IADR Abstract Archives
Host-lysosomal pH role in the Aggregatibacter actinomycetemcomitans infection
Objectives: The intracellular bacteria Aggregatibacter actinomycetemcomitans (serotype b) is the most prevalent pathogen associated with aggressive periodontitis. Host-lysosomes are crucial for control and degradation of intracellular pathogens. Herein, we decided to investigate the role of host-cell lysosomal pH in the infection and dissemination of the periodontal pathogen A. actinomycetemcomitans in gingival epithelium and its correlation with the pathogenesis of aggressive periodontitis.
Methods: We generate an in vitro infection model, using human keratinocytes from the gingival junctional epithelium (OKF6/TERT-2 cells) to recreate the initial stages of periodontitis pathogenesis. The role of lysosomal degradative enzymes was addressed by treatment with alkalinizing drugs such bafilomycin A1, chloroquine and NH4Cl. The effects of host-lysosomes alkalinization on A. actinomycetemcomitans infection were analyzed by high-resolution confocal microscopy, gentamicin protection assay and flow cytometry.
Results: Alkalinization of lysosomes before bacterial invasion did not alter the cell structure nor subcellular localization or lysosomal number on the host-cell as determined by the confocal microscopy. Accordingly, host-cell invasion by A. actinomycetemcomitans was not affected upon lysosome alkalinization. Additionally, lysosome alkalinization post-invasion increased the number of intracellular bacteria, suggesting that alkalinization enhanced bacterial survival, and intracellular multiplication. Consequently, the increase in bacterial survival enhanced the intercellular dissemination raising the percentage of infected cells, a major step to induce bacterial persistence in periodontal tissues.
Conclusions: Taken together our results, strongly suggest that lysosomes play a critical role in the periodontitis pathogenesis following the cell invasion. Although the lysosome alkalinization did not alter the bacterial invasion rate we cannot rule out involvement of this cellular organelle in the initial steps leading to bacterial invasion. Our data reinforces that maintenance of acidic lysosomal pH is crucial for host-cell clearance of pathogenic intracellular bacteria. Further investigation are ongoing to determine the lysosomal pH role during bacterial persistence
Methods: We generate an in vitro infection model, using human keratinocytes from the gingival junctional epithelium (OKF6/TERT-2 cells) to recreate the initial stages of periodontitis pathogenesis. The role of lysosomal degradative enzymes was addressed by treatment with alkalinizing drugs such bafilomycin A1, chloroquine and NH4Cl. The effects of host-lysosomes alkalinization on A. actinomycetemcomitans infection were analyzed by high-resolution confocal microscopy, gentamicin protection assay and flow cytometry.
Results: Alkalinization of lysosomes before bacterial invasion did not alter the cell structure nor subcellular localization or lysosomal number on the host-cell as determined by the confocal microscopy. Accordingly, host-cell invasion by A. actinomycetemcomitans was not affected upon lysosome alkalinization. Additionally, lysosome alkalinization post-invasion increased the number of intracellular bacteria, suggesting that alkalinization enhanced bacterial survival, and intracellular multiplication. Consequently, the increase in bacterial survival enhanced the intercellular dissemination raising the percentage of infected cells, a major step to induce bacterial persistence in periodontal tissues.
Conclusions: Taken together our results, strongly suggest that lysosomes play a critical role in the periodontitis pathogenesis following the cell invasion. Although the lysosome alkalinization did not alter the bacterial invasion rate we cannot rule out involvement of this cellular organelle in the initial steps leading to bacterial invasion. Our data reinforces that maintenance of acidic lysosomal pH is crucial for host-cell clearance of pathogenic intracellular bacteria. Further investigation are ongoing to determine the lysosomal pH role during bacterial persistence