Effect of extracytoplasmic function sigma factors on autoaggregation, hemagglutination, and cell surface properties of Porphyromonas gingivalis

Abstract

Porphyromonas gingivalis is a bacterium frequently isolated from chronic periodontal lesions and is involved in the development of chronic periodontitis. To colonize the gingival crevice, P. gingivalis has to adapt to environmental stresses. Microbial gene expression is regulated by transcription factors such as those in two-component systems and extracytoplasmic function (ECF) sigma factors. ECF sigma factors are involved in the regulation of environmental stress response genes; however, the roles of individual ECF sigma factors are largely unknown. The purpose of this study was to investigate the functions, including autoaggregation, hemagglutination, gingipain activity, susceptibility to antimicrobial agents, and surface structure formation, of P. gingivalis ECF sigma factors encoded by SigP (PGN_0274), SigCH (PGN_0319), PGN_0450, PGN_0970, and SigH (PGN_1740). Various physiological aspects of the sigP mutant were affected; autoaggregation was significantly decreased at 60 min (p < 0.001), hemagglutination activity was markedly reduced, and enzymatic activities of Kgp and Rgps were significantly decreased (p < 0.001). The other mutants also showed approximately 50% reduction in Rgps activity. Kgp activity was significantly reduced in the sigH mutant (p < 0.001). No significant differences in susceptibilities to tetracycline and ofloxacin were observed in the mutants compared to those of the wild-type strain. However, the sigP mutant displayed an increased susceptibility to ampicillin, whereas the PGN_0450 and sigH mutants showed reduced susceptibility. Transmission electron microscopy images revealed increased levels of outer membrane vesicles formed at the cell surfaces of the sigP mutant. These results indicate that SigP is important for bacterial surface-associated activities, including gingipain activity, autoaggregation, hemagglutination, vesicle formation, and antimicrobial susceptibility.

Fig 1. Autoaggregation of Porphyromonas gingivalis wild type and extracytoplasmic function (ECF) sigma factor mutants.

(A) Representative images of strains at 60 min. The OD₆₆₀ of ECF sigma factor mutants (B) or the sigP mutant and sigP-complemented strain (C) were measured at 0, 10, 20, 30, and 60 min. Relative turbidities were calculated as follows: relative turbidity (%) = (turbidity at each time point) / (turbidity at 0 min) × 100. (D) Hydrophobicity. All assays were performed three times, and means ± SD (standard deviations) are shown.

Fig 2. Morphology and hemagglutinin activity of Porphyromonas gingivalis wild type and ECF sigma factor mutant strains.

(A) Colony pigmentation. The strains were grown anaerobically on enriched tryptic soy agar plates supplemented with 5% defibrinated horse blood at 37°C for 7 days. (B and C) Hemagglutination. The OD₆₀₀ of P. gingivalis ECF sigma factor mutant (B) or the sigP mutant and sigP-complemented strain (C) cultures were adjusted to 1.8, and the cultures were diluted 1 to 32 times. After diluting, horse erythrocyte suspensions (1% in PBS) were added to each of the wells.

Fig 3. Gingipain activities of the indicated Porphyromonas gingivalis strains.

Following 48 h of growth, P. gingivalis cultures were centrifuged. Gingipain activities in “culture supernatants” and “whole cells” of ECF sigma factor mutants (A) or the sigP mutant and its complemented strain (B) were normalized to those of P. gingivalis ATCC 33277, with the latter considered 100%. Data are expressed as means ± SD of nine independent experiments. *, p < 0.05; ***, p < 0.001.

Fig 4. Expression levels of rgpA, rgpB, kgp, and hagA in the indicated Porphyromonas gingivalis strains.

qRT-PCR analysis of the rgpA, rgpB, kgp, and hagA genes in ECF sigma factor mutants (A) or the sigP mutant and its complemented strain (B). Total RNA was extracted from P. gingivalis cells grown to an OD₆₆₀ of 0.6–0.8. All assays were performed nine times, and means ± SD (standard deviations) are shown. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Fig 5. Transmission electron microscopy images of Porphyromonas gingivalis wild-type and ECF sigma factor mutant strains.

The cells were stained for less than 1 min in 4% uranyl acetate and 0.2% lead citrate solutions. Scale bars are shown. The arrows indicate outer membrane vesicles.

Fig 6. Vesicle numbers and intermembrane distances in Porphyromonas gingivalis wild-type and ECF sigma factor mutant strains.

The number of vesicles per 1.4 μm² (15 specimen counted) (A) and inter-membrane distance (distance between the outer and cytoplasmic membranes) (at least 20 specimen counted) (B) were established from TEM images. Data are expressed as means ± SD. *, p < 0.05; ***, p < 0.001.

Fig 7. Virulence of Porphyromonas gingivalis W83 and the sigP mutant strain in a murine model.

Five BALB/c mice were inoculated subcutaneously with 0.1 mL of bacterial suspension at two sites on the depilated dorsal surface (0.2 mL per mouse), and the survival of the mice was monitored daily for up to 10 days. Three sets of experiments were carried out (15 mice in total). For the data analysis, Kaplan-Meier plots were constructed, and the log rank test was used to evaluate the differences in mean survival rates between mice infected with the W83 parent strain, sigP mutant, and complemented strain in three experiments.