.8405

Andreas Peschel, Michael Otto + Show 4 more

Open Access

https://doi.org/10.1074/jbc.274.13.8405

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Abstract

Positively charged antimicrobial peptides with membrane-damaging activity are produced by animals and humans as components of their innate immunity against bacterial infections and also by many bacteria to inhibit competing microorganisms. Staphylococcus aureus and Staphylococcus xylosus, which tolerate high concentrations of several antimicrobial peptides, were mutagenized to identify genes responsible for this insensitivity. Several mutants with increased sensitivity were obtained, which exhibited an altered structure of teichoic acids, major components of the Gram-positive cell wall. The mutant teichoic acids lacked D-alanine, as a result of which the cells carried an increased negative surface charge. The mutant cells bound fewer anionic, but more positively charged proteins. They were sensitive to human defensin HNP1-3, animal-derived protegrins, tachyplesins, and magainin II, and to the bacteria-derived peptides gallidermin and nisin. The mutated genes shared sequence similarity with the dlt genes involved in the transfer of D-alanine into teichoic acids from other Gram-positive bacteria. Wild-type strains bearing additional copies of the dlt operon produced teichoic acids with higher amounts of D-alanine esters, bound cationic proteins less effectively and were less sensitive to antimicrobial peptides. We propose a role of the D-alanine-esterified teichoic acids which occur in many pathogenic bacteria in the protection against human and animal defense systems.

Highlights

Charged antimicrobial peptides with membrane-damaging activity are produced by animals and humans as components of their innate immunity against bacterial infections and by many bacteria to inhibit competing microorganisms
We propose a role of the D-alanine-esterified teichoic acids which occur in many pathogenic bacteria in the protection against human and animal defense systems
To investigate the resistance mechanism, S. xylosus C2a was mutagenized with Tn917 as described under “Experimental Procedures,” and the resulting transposon insertion mutants were analyzed for reduced growth on agar plates containing the antimicrobial peptide gallidermin

Summary

EXPERIMENTAL PROCEDURES

The culture was subsequently diluted 100-fold in BM broth containing 2.5 ␮g of erythromycin/ml and incubated for 14 h at 42 °C to select for transposon insertion mutants. This culture was diluted again in the same way and grown for another 14 h at 42 °C. Isolation of WTA and LTA—Bacteria were grown overnight in 500 ml of BM broth containing 0.25% (S. xylosus) or 0.3% glucose (S. aureus), harvested by centrifugation, and washed in 100 ml of sodium acetate buffer (20 mM, pH 4.6). In order to isolate WTA, 500-␮l aliquots of the crude cell extracts were diluted 4-fold in sodium acetate buffer containing 2% SDS, sonicated for 15 min, and vigorously shaken for 1 h at 60 °C. The amount of gallidermin in the supernatant was determined by RP-HPLC analysis using a linear gradient from 30 to 60% acetonitrile in 0.1% trifluoroacetic acid over 20 column volumes on a Spherisorb ODS2 column (Grom Analytik, Herrenberg, Germany)

RESULTS

DISCUSSION

19 NDa NDa