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The Porphyromonas gingivalis O antigen is required for inhibition of apoptosis in gingival epithelial cells following bacterial infection
Author(s) -
Soto C.,
Bugueño I.,
Hoare A.,
Gonzalez S.,
Venegas D.,
Salinas D.,
MelgarRodríguez S.,
Vernal R.,
Gamonal J.,
Quest A. F. G.,
PérezDonoso J. M.,
Bravo D.
Publication year - 2016
Publication title -
journal of periodontal research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.31
H-Index - 83
eISSN - 1600-0765
pISSN - 0022-3484
DOI - 10.1111/jre.12331
Subject(s) - porphyromonas gingivalis , antigen , biology , microbiology and biotechnology , apoptosis , lipopolysaccharide , viability assay , immunology , bacteria , biochemistry , genetics
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 evaluate 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 ( OKF 6/ TERT 2 line) were infected by different freshly prepared P. gingivalis clinical isolates, obtained from subjects with chronic periodontitis ( CP 3 and CP 4) 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 TLR 4 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 TLR 4 (nearly sixfold), which correlated with inhibition of apoptosis. Conclusion 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 TLR 4 expression, suggesting a role for TLR 4 in this process.