Porphyromonas Evades Ubiquitination-NDP52/p62 Machinery and Induces Autophagosome-like Vacuoles for Survival

P. gingivalis is a major etiological agent in the development of periodontal disease. The ability to survive and replicate in gingival epithelial cells (GECs), which are the first line of defense of oral mucosa, is critical to the success of P. gingivalis as a pathogen. Our recent studies showed activation of autophagy is an important mechanism of P. gingivalis’ survival in GECs where the organism rapidly positions itself in endoplasmic-reticulum (ER) and induces autophagosome-like vacuoles for affluent intracellular life. Nevertheless, detailed characterization of the intracellular fate of P. gingivalis in GECs has yet to be fully elucidated. Objective: To study the spatio-temporal fate of intracellular P. gingivalis in GECs and examine the role of ubiquitin-binding-adaptor proteins NDP52/p62 for targeting of P. gingivalis to lysosomal degradation pathway.Method: Primary GECs were infected with wild-type-strain or FMN-green-fluorescent-strain (PgFbFP) over 24-hours. ER-Tracker or Lysosome-marker LAMP-1 were used to determine subcellular localization of the organism in GECs by confocal microscopy. Use of PgFbFP-strain in conjunction with digitonin treatment and anti-P. gingivalis red-fluorescent-antibody staining followed by anti-NDP52 or anti-p62 blue-fluorescent-antibody staining permitted measurement of the co-localization of vacuolar versus cytosolic bacteria with the ubiquitination markers. Result: Concordant with our recent findings, confocal analyses displayed steady-state level of co-localizations between P. gingivalis and ER (~90%) over 24-hours, whereas less than 25% of the bacteria were associated with lysosomes. P. gingivalis appeared to predominantly reside in the vacuoles, while small percentage of bacteria was found in the cytosol, distinctly marked by the NDP52/p62. This was statistically significant at P<0.01 t-test compared to P. gingivalisin the vacuoles. Conclusion: These results indicate P. gingivalis utilize ER-rich-autophagosomes for successful persistence and evade anti-microbial ubiquitin-lysosomal-degradation pathway. This new knowledge may lead to highly targeted therapeutic interventions for controlling P. gingivalis’ colonization in the oral mucosa by using specific autophagy inhibitors.