Degradation of human antimicrobial peptide LL-37 by Staphylococcus aureus-derived proteinases

Abstract

Cathelicidin LL-37 is one of the few human bactericidal peptides with potent antistaphylococcal activity. In this study we examined the susceptibility of LL-37 to proteolytic degradation by two major proteinases produced by Staphylococcus aureus, a metalloproteinase (aureolysin) and a glutamylendopeptidase (V8 protease). We found that aureolysin cleaved and inactivated LL-37 in a time- and concentration-dependent manner. Analysis of the generated fragments by mass spectroscopy revealed that the initial cleavage of LL-37 by aureolysin occurred between the Arg19-Ile20, Arg23-Ile24, and Leu31-Val32 peptide bonds, instantly annihilating the antibacterial activity of LL-37. In contrast, the V8 proteinase hydrolyzed efficiently only the Glu16-Phe17 peptide bond, rendering the C-terminal fragment refractory to further degradation. This fragment (termed LL-17-37) displayed antibacterial activity against S. aureus at a molar level similar to that of the full-length LL-37 peptide, indicating that the antibacterial activity of LL-37 resides in the C-terminal region. In keeping with LL-37 degradation by aureolysin, S. aureus strains that produce significant amounts of this metalloprotease were found to be less susceptible to LL-17-37 than strains expressing no aureolysin activity. Taken together, these data suggest that aureolysin production by S. aureus contributes to the resistance of this pathogen to the innate immune system of humans mediated by LL-37.

FIG. 1.

Concentration-dependent effect of staphylococcal proteinases on the antibacterial activity of LL-37. LL-37 (3 mg/ml) was mixed with an equal volume of aureolysin (A and B) or the V8 protease (C and D) in 20 mM Tris, pH 7.8, with 1 mM CaCl₂ to yield indicated concentrations of both proteinases. The digested samples were tested for antibacterial action against S. aureus strain Newman (A and C) and E. coli ATCC 33694 (B and D). The diameter of the bacterial growth inhibition zones was measured and converted into units (0.1 mm = 1 U of inhibition growth). Each point in the figure represents a mean ± standard deviation of at least two independent triplicate assays. Insets in panels C and D show representative results of bacterial growth inhibition by LL-37 alone and after preincubation with two different concentrations of the V8 protease.

FIG. 2.

Cleavage sites of LL-37 and its N-terminally truncated form (LL-17-37) by aureolysin (A and B) and the V8 proteinase (C). Solid and dotted arrows indicate primary and secondary cleavage sites, respectively.

FIG. 3.

Comparison of bacteriostatic and bactericidal activity of LL-37 and LL-17-37. The Newman strain of S. aureus in the logarithmic phase was mixed with basic agar and poured on the sterile petri dishes. After agar solidification, the wells were punched and 2.5-μl aliquots of the peptide at concentrations of 4, 3, 2, 1, 0.5, and 0.25 mg/ml were added. Plates were incubated for 3 h at 37°C, and then nutritious TSA was poured on the basic agar. Plates were incubated overnight at 37°C, dried, and stained with Coomassie brilliant blue, and the diameters of zones of inhibition growth were measured (inset) and plotted against the log of peptide concentration (in micrograms/milliliter). Each point represents a mean ± standard deviation of three independent duplicate assays. From the intercept with the x axis, the MIC was calculated at 30 and 152 μg/ml for LL-17-37 and LL-37, respectively. The inset shows representative results of bacterial growth inhibition by different concentrations of LL-37 and LL-17-37.

FIG. 4.

Proteolytic activity in media from the late-logarithmic phase of growth of isogenic mutants of the S. aureus strains Newman and SH1000. The activity was visualized either by gelatin-zymography analysis (A) or was quantified using azocasein as the substrate (B). The contribution of V8 proteinase (full arrowhead in panel A and stippled bars in panel B) and aureolysin (empty arrowhead and crossed bars) to total proteolytic activity was determined with the specific inhibitors 3,4-dichloroisocoumarin and o-phenanthroline, respectively.

FIG. 5.

Comparison of antistaphylococcal activity of LL-17-37 against S. aureus strains Newman and SH1000 and their sarA mutants. The bacteriostatic and bactericidal activity of LL-17-37 was determined using the radial diffusion assay. The diameter of the bacterial growth inhibition zones was measured before and after staining the agarose plates with Coomassie brilliant blue (inset) and was converted into units (0.1 mm = 1 U of inhibition growth). The units were plotted against the log of peptide concentration (in micrograms/milliliter), and the MIC was calculated. Each point represents a mean ± standard deviation of at least six independent duplicate assays. The statistically significant differences (Student's test) in MIC in comparison to that of the wild-type strain within the same group of isogenic mutants are indicated by asterisks: *, P < 0.01; **, P < 0.001.