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. 2013 Oct;81(10):3577-85.
doi: 10.1128/IAI.01288-12. Epub 2013 Jul 8.

LL-37 opsonizes and inhibits biofilm formation of Aggregatibacter actinomycetemcomitans at subbactericidal concentrations

Asaf Sol  1 , Ofir Ginesin, Stella Chaushu, Laila Karra, Shunit Coppenhagen-Glazer, Isaac Ginsburg, Gilad Bachrach
Affiliations

LL-37 opsonizes and inhibits biofilm formation of Aggregatibacter actinomycetemcomitans at subbactericidal concentrations

Asaf Sol et al. Infect Immun. 2013 Oct.

Abstract

Host defense peptides are immediate responders of the innate immunity that express antimicrobial, immunoregulatory, and wound-healing activities. Neutrophils are a major source for oral host defense peptides, and phagocytosis by neutrophils is a major mechanism for bacterial clearance in the gingival tissue. Dysfunction of or reduction in the numbers of neutrophils or deficiency in the LL-37 host defense peptide was each previously linked with proliferation of oral Aggregatibacter actinomycetemcomitans which resulted in an aggressive periodontal disease. Surprisingly, A. actinomycetemcomitans shows resistance to high concentrations of LL-37. In this study, we demonstrated that submicrocidal concentrations of LL-37 inhibit biofilm formation by A. actinomycetemcomitans and act as opsonins and agglutinins that greatly enhance its clearance by neutrophils and macrophages. Improved uptake of A. actinomycetemcomitans by neutrophils was mediated by their opsonization with LL-37. Enhanced phagocytosis and killing of A. actinomycetemcomitans by murine macrophage-like RAW 264.7 cells were dependent on their preagglutination by LL-37. Although A. actinomycetemcomitans is resistant to the bactericidal effect of LL-37, our results offer a rationale for the epidemiological association between LL-37 deficiency and the expansion of oral A. actinomycetemcomitans and indicate a possible therapeutic use of cationic peptides for host defense.

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Figures

Fig 1
Fig 1
Resistance of A. actinomycetemcomitans to LL-37. (A) MICs of A. actinomycetemcomitans strains tested in this study. (B and C) Growth inhibition (see Materials and Methods) of A. actinomycetemcomitans strains and E. coli ATCC 25922 grown in the presence of increasing concentrations of LL-37 (B) or scrambled LL-37 (C) (22.2 μM). *, P < 0.05 compared to untreated control (no LL-37) using the Student t test.
Fig 2
Fig 2
LL-37 binds A. actinomycetemcomitans at submicrocidal concentrations. (A) Fluorescence microscopy analysis of binding of 4.4 μM and 11.1 μM tetramethylrhodamine-labeled LL-37 (red) to FITC-stained A. actinomycetemcomitans JP2 (green). (B) Flow cytometry analysis of A. actinomycetemcomitans strains incubated with increasing concentrations of tetramethylrhodamine-labeled LL-37. Graphs indicate mean cell percentages ± standard deviations from three independent experiments.
Fig 3
Fig 3
Binding of LL-37 to A. actinomycetemcomitans is sequence specific and sensitive to high ionic strength. (A) Flow cytometry analysis of A. actinomycetemcomitans JP2 incubated with increasing concentrations of tetramethylrhodamine-labeled LL-37 and 6-FAM-labeled scrambled LL-37. (B) A. actinomycetemcomitans incubated with tetramethylrhodamine-labeled LL-37 in the presence (+sLL-37) or absence (no sLL-37) of unlabeled 11.1 μM scrambled LL-37. (C) Flow cytometry histogram of binding of 11.1 μM tetramethylrhodamine-labeled LL-37 (TMR-LL-37) to A. actinomycetemcomitans JP2 at physiological ionic strength (PBS) (red) or high ionic strength (200 mM NaCl [black line] or 50 mM MgCl2 [blue line]). The gray filled line represents JP2 without LL-37. Graphs and numbers indicate cell percentages ± standard deviations from two independent experiments.
Fig 4
Fig 4
LL-37 inhibits biofilm formation of A. actinomycetemcomitans. Biofilm formation of A. actinomycetemcomitans strains on 96-well microtiter plates grown in the presence of increasing concentrations of LL-37 (A) or 22.2 μM scrambled LL-37 (B) for 24 h (see Materials and Methods) is shown.
Fig 5
Fig 5
LL-37 acts as an opsonin of A. actinomycetemcomitans. (A) FACS analysis (fold increase of mean fluorescence intensity [MFI]) of human neutrophils incubated with FITC-labeled A. actinomycetemcomitans JP2 in the absence (black lines) or presence (red lines) of 6.6 μM, 8.8 μM, and 11.1 μM LL-37 and 11.1 μM scrambled LL-37. Neutrophils without labeled bacteria and without LL-37 were used as baseline, which is represented by a filled blue histogram. (B) Fold increase of mean fluorescence intensity of A. actinomycetemcomitans (A. a) opsonization in the presence of increasing concentrations of LL-37. Neutrophils with A. actinomycetemcomitans only were used as a baseline. (C) Fold increase of mean fluorescence intensity of A. actinomycetemcomitans VT726S Ltx− opsonization after incubation with LL-37 or scrambled LL-37. Neutrophils with labeled bacteria and no LL-37 were used as a baseline. (D) Fluorescence microscopy of human neutrophils (arrows) incubated with FITC-labeled A. actinomycetemcomitans JP2 (A. a) in the absence or presence of LL-37. *, P < 0.05 compared to baseline using the Student t test.
Fig 6
Fig 6
LL-37 has only a moderate effect on binding of A. actinomycetemcomitans by murine macrophages. Macrophage-like RAW 264.7 cells were incubated with FITC-labeled A. actinomycetemcomitans in the absence (black lines) or presence (red lines) of increasing concentrations of LL-37 (6.6 μM, 8.8 μM, and 11.1 μM). The fold increase in mean fluorescence intensity of A. actinomycetemcomitans JP2 binding to RAW cells in the presence of LL-37 is shown. RAW cells without labeled bacteria and with no LL-37 are represented by a filled blue line. RAW cells incubated with labeled bacteria without LL-37 were used as a baseline for fold increase calculations.
Fig 7
Fig 7
Preincubation of A. actinomycetemcomitans JP2 with LL-37 enhances its phagocytosis by murine macrophages. FITC-labeled A. actinomycetemcomitans (A. a) was incubated in the absence of tetramethylrhodamine-labeled LL-37 or in the presence of increasing concentrations of tetramethylrhodamine-labeled LL-37 (6.6 μM, 8.8 μM, and 11.1 μM) for 10 min at room temperature, washed twice, incubated with macrophage-like RAW 264.7 cells for 2 h, washed twice, and analyzed using fluorescence microscopy. Colocalization analysis appears above the colocalization panels for each concentration. Mean fluorescence intensities from two independent experiments are shown in the bottom panel.
Fig 8
Fig 8
LL-37 enhances killing of A. actinomycetemcomitans by murine macrophages. A. actinomycetemcomitans JP2 was incubated with macrophage-like RAW 264.7 cells at a multiplicity of infection of 10 for 2 h in the presence of LL-37 or scrambled LL-37 at increasing concentrations. Murine macrophage killing of A. actinomycetemcomitans JP2 was determined after 2 h of incubation by plating the samples on blood agar. A. actinomycetemcomitans JP2 without macrophages was used as a reference of bacteria number and no killing (0%).
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