Porphyromonas gingivalisis a crucial component of complex plaque biofilms that form in the oral cavity, resulting in the progression of periodontal disease. To elucidate the mechanism of periodontal biofilm formation, we analyzed the involvement of several genes related to the synthesis of polysaccharides inP. gingivalis. Gene knockoutP. gingivalismutants were constructed by insertion of anermF-ermAMcassette; among these mutants, thegalEmutant showed some characteristic phenotypes involved in the loss of GalE activity. As expected, thegalEmutant accumulated intracellular carbohydrates in the presence of 0.1% galactose and did not grow in the presence of galactose at a concentration greater than 1%, in contrast to the parental strain. Lipopolysaccharide (LPS) analysis indicated that the length of the O-antigen chain of thegalEmutant was shorter than that of the wild type. It was also demonstrated that biofilms generated by thegalEmutant had an intensity 4.5-fold greater than those of the wild type. Further, thegalEmutant was found to be significantly susceptible to some antibiotics in comparison with the wild type. In addition, complementation of thegalEmutation led to a partial recovery of the parental phenotypes. We concluded that thegalEgene plays a pivotal role in the modification of LPS O antigen and biofilm formation inP. gingivalisand considered that our findings of a relationship between the function of theP. gingivalis galEgene and virulence phenotypes such as biofilm formation may provide clues for understanding the mechanism of pathogenicity in periodontal disease.

Propolis, a resinous substance produced by bees, is used as a folk medicine for treatment of periodontal diseases. However, its mode of the action and the compounds responsible for its activities remain obscure. In the present study, we comprehensively investigated the antibacterial activities of ethanol-extracted propolis (EEP) and EEP-derived compounds toward Porphyromonas gingivalis, a keystone pathogen for periodontal diseases. Broth microdilution and agar dilution assays were used to determine the minimum inhibitory concentrations of EEP against a range of oral bacterial species, of which P. gingivalis showed a higher level of sensitivity than oral commensals such as streptococci. Its antibacterial activity toward P. gingivalis was maintained even after extensive heat treatment, demonstrating a high level of thermostability. EEP also induced death of P. gingivalis cells by increasing membrane permeability within 30 min. Spatiotemporal analysis based on high-speed atomic force microscopy revealed that EEP immediately triggered development of aberrant membrane blebs, followed by bleb fusion events on the bacterial surface. Furthermore, we isolated artepillin C, baccharin, and ursolic acid from EEP as antibacterial compounds against P. gingivalis. Of those, artepillin C and baccharin showed bacteriostatic activities with membrane blebbing, while ursolic acid showed bactericidal activity with membrane rupture. In particular, ursolic acid demonstrated a greater ability to affect bacterial membrane potential with increased membrane permeability, probably because of its highly lipophilic nature as compared with other compounds. Taken together, these findings provide mechanistic insight into the antibacterial activities of EEP and its exquisite membrane-targeting antibacterial compounds and imply the applicability of narrow-spectrum therapeutics with EEP for treatment of periodontitis. In addition, the advanced technology utilized in the present study to visualize the nanometer-scale dynamics of microorganisms will contribute to expanding our understanding of the activities of antimicrobials and the mechanism of drug resistance in bacteria.

Summary: Bacterial membrane vesicles (MVs) have attracted strong interest in recent years as novel nanoparticle delivery platforms. Glycine is known to induce morphological changes in the outer layer of bacteria. We report here that glycine dramatically facilitates MV production in a flagella‐deficient mutant of the non‐pathogenic probiotic Escherichia coli strain Nissle 1917. Supplementation of culture medium with 1.0% glycine induced cell deformation at the early exponential phase, eventually followed by quasi‐lysis during the late exponential to stationary phase. Glycine supplementation also significantly increased the number of MVs with enlarged particle size and altered the protein profile with an increase in the inner membrane and cytoplasmic protein contents as compared to non‐induced MVs. Of note, the endotoxin activity of glycine‐induced MVs was approximately eightfold or sixfold lower than that of non‐induced MVs when compared at equal protein or lipid concentrations respectively. Nevertheless, glycine‐induced MVs efficiently induced both immune responses in a mouse macrophage‐like cell line and adjuvanticity in an intranasal vaccine mouse model, comparable to those of non‐induced MVs. We propose that the present method of inducing MV production with glycine can be used for emerging biotechnological applications of MVs that have immunomodulatory activities, while dramatically reducing the presence of endotoxins.