OmpA-Like Proteins of Porphyromonas gingivalis Mediate Resistance to the Antimicrobial Peptide LL-37

Abstract

Subgingival bacteria are continually exposed to gingival crevicular fluids that are derived from serum, which contain various bactericidal agents. The periodontopathic bacterium Porphyromonas gingivalis has been demonstrated to possess a variety of abilities to resist bactericidal agents, due to which it is able to propagate in the subgingival environment. We previously demonstrated that the major surface glycoproteins of P. gingivalis-Pgm6 and Pgm7, also called outer membrane protein A-like proteins (OmpALPs)-mediate resistance to the bactericidal activity of human serum, but their precise role remains unknown. In this study, we investigated the sensitivity of the wild-type and Pgm6/Pgm7-deficient P. gingivalis strains toward major antimicrobial peptides in the oral cavity, human β-defensins (hBDs) 1-3, and human cathelicidin LL-37. hBDs showed a considerably weak bactericidal activity against both bacterial strains. LL-37 also showed a weak activity against the wild-type strain; however, it showed a significant activity against the Pgm6/Pgm7-deficient strain. In the Pgm6/Pgm7-deficient strain, LL-37 remarkably accumulated on the bacterial cell surface, which may result in the destruction of the outer membrane. Additionally, the bactericidal activity of hBDs against the Pgm6/Pgm7-deficient strain was found to be synergistically promoted in the presence of LL-37. Our results suggest that OmpALPs specifically protect P. gingivalis from the bactericidal activity of LL-37; thus, P. gingivalis may adeptly survive in LL-37-producing subgingival environments.

Figure 1

Sensitivity of the wild-type and OmpALP-deficient strains of P. gingivalis to the bactericidal activities of hBD1, hBD2, hBD3, and LL-37. (a) Bacterial cells (10⁷) of the wild-type and Pgm6/Pgm7-deficient strains, suspended in sTSB containing the 2-fold serial concentrations of the indicated antimicrobial peptides (0.156–5 μM), were anaerobically cultured for 6 h. Bacterial survival was assessed by ATP production. Data are expressed as mean ± SD (n= 3). The statistical significance of differences was determined by the paired Student's t-test. ∗p < 0.05. (b) Approximately 10⁷ wild-type and Pgm6/Pgm7-deficient bacterial cells were anaerobically cultured for the indicated periods (6–48 h) in the presence of hBD1 or LL-37 (5 μM). Bacterial survival was assessed by ATP production. Data are expressed as mean ± SD (n= 3). The statistical significance of differences was determined by the paired Student's t-test. ∗p < 0.05. (c) Approximately 10⁷ wild-type or Pgm6/Pgm7-deficient bacterial cells were anaerobically cultured for 24 h in the presence of LL-37 (5 μM). The integrity of the outer membranes was assessed by fluorescence staining of bacterial cells with DMAO and EthD-III and then analyzed by fluorescence microscopy.

Figure 2

Sensitivity of the single OmpALP-deficient strains of P. gingivalis to the bactericidal activities of hBD1 and LL-37. (a, b) Approximately 10⁷ wild-type, Pgm6-deficient, Pgm7-deficient, or Pgm6/Pgm7-deficient bacterial cells were suspended in sTSB containing hBD1 or LL-37 (5 μM) and anaerobically cultured for 6 and 24 h. Bacterial survival was assessed by ATP production. Data are expressed as mean ± SD (n= 3). ∗p < 0.05, one-way ANOVA and Dunnett's test for post hoc comparisons (μc≠μi).

Figure 3

LL-37 on the cell surface was visualized by immunofluorescence staining in the LL-37-treated wild-type and OmpALP-deficient P. gingivalis cells. Approximately 10⁷ wild-type or Pgm6/Pgm7-deficient bacterial cells were treated with 5 μM LL-37 for 1 h. The presence of LL-37 on the surface of fixed bacterial cells was visualized by immunofluorescence staining and analyzed by fluorescence microscopy.

Figure 4

Sensitivity of the wild-type and Pgm6/Pgm7-deficient strains of P. gingivalis to the bactericidal activities of combinational treatment of hBD with LL-37. Bacterial cells (10⁷) of the wild-type and Pgm6/Pgm7-deficient strains, suspended in sTSB containing the indicated antimicrobial peptides (5 μM), were anaerobically cultured for 6 h. Bacterial survival was assessed by ATP production. Data are expressed as mean ± SD (n= 3). ∗p < 0.05, one-way ANOVA and Dunnett's test for post hoc comparisons (μc≠μi).