Human beta-defensin 3 inhibits cell wall biosynthesis in Staphylococci

Abstract

Human beta-defensin 3 (hBD3) is a highly charged (+11) cationic host defense peptide, produced by epithelial cells and neutrophils. hBD3 retains antimicrobial activity against a broad range of pathogens, including multiresistant Staphylococcus aureus, even under high-salt conditions. Whereas antimicrobial host defense peptides are assumed to act by permeabilizing cell membranes, the transcriptional response pattern of hBD3-treated staphylococcal cells resembled that of vancomycin-treated cells (V. Sass, U. Pag, A. Tossi, G. Bierbaum, and H. G. Sahl, Int. J. Med. Microbiol. 298:619-633, 2008) and suggested that inhibition of cell wall biosynthesis is a major component of the killing process. hBD3-treated cells, inspected by transmission electron microscopy, showed localized protrusions of cytoplasmic contents, and analysis of the intracellular pool of nucleotide-activated cell wall precursors demonstrated accumulation of the final soluble precursor, UDP-MurNAc-pentapeptide. Accumulation is typically induced by antibiotics that inhibit membrane-bound steps of cell wall biosynthesis and also demonstrates that hBD3 does not impair the biosynthetic capacity of cells and does not cause gross leakage of small cytoplasmic compounds. In in vitro assays of individual membrane-associated cell wall biosynthesis reactions (MraY, MurG, FemX, and penicillin-binding protein 2 [PBP2]), hBD3 inhibited those enzymes which use the bactoprenol-bound cell wall building block lipid II as a substrate; quantitative analysis suggested that hBD3 may stoichiometrically bind to lipid II. We report that binding of hBD3 to defined, lipid II-rich sites of cell wall biosynthesis may lead to perturbation of the biosynthesis machinery, resulting in localized lesions in the cell wall as demonstrated by electron microscopy. The lesions may then allow for osmotic rupture of cells when defensins are tested under low-salt conditions.

FIG. 1.

Cartoon of the membrane-associated cell wall biosynthesis reactions in S. aureus. The biosynthesis reactions at the inner face of the cytoplasmic membrane catalyzed by MraY, MurG, and FemXAB produce the membrane-bound precursor molecule lipid II (undecaprenylpyrophosphate-MurNAc[pentapeptide]-GlcNAc) with the pentaglycine interpeptide bridge attached. The cell wall building block is then translocated across the membrane by an unknown mechanism and subsequently incorporated into the growing peptidoglycan network. When bactoprenol cycling is inhibited by antibiotics at any stage, the ultimate soluble precursor UDP-MurNAc-pentapeptide (boxed) accumulates in the cytoplasm.

FIG. 2.

Intracellular accumulation of the ultimate soluble cell wall precursor UDP-N-acetylmuramyl pentapeptide (UDP-MurNAc-pentapeptide) in S. aureus SG511. Cells were treated with 5× MIC of the respective antibiotic compound, incubated for 30 min, and subsequently extracted with boiling water. The intracellular nucleotide pool was analyzed by applying standardized aliquots to reversed-phase HPLC. (A) Untreated control cells. (B) Vancomycin-treated (4 mg/liter) positive-control cells. (C) LL37-treated (73.625 mg/liter) cells. (D) hBD3-treated (27.8 mg/liter) cells. (E) hBD3-treated cells growing in cell culture broth (27.8 mg/liter). (Inset) Mass spectrum of the peak containing UDP-MurNAc-pentapeptide; the calculated mass is 1,149.43; in addition, the mono- and disodium salts are detectable.

FIG. 3.

Transmission electron microscopy micrograph of S. aureus SG511 treated with 15 μM hBD3. (Panel 1) Cells treated for 10 min; cells did not show any sign of damage and did not differ from untreated controls (not shown). (Panels 2 to 5) Cells treated for 30 min; in approximately 5 to 10% of cells, membrane protrusions with cytoplasmic contents were observed (panels 3 and 4; panel 4 shows a magnification of the area indicated by an arrow in panel 2); additionally, 5 to 10% of the cells showed aberrant septum formation, as in panel 5. (Panels 6 to 9) Cells treated for 60 min; the number of cells with membrane protrusions (panels 6 and 7) and with additional septa (panel 8) increased, and more damaged cells with signs of general lysis (panel 9) occurred. Scale bar, 200 nm.

FIG. 4.

Impact of detergent on the lipid II-hBD3 interactions. Effect of hBD3 on the FemX-catalyzed addition of glycine to lipid II in vitro in the presence (dark gray bars) or absence (light gray bars) of Triton X-100.

FIG. 5.

Inhibition of the PBP2-catalyzed reaction by hBD3 in vitro. (A) The conversion of lipid II into polymeric peptidoglycan in the presence of increasing concentrations of hBD3 was qualitatively analyzed using TLC and PMA staining. (B) For quantitative analysis of the reaction, [¹⁴C]lipid II was used as a substrate; reaction mixtures were applied directly onto TLC plates and developed in solvent B (butanol-acetic acid-water-pyridine [15:3:12:10, vol/vol/vol/vol]) with subsequent detection and quantification of residual [¹⁴C]lipid II using storage phosphor technology.

FIG. 6.

Impact of cationic peptides on the lipid II-hBD3 interactions. In vitro inhibition of the PBP2-catalyzed reaction by Pep5 (+8) or Lys-Lys-Lys (+3) and by hBD3 (+11) in the presence of Pep5 or Lys-Lys-Lys.