Date of Award

Fall 8-29-2024

Embargo Period

8-29-2029

Document Type

Dissertation - MUSC Only

Degree Name

Doctor of Philosophy (Medical Science)

Department

Oral Health Sciences

College

College of Graduate Studies

First Advisor

Ozlem Yilmaz

Abstract

Porphyromonas gingivalis, a major, host-adapted, oral pathobiont, evades canonical host pathogen clearance in human-primary-gingival-epithelial-cells (GECs) by initiating a non-canonical variant of autophagy consisting of Microtubule-associated-protein-1A/1B-light-chain-3 (LC3)-rich replicative autophagosomes. Simultaneously, P. gingivalis inhibits oxidative-stress, including extracellular-ATP (eATP)-mediated reactive-oxygen-species (ROS) production, via phosphorylating Heat-Shock-Protein-27 (HSP27) with the bacterial nucleoside-diphosphate-kinase (Ndk) and via inducing Glutathione-Peroxidase-1 (GpX1) in a glutamine (Gln) metabolism-mediated manner. Here, we have mechanistically identified how the host anti-stress molecules HSP27 and GpX1 abet P. gingivalis’ autophagic survival in GECs. Specifically, we define that P. gingivalis-mediated induction of HSP27 is crucial for the recruitment of the LC3-isoform, LC3C, to drive the formation of live P. gingivalis-containing, Beclin-1-ATG14-rich, autophagosomes that are redox-sensitive and non-degrading. Additionally, HSP27 depletion of infected GECs, accompanied by eATP-treatment, removed protracted Beclin-1-ATG14 partnering and significantly decreased live intracellular P. gingivalis levels. These events were partially restored via treatments with the antioxidant N-acetyl cysteine (NAC), which rescued the cellular redox-environment independent of HSP27. Moreover, the temporal-phosphorylation of HSP27 by the bacterial Ndk resulted in HSP27 tightly binding with LC3C, hindering LC3C canonical-cleavage, extending Beclin-1-ATG14 associations, and halting canonical-autophagosomal-maturation. Separately, P. gingivalis was found to induce GpX1 to partner with LC3C following autophagosomal formation. This partnering induces Akt phosphorylation on Ser473, which then phosphorylated the maturation modulator UV-Radiation-Resistant-Associated-Gene-Protein (UVRAG) to further inhibit canonical maturation of P. gingivalis-specific autophagosomes. Additionally, the negative effects of GpX1 depletion of infected GECs, accompanied by eATP-treatment, reverted when concurrently treated with exogenous-Gln, the major precursor of Gln metabolism, which P. gingivalis also alters to increase Glutathione (GSH) synthesis in GECs. HSP27 and GpX1 depletions of infected GECs and gingiva-mimicking organotypic-culture-systems resulted in the collapse of P. gingivalis-mediated autophagosomes. GpX1-KO mouse models confirmed that the depletion of these host molecules abolished P. gingivalis-induced LC3C-specific autophagic-flux and human-gingival-biopsy-specimens were utilized to reconfirm the pro-bacterial interactions in P. gingivalis-associated periodontitis. These findings pinpoint how HSP27 and GpX1 are influenced by P. gingivalis to pleiotropically serve as major platform-molecules, redox-regulators, and stepwise-modulators of LC3C to mediate pro-bacterial autophagy. Thus, our findings can determine specific molecular strategies for interfering with the host-adapted P. gingivalis’ successful mucosal colonization and oral dysbiosis.

Rights

Copyright is held by the author. All rights reserved.

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Available for download on Wednesday, August 29, 2029

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