title: The Porphyromonas gingivalis O antigen is required for inhibition of apoptosis in gingival epithelial cells following bacterial infection.
abstract: OBJECTIVE
  Porphyromonas gingivalis infection induces apoptosis inhibition in gingival epithelial cells; however, it is not fully understood which bacterial effectors are involved in this process. The aim of this study is to evaluate whether the P. gingivalis lipopolysaccharide (LPS), specifically the O-antigen region, affects adherence, invasion, viability and apoptosis of gingival epithelial cells.
  
  
  METHODS
  Gingival epithelial cells (OKF6/TERT2 line) were infected by different freshly prepared P. gingivalis clinical isolates, obtained from subjects with chronic periodontitis (CP3 and CP4) and healthy individuals (H1 and H3). Periodontitis and healthy isolates show differences in O-antigen production, as healthy isolates lack the O-antigen region. In addition, cells were infected by a site-specific mutant lacking the O-antigen portion. After 24 h postinfection, cell proliferation, viability and apoptosis were evaluated by Trypan blue, MTS and annexin V assays, respectively. Bacterial invasion, adhesion and proliferation were measured by gentamicin/metronidazole protection assays. Finally, toll-like receptor (TLR)2 and TLR4 mRNA expression was evaluated by quantitative reverse transcription-polymerase chain reaction. Statistical analysis was performed using ANOVA, Tukey's or Dunnett's tests (p < 0.05).
  
  
  RESULTS
  At 24 h postinfection, strains lacking the O-antigen region (healthy isolates and O-antigen ligase-deficient strain) were unable to increase proliferation and viability, or decrease apoptosis as compared with strains producing intact LPS (periodontitis isolates and reference strain). However, the presence of the O-antigen neither contributed to changes in the ability of the bacteria to adhere to or invade cells, nor to intracellular survival. The presence of O-antigen also increased the expression of TLR4 (nearly sixfold), which correlated with inhibition of apoptosis.
  
  
  CONCLUSIONS
  The O-antigen region of P. gingivalis LPS is required to increase gingival epithelial cell viability upon infection by bacteria and this increase is attributable to a reduction in apoptosis. Moreover, although bacterial internalization is required, the effects observed are not due to alterations in P. gingivalis adherence, invasion or intracellular survival. Interestingly, inhibition of apoptosis correlates with increased TLR4 expression, suggesting a role for TLR4 in this process.
authors.affiliation: Oral Microbiology Laboratory, Department of Pathology and Oral Medicine, Faculty of Dentistry, Universidad de Chile, Santiago, Chile.
authors.forename: C
authors.initials: C
authors.lastname: Soto
authors.affiliation: Oral Microbiology Laboratory, Department of Pathology and Oral Medicine, Faculty of Dentistry, Universidad de Chile, Santiago, Chile.
authors.forename: I
authors.initials: I
authors.lastname: Bugueño
authors.affiliation: Oral Microbiology Laboratory, Department of Pathology and Oral Medicine, Faculty of Dentistry, Universidad de Chile, Santiago, Chile.
authors.forename: A
authors.initials: A
authors.lastname: Hoare
authors.affiliation: Oral Microbiology Laboratory, Department of Pathology and Oral Medicine, Faculty of Dentistry, Universidad de Chile, Santiago, Chile.
authors.forename: S
authors.initials: S
authors.lastname: Gonzalez
authors.affiliation: Oral Microbiology Laboratory, Department of Pathology and Oral Medicine, Faculty of Dentistry, Universidad de Chile, Santiago, Chile.
authors.forename: D
authors.initials: D
authors.lastname: Venegas
authors.affiliation: Oral Microbiology Laboratory, Department of Pathology and Oral Medicine, Faculty of Dentistry, Universidad de Chile, Santiago, Chile.
authors.forename: D
authors.initials: D
authors.lastname: Salinas
authors.affiliation: Laboratory of Periodontal Biology, Department of Conservative Dentistry, Faculty of Dentistry, Universidad de Chile, Santiago, Chile.
authors.forename: S
authors.initials: S
authors.lastname: Melgar-Rodríguez
authors.affiliation: Laboratory of Periodontal Biology, Department of Conservative Dentistry, Faculty of Dentistry, Universidad de Chile, Santiago, Chile.
authors.forename: R
authors.initials: R
authors.lastname: Vernal
authors.affiliation: Laboratory of Periodontal Biology, Department of Conservative Dentistry, Faculty of Dentistry, Universidad de Chile, Santiago, Chile.
authors.forename: J
authors.initials: J
authors.lastname: Gamonal
authors.affiliation: Advanced Center for Chronic Diseases (ACCDiS), Universidad de Chile, Santiago, Chile.|Laboratory of Cell Communication, Center for Molecular Studies of the Cell, Institute of Biomedical Sciences, Faculty of Medicine, Universidad de Chile, Santiago, Chile.
authors.forename: A F G
authors.initials: AF
authors.lastname: Quest
authors.affiliation: BioNanotechnology and Microbiology Laboratory, Center for Bioinformatics and Integrative Biology (CBIB), Faculty of Biological Sciences, Universidad Andres Bello, Santiago, Chile.
authors.forename: J M
authors.initials: JM
authors.lastname: Pérez-Donoso
authors.affiliation: Oral Microbiology Laboratory, Department of Pathology and Oral Medicine, Faculty of Dentistry, Universidad de Chile, Santiago, Chile.
authors.forename: D
authors.initials: D
authors.lastname: Bravo
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D001424:Bacterial Infections; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D015870:Gene Expression; D005881:Gingiva / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D019081:O Antigens / Q000494:pharmacology*; D010518:Periodontitis; D016966:Porphyromonas gingivalis / Q000302:isolation & purification / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism; D051195:Toll-Like Receptor 2 / Q000235:genetics / Q000378:metabolism; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism
chemical_list: D008070:Lipopolysaccharides; D019081:O Antigens; D012333:RNA, Messenger; C495342:TLR2 protein, human; C495345:TLR4 protein, human; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4
keywords: O-antigen; Porphyromonas gingivalis; apoptosis; gingival epithelial cells; lipopolysaccharide