ABSTRACT
<p><b>OBJECTIVE</b>This work aims to determine the effect of cytosolic bacteria on the expression of cyclic GMP-AMP synthase (cGAS) in human periodontal ligament cells (hPDLCs) and gingival tissues.</p><p><b>METHODS</b>The ability of Porphyromonas gingivalis (P. gingivalis) to invade hPDLCs was detected using laser scanning confocal microscope assay at a multiplicity of infection of 10. P. gingivalis-infected cells were sorted by fluorescence-activated cell sorting (FACS). Then, quantitative real time reverse transcription polymerase chain reaction (qRT-PCR) and Western blot were used to detect cGAS expression in infected cells. Finally, the location and expression of cGAS in inflammatory and normal gingival tissues were investigated by immunohistochemistry.</p><p><b>RESULTS</b>P. gingivalis actively invaded hPDLCs. Moreover, cGAS expression significantly increased in P. gingivalis-infected cells. Although cGAS was expressed in the epithelial and subepithelial cells of both inflamed and normal gingival tissues, cGAS expression significantly increased in inflamed gingival tissues.</p><p><b>CONCLUSIONS</b>Cytosolic bacteria can upregulate cGAS expression in infected cells. These data suggest that cGAS may act as pattern-recognition receptors and participate in recognizing cytosolic nucleic acid pathogen-associated molecular patterns. .</p>
Subject(s)
Humans , Blotting, Western , Cells, Cultured , Flow Cytometry , Gingiva , Nucleotides, Cyclic , Periodontal Ligament , Porphyromonas gingivalis , Real-Time Polymerase Chain ReactionABSTRACT
<p><b>OBJECTIVE</b>To test whether Porphyromonas gingivalis (P. gingivalis) could produce bacterial signal molecule, bis-(3'-5')-cyclic dimeric adenosine monophosphate (c-di-AMP) and lay the foundation for explorations of its roles in life metabolism and periodontitis immunity of P. gingivalis.</p><p><b>METHODS</b>P. gingivalis standard strain ATCC33277 was used as the experimental strain to extract nucleic acids from the bacteria. Then, c-di-AMP was detected using high performance liquid chromatography coupled with mass spectrometry (HPLC-MS/MS). Subsequently, HPLC was used to validate the sample further.</p><p><b>RESULTS</b>Based on the signal/noise (S/N) for 3 : 1, the limit of determination of HPLC-MS/MS for peak time of c-di-AMP standard substances was 7.49 min and nucleic acid extractions from P. gingivalis was 8.82 min (S/N > 3). Further confirmation of HPLC showed that nucleic acid extractions from both P. gingivalis and c-di-AMP standard substances pre- sented goal absorbent peaks at 15.7 min, with the same ultraviolet absorbent spectrum.</p><p><b>CONCLUSION</b>The nucleic acid extrac- tions from P. gingivalis contained c-di-AMP, which shows that P. gingivalis could produce c-di-AMP.</p>