Autophagy in Host Response to Porphyromonas gingivalis

Objectives: Numerous bacterial species have been implicated in the progression of chronic periodontitis, yet the strongest evidence supports the notion that Porphyromonas gingivalis is the keystone pathogen in disease. Previous studies suggest that lipopolysaccharide (LPS) variants alter autophagy processes in primary human gingival cells from patients with periodontal disease. In these studies, we tested the hypothesis that P. gingivalis (strain 33277) modulates gingival keratinocyte autophagy host response.
Methods: OKF6-TERT-1 cells (human keratinocytes) were cultured and allowed to reach confluence in a 1:1 mixture of K-sfm and DF-K media. Cells were challenged with P. gingivalis (MOI=100) and incubated for 30, 60 and 180 minutes at 37^oC. Cells were washed with DPBS and subsequently lysed with RIPA buffer. Proteins were separated via electrophoresis and quantified by immunoblot. Levels of the autophagy proteins; MREG, Rubicon, LC3B, LAMP1, Beclin1, Atg5, and Atg3 as well as gasdermin, a substrate for inflammatory caspases and executioner of pyroptotic cell death, were determined.
Results: We assessed both early and late host response to P. gingivalis; within the first 30 minutes, the lysosome protein LAMP1 decreased while all autophagy-associated protein levels increased. Moreover, gasdermin levels increased, albeit no gasdermin cleavage was detected. Cleavage, to a 30kDa product is indicative of potential for membrane pore formation. After 180 minutes of incubation with P. gingivalis, LAMP1 levels remained low, while autophagy associated proteins; MREG, LC3B, Beclin1, and Atg5 were increased. Also, gasdermin cleavage products were observed and correlated with increased LDH release and enhanced cytotoxicity.
Conclusions: Collectively, our results suggest that P. gingivalis likely persists in gingival keratinocytes. Research has provided evidence that P. gingivalis may contribute to the development and progression of diseases beyond periodontitis including rheumatoid arthritis, atherosclerosis, and cancer. To better manage these diseases, a more complete understanding of how P. gingivalis interacts with host cells is essential.