The antibacterial activity of LL-37 against Treponema denticola is dentilisin protease independent and facilitated by the major outer sheath protein virulence factor

Abstract

Host defense peptides are innate immune effectors that possess both bactericidal activities and immunomodulatory functions. Deficiency in the human host defense peptide LL-37 has previously been correlated with severe periodontal disease. Treponema denticola is an oral anaerobic spirochete closely associated with the pathogenesis of periodontal disease. The T. denticola major surface protein (MSP), involved in adhesion and cytotoxicity, and the dentilisin serine protease are key virulence factors of this organism. In this study, we examined the interactions between LL-37 and T. denticola. The three T. denticola strains tested were susceptible to LL-37. Dentilisin was found to inactivate LL-37 by cleaving it at the Lys, Phe, Gln, and Val residues. However, dentilisin deletion did not increase the susceptibility of T. denticola to LL-37. Furthermore, dentilisin activity was found to be inhibited by human saliva. In contrast, a deficiency of the T. denticola MSP increased resistance to LL-37. The MSP-deficient mutant bound less fluorescently labeled LL-37 than the wild-type strain. MSP demonstrated specific, dose-dependent LL-37 binding. In conclusion, though capable of LL-37 inactivation, dentilisin does not protect T. denticola from LL-37. Rather, the rapid, MSP-mediated binding of LL-37 to the treponemal outer sheath precedes cleavage by dentilisin. Moreover, in vivo, saliva inhibits dentilisin, thus preventing LL-37 restriction and ensuring its bactericidal and immunoregulatory activities.

Fig 1

Inhibition of T. denticola growth by LL-37. T. denticola TD4 (ATCC 35404) (1 × 10⁸ cells/ml) was inoculated in GM-1 medium under anaerobic conditions at 37°C with or without increasing concentrations of LL-37. Bacterial growth was measured spectrophotometrically at 660 nm after 4 days. The optical density at time zero was considered 100% growth inhibition. The optical density of bacterial growth in the absence of LL-37 was considered 0% growth inhibition. The data are means and standard deviations for a representative experiment performed in triplicate and repeated twice.

Fig 2

Effects of exposure time, dentilisin protease, and the major outer sheath protein on the susceptibility of T. denticola to LL-37. T. denticola TD4 and T. denticola mutants K1 (lacking the outer sheath protease dentilisin) and MHE (lacking the major outer sheath protein) were incubated with 50 μg/ml LL-37 for the indicated times and were analyzed for intracellular ATP levels. The data are means and standard deviations for three independent experiments performed in duplicate. *, P < 0.0001 compared with T. denticola TD4 and T. denticola K1.

Fig 3

The binding of Alexa Fluor 350-labeled LL-37 to T. denticola is enhanced by the major outer sheath protein. Wild-type TD4 (A) and MHE mutant (lacking the major outer sheath protein) (B) cells were incubated with Alexa Fluor 350-labeled LL-37 and were analyzed by flow cytometry. Light shaded histograms, unlabeled bacteria; dark shaded histograms, bacteria reacted with Alexa Fluor 350-labeled LL-37. The geometric mean number for each histogram is given. The results shown are representative of three separate experiments.

Fig 4

LL-37 binds MSP. Bovine serum albumin (A), MSP (B), and fibrinogen (C), in the amounts indicated, were bound to nitrocellulose membranes and were reacted with LL-37 (1 μg/ml). Bound LL-37 was detected using anti-LL-37 rabbit serum as described in Materials and Methods.

Fig 5

LL-37 degradation by the T. denticola dentilisin. (A) LL-37 (10 μg) was incubated for 2 h at 37°C without (a) or with T. denticola TD4 (wild-type) supernatant (b), T. denticola K1 (dentilisin activity-deficient) supernatant (c), or T. denticola TD4 supernatant preincubated (20 min) with either 1 mM PMSF (d), 0.4 mM chymostatin (e), or saliva (13 μl cleared saliva/10 μl supernatant) (f). (B) LL-37 was incubated as described above without (a) or with purified dentilisin in the absence (b) or presence (c) of saliva (13 μl cleared saliva/2 × 10⁻³ U purified dentilisin).

Fig 6

LL-37 sites cleaved by dentilisin. (A) ESI-MS of LL-37-derived peptides. (B) MS-MS identification of the peaks generated by hydrolysis with purified dentilisin. (C) Intact LL-37 sequence. Arrows indicate the cleavage sites identified, and the proposed antibacterial region is underlined.

Fig 7

Inactivation of LL-37 by T. denticola supernatant. LL-37 incubated for 2 h without or with T. denticola supernatant or with heat-inactivated supernatant (boiled) was added to a growing culture of T. denticola TD4 (50 μg/ml) (A) or E. coli (20 μg/ml) (B). (A) Intracellular ATP was measured to calculate the percentage of treponeme survival. ATP levels of bacteria not treated with LL-37 were considered to represent 100% survival, and ATP levels of heat-killed bacteria (exposure to 100°C for 5 min) were defined as 0% survival. Data are means and standard deviations for three independent experiments performed in duplicate. *, P < 0.0001 compared with LL-37 incubated without T. denticola supernatant or with heat-inactivated (boiled) supernatant. (B) Incubations with untreated LL-37 and with no LL-37 were used as controls. The data are means and standard deviations for a representative experiment performed in duplicate and repeated 3 times.

Fig 8

Inhibition of dentilisin by saliva is dose dependent. A total of 2 × 10⁻³ units of purified dentilisin were preincubated with increasing volumes of cleared saliva at room temperature for 20 min. Dentilisin activity was determined by cleavage of the chromogenic substrate SAAPNA. The data are means and standard deviations for two independent experiments performed in duplicate.