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Porphyromonas gingivalis is a host-adapted oral pathogen associated with chronic periodontitis that successfully survives and persists in the oral epithelium. Recent studies have positively correlated periodontitis with increased risk and severity of oral squamous cell carcinoma (OSCC). Intriguingly, the presence of P. gingivalis enhances tumorigenic properties independently of periodontitis and has therefore been proposed as a potential etiological agent for OSCC. However, the initial host molecular changes induced by P. gingivalis infection which promote predisposition to cancerous transformation through EMT (epithelial-mesenchymal-transition), has never been studied in human primary cells which more closely mimic the physiological state of cells in vivo. In this study, we examine for the first time in primary oral epithelial cells (OECs) the expression and activation of key EMT mediators during long-term P. gingivalis infection in vitro. We examined the inactive phosphorylated state of glycogen synthase kinase-3 beta (p-GSK3β) over 120 h P. gingivalis infection and found p-GSK3β, an important EMT regulator, significantly increases over the course of infection (p < 0.01). Furthermore, we examined the expression of EMT-associated transcription factors, Slug, Snail, and Zeb1 and found significant increases (p < 0.01) over long-term P. gingivalis infection in protein and mRNA expression. Additionally, the protein expression of mesenchymal intermediate filament, Vimentin, was substantially increased over 120 h of P. gingivalis infection. Analysis of adhesion molecule E-cadherin showed a significant decrease (p < 0.05) in expression and a loss of membrane localization along with β-catenin in OECs. Matrix metalloproteinases (MMPs) 2, 7, and 9 are all markedly increased with long-term P. gingivalis infection. Finally, migration of P. gingivalis infected cells was evaluated using scratch assay in which primary OEC monolayers were wounded and treated with proliferation inhibitor, Mitomycin C. The cellular movement was determined by microscopy. Results displayed P. gingivalis infection promoted cell migration which was slightly enhanced by co-infection with Fusobacterium nucleatum, another oral opportunistic pathogen. Therefore, this study demonstrates human primary OECs acquire initial molecular/cellular changes that are consistent with EMT induction during long-term infection by P. gingivalis and provides a critically novel framework for future mechanistic studies.

Porphyromonas gingivalis, a major opportunistic pathogen in the etiology of chronic periodontitis, successfully survives in human gingival epithelial cells (GECs). P. gingivalis abrogates the effects of a host danger molecule, extracellular ATP (eATP)/P2X7 signaling, such as the generation of reactive oxygen species (ROS) via the mitochondria and NADPH oxidases (NOX) from primary GECs. However, antimicrobial functions of ROS production are thoroughly investigated in myeloid-lineage immune cells and have not been well-understood in epithelial cells. Therefore, this study characterizes antibacterial NOX2 generated ROS and host downstream effects in P. gingivalis infected human primary GECs. We examined the expression of NOX isoforms in the GECs and demonstrate eATP stimulation increased the mRNA expression of NOX2 (p < 0.05). Specific peptide inhibition of NOX2 significantly reduced eATP-mediated ROS as detected by DCFDA probe. The results also showed P. gingivalis infection can temporally modulate NOX2 pathway by reorganizing the localization and activation of cytosolic molecules (p47phox, p67phox, and Rac1) during 24 h of infection. Investigation into downstream biocidal factors of NOX2 revealed an eATP-induced increase in hypochlorous acid (HOCl) in GECs detected by R19-S fluorescent probe, which is significantly reduced by a myeloperoxidase (MPO) inhibitor. MPO activity of the host cells was assayed and found to be positively affected by eATP treatment and/or infection. However, P. gingivalis significantly reduced the MPO product, bactericidal HOCl, in early times of infection upon eATP stimulation. Analysis of the intracellular levels of a major host-antioxidant, glutathione during early infection revealed a substantial decrease (p < 0.05) in reduced glutathione indicative of scavenging of HOCl by P. gingivalis infection and eATP treatment. Examination of the mRNA expression of key enzymes in the glutathione synthesis pathway displayed a marked increase (p < 0.05) in glutamate cysteine ligase (GCL) subunits GCLc and GCLm, glutathione synthetase, and glutathione reductase during the infection. These suggest P. gingivalis modulates the danger signal eATP-induced NOX2 signaling and also induces host glutathione synthesis to likely avoid HOCl mediated clearance. Thus, we characterize for the first time in epithelial cells, an eATP/NOX2-ROS-antibacterial pathway and demonstrate P. gingivalis can circumvent this important antimicrobial defense system potentially for successful persistence in human epithelial tissues.