Date of Award

2018

Embargo Period

12-31-2023

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Oral Health Sciences

College

College of Graduate Studies

First Advisor

Özlem Yilmaz

Second Advisor

Besim Ogretmen

Third Advisor

Andrew Jakymiw

Fourth Advisor

Jennifer Mulligan

Fifth Advisor

Meenal Mehrotra

Abstract

Porphyromonas gingivalis, an opportunistic pathogen usurps gingival epithelial cells (GECs) as a primary intracellular niche for its colonization in the oral mucosa. P. gingivalis abrogates the effects of 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. Furthermore, P. gingivalis harnesses autophagic machinery to establish a replicative niche inside LC3-positive ER-rich autophagosomes. However, the antimicrobial functions of ROS production and precise characterization of the intracellular trafficking and fate of P. gingivalis in GECs are not well understood. Therefore, using a variety of phenotypic and molecular analyses (e.g. TEM, Confocal microscopy, Western-Blotting, ELISA, qPCR, enzyme-specific-activity assays), this study characterized an early host response (antibacterial-NOX2-ROS) and a mechanism of long-term survival (autophagy) which are utilized by P. gingivalis in human primary GECs. P. gingivalis infection can temporally inhibit eATP-induced NOX2 activation by inhibiting the organization of its key cytosolic molecules. However, prior to this inhibition, we observe the generation of host biocide, hypochlorous acid (HOCl), produced downstream of eATP-induced NOX2-ROS via myeloperoxidase. P. gingivalis is able to protect itself from the noxious effects of HOCl by utilizing a major host-antioxidant, glutathione, which scavenges ROS/HOCl. Therefore, P. gingivalis is able to successfully evade early bacterial clearance mechanisms of primary GECs. We further investigate survival and persistence of P. gingivalis through western-blotting and confocal microscopy. The results demonstrated that P. gingivalis co-localizes with autophagy marker, LC3, but not with lysosomal marker, LAMP-1. However, an ndk-deficient-mutant, less capable of surviving in GECs, differentially traffics to lysosomes during infection and is degraded. The survival of this mutant is increased through inhibition of ROS, LC3B, lysosomal-degradation, and the overexpression of phospho-HSP27, suggesting an important role for these pathways in P. gingivalis survival. Moreover, this ndk-deficient-mutant may be a model to delineate important autophagy and ROS-related P. gingivalis survival mechanisms. Thus, we characterize for the first time, in epithelial cells, an eATP/NOX2-ROS-antibacterial pathway and demonstrate a novel mechanism for P. gingivalis survival in GECs by commandeering host autophagy machinery to establish a successful replicative niche and persistence in the oral mucosa.

Rights

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