Date on Master's Thesis/Doctoral Dissertation

12-2017

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Interdisciplinary and Graduate Studies

Degree Program

Interdisciplinary Studies (Individualized Degree), PhD

Committee Chair

Lamont, Richard J.

Committee Member

Scott, David A.

Committee Member

Abu-kwaik, Yousef

Committee Member

Demuth, Donald R.

Author's Keywords

P. gingivalis; serine phosphatase SerB; Ptk1 tyrosine kinase; NF-kappaB; gingival epithelial cells; periodontal disease

Abstract

Periodontal diseases result from the interplay between the dysregulation of the host inflammatory response and the actions of a dysbiotic bacterial community. These chronic inflammatory conditions affect persons on a worldwide scale. P. gingivalis is strongly implicated as a key periodontal pathogen in the more severe manifestations of periodontal diseases such as periodontitis. A core element of P. gingivalis pathogenicity is its dysregulation of innate immunity, including suppression of IL-8 production by gingival epithelial cells. The NF-κB family of related transcription factors plays a central role in regulating many important aspects of innate immune responses. NF-κB RelA/p65 homodimers regulate transcription of IL-8. Here we show that SerB, a serine phosphatase secreted by P. gingivalis, is produced intracellularly and can specifically dephosphorylate S536 of p65 in gingival epithelial cells. A P. gingivalis mutant lacking SerB showed diminished ability to dephosphorylate p65 S536. Ectopically expressed SerB bound to p65 and co-localized with p65 in the cytoplasm. Ectopic expression of SerB also resulted in dephosphorylation of p65 with reduced nuclear translocation in TNF-stimulated epithelial cells. Conversely, the p105/50 subunit of NF-κB was unaffected by the presence of SerB. Co-expression of a constitutively active p65 mutant (S536D) relieved inhibition of nuclear translocation. IL-8 promoter activity and the production of IL-8 were both found to be inhibited by SerB. This study also characterized a bacterial tyrosine kinase in P. gingivalis, designated Ptk1, and demonstrated its involvement in interspecies signaling in the context of dual species P. gingivalis-S. gordonii communities. Our findings show that Ptk1 is capable of autophosphorylation, and is one of the substrates of P. gingivalis tyrosine phosphatase Ltp1, as it is dephosphorylated by Ltp1. In the Δptk1 mutant (no active Ptk1) there was significantly reduced accumulation of P. gingivalis on the S. gordonii substrate indicating that tyrosine kinase activity is required for optimal polymicrobial synergy. Collectively these findings illustrate (1) the molecular mechanisms underlying a bacterial strategy to manipulate the innate host response and host inflammatory pathways, and (2) that Ptk1 is an important component in the signaling networks that regulate synergistic interactions between P. gingivalis and S. gordonii.

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