Heparin-LL37 complexes are less cytotoxic for human dental pulp cells and have undiminished antimicrobial and LPS-neutralizing abilities.

AIM: To investigate whether glycosaminoglycans (GAGs) binding to high-dose LL37 eliminates its cytotoxicity to dental pulp cells (hDPCs) whilst retaining undiminished antimicrobial and LPS-neutralizing abilities. METHODOLOGY: hDPCs were stimulated with varying concentrations of LL37, and their cell viability was analysed by MTT. Then, high-dose LL37 (10 µmol L-1 ) was bound to varying concentrations of three GAGs, heparin, chondroitin sulphate and hyaluronic acid, and their cytotoxic effects on hDPCs and antimicrobial effects were evaluated and compared. Furthermore, the LPS-neutralizing ability of heparin (5 µg mL-1 )-LL37 (10 µmol L-1 ) complexes, which were found to be less cytotoxic for hDPCs with undiminished antimicrobial ability, was investigated. Statistical analysis was performed using one-way analysis of variance (anova), followed by Dunnett's test. P values below 0.05 were considered significant. RESULTS: LL37 significantly reduced the cell viability of hDPCs in a dose-dependent manner (P < 0.01). LL37 (10 µmol L-1 ) binding to heparin within a limited concentration range (2~6 µg mL-1 ) eliminated the cytotoxicity for hDPCs (P < 0.01) whilst exerting potent antimicrobial effects against Streptococcus mutans, Streptococcus sobrinus, Streptococcus salivarius, Aggegatibacter actinomycetemcomitans and Escherichia coli. LL37 (10 µmol L-1 ) binding to chondroitin sulphate exhibited similar functions (P < 0.01); however, the effective chondroitin sulphate concentration was highly restricted (3 µg mL-1 ). LL37 (10 µmol L-1 ) binding to hyaluronic acid was unable to abrogate the cytotoxicity of LL37 even at higher concentrations (10 and 100 µg mL-1 ). Moreover, exogenous addition of LPS dose-dependently reduced the amount of LL37 precipitated with the heparin-LL37 agarose beads (P < 0.01), and the released LL37 simultaneously neutralized the pro-inflammatory ability of LPS in macrophages (P < 0.01). CONCLUSIONS: Heparin-LL37 complexes generated at suitable concentration ratios are easy to make, are less cytotoxic and are broad-range antimicrobial materials that can neutralize LPS by providing LL37 in accordance with the amount of free LPS. They may be a potential treatment to save dental pulp tissue from the acute inflammation exacerbated by invading bacteria and the LPS they release.

Association of CiaRH with resistance of Streptococcus mutans to antimicrobial peptides in biofilms.

Streptococcus mutans is a cariogenic pathogen in humans. To persist in the oral cavity, S. mutans is resistant against several antibacterial factors derived from the host. In this study, we investigated the mechanism of resistance to cationic antimicrobial peptides (AMPs), which are innate immune factors in humans. Because dltA-D (teichoic acid biosynthesis) was reported to affect the susceptibility to AMPs in other bacterial species, we evaluated the susceptibility of a dltC knockout mutant of S. mutans to the AMPs human beta-defensin-1 (hBD1), hBD2, hBD3 and LL37. The dltC mutant exhibited significantly increased susceptibility to AMPs. Regulation of dltC expression involved CiaRH, a two-component system. Expression of dltC in the wild-type strain was significantly increased in biofilm cells compared with that in planktonic cells, whereas expression was not increased in a ciaRH knockout mutant. In biofilm cells, we found that susceptibility to LL37 was increased in the ciaRH mutant compared with that in the wild type. From these results, it is concluded that Dlt is involved in the susceptibility of S. mutans to AMPs and is regulated by CiaRH in biofilm cells.

Role of two component signaling response regulators in acid tolerance of Streptococcus mutans.

INTRODUCTION: In bacteria, two-component systems (TCS) involving the products of a histidine kinase gene (hk) and a response regulator gene (rr) play important roles in adaptation to environmental changes. Fourteen hk-rr homologs and one orphan rr homolog were identified in the Streptococcus mutans UA159 genome database. There have been no comprehensive evaluations of the roles of rr homologs in the acid tolerance of S. mutans. METHODS: The TCS genes (tcs) of S. mutans were designated smtcs01-15. Mutants of S. mutans UA159 with deletions of rr and hk-rr were constructed. Acid tolerance was evaluated by comparing the doubling times at pH 7.2 and pH 5.5 between the wild-type and mutant strains. RESULTS: Excluding smtcs10 and 12, for which viable mutants could not be obtained, a total of 13 rr deletion mutants were constructed. The rr deletions in smtcs03, 05, 08, and 13 resulted in diminished acid tolerance in comparison with UA159. The hk-rr double-mutants exhibited acid sensitivity levels similar to those of the corresponding rr mutants. The results of the present study reveal the involvement of the rr genes of smtcs03 and 05 in acid tolerance. Deletion of hk and/or rr in smtcs03 generated an acid-sensitive phenotype. In contrast, for smtcs05, while deletion of rr resulted in reduced acid tolerance, a single-deletion of hk had no effect on acid tolerance. CONCLUSIONS: We implicated two rr genes in the acid tolerance of S. mutans. In particular, smtcs05 is a novel tcs, the sole rr of which is involved in the acid tolerance of S. mutans.