The Porphyromonas gingivalis O antigen is required for inhibition of apoptosis in gingival epithelial cells following bacterial infection Soto C, Buguen ˜o I, Hoare A, Gonzalez S, Venegas D, Salinas D, Melgar- Rodr  ıguez S, Vernal R, Gamonal J., Quest A. F. G, Pe ´rez-Donoso J. M, Bravo D. The Porphyromonas gingivalis O antigen is required for inhibition of apoptosis in gingival epithelial cells following bacterial infection. J Periodont Res 2015; doi:10.1111/jre.12331. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Background and 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 evalu- ate whether the P. gingivalis lipopolysaccharide (LPS), specifically the O-antigen region, affects adherence, invasion, viability and apoptosis of gingival epithelial cells. Material and 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 Try- pan 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 anal- ysis 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 iso- lates 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 inhibi- tion of apoptosis. C. Soto 1 , I. Bugue ~ no 1 , A. Hoare 1 , S. Gonzalez 1 , D. Venegas 1 , D. Salinas 1 , S. Melgar-Rodr  ıguez 2 , R. Vernal 2 , J. Gamonal 2 , A. F. G. Quest 3,4 , J. M. P erez- Donoso 5 , D. Bravo 1 1 Oral Microbiology Laboratory, Department of Pathology and Oral Medicine, Faculty of Dentistry, Universidad de Chile, Santiago, Chile, 2 Laboratory of Periodontal Biology, Department of Conservative Dentistry, Faculty of Dentistry, Universidad de Chile, Santiago, Chile, 3 Advanced Center for Chronic Diseases (ACCDiS), Universidad de Chile, Santiago, Chile, 4 Laboratory of Cell Communication, Center for Molecular Studies of the Cell, Institute of Biomedical Sciences, Faculty of Medicine, Universidad de Chile, Santiago, Chile and 5 BioNanotechnology and Microbiology Laboratory, Center for Bioinformatics and Integrative Biology (CBIB), Faculty of Biological Sciences, Universidad Andres Bello, Santiago, Chile Denisse Bravo Rodr  ıguez, PhD, Oral Microbiology Laboratory, Department of Pathology and Oral Medicine, Faculty of Dentistry, Universidad de Chile, Sergio Livingstone Pohlhammer 943, Independencia, Santiago, Chile Tel: 56 2 29781832 e-mail: denbravo@uchile.cl Key words: apoptosis; gingival epithelial cells; lipopolysaccharide; O-antigen; Porphyromonas gingivalis Accepted for publication September 5, 2015 J Periodont Res 2015 All rights reserved © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd JOURNAL OF PERIODONTAL RESEARCH doi:10.1111/jre.12331