PG0717 in Porphyromonas gingivalis is Essential to Induce Endothelial Autophagy

Objectives: Porphyromonas gingivalis (Pg) has been associated with multiple systemic diseases, such as atherosclerosis. Our group has reported that Pg strain W83 utilizes the autophagic pathway to persist within autophagosomes in human coronary artery endothelial cells (HCAEC). Deletion of PG0717 produced an altered virulence phenotype mutant with diminished gingipain activity and an inability to activate autophagy, thereby surviving within host endothelial cells. The objective of this study was to restore expression of PG0717 in W83Δ717, assess the restoration of the mutant phenotype, and establish a function role for PG0717.
Methods: W83Δ717 was complemented by knock-in of PG0717 with a TetQ resistance gene. PG0717 gene
expression was confirmed by qRT-PCR. Overnight cultures of W83, W83Δ717, or W83Δ717::717 were suspended in a protease reaction buffer containing cysteine. Arginine and lysine gingipain activities were measured using Nα-Benzoyl-DL-arginine-p-nitroanilide or Ac-lysine-p-nitroanilide HCL, respectively. Gingipain activities were calculated from A₄₀₅ vs. time curves using a picomolar extinction coefficient of 9200 for the product, p-nitroanilide. To assess the autophagy response, HCAECs were transduced with adenoviral-mCherry-GFP-LC3, a marker for autophagosomes and autolysosomes. The cells were infected with W83, W83Δ717, or W83Δ717::717 at an MOI of 100. At 6 and 24 hours post-infection, the cells were fixed and imaged by fluorescence microscopy.
Results: Both arginine and lysine gingipain activities in W83Δ717::717 were restored to wild-type levels. We have previously shown that W83 traffics to autophagosomes and W83Δ717 does not traffic to the autophagosomes. The complemented mutant, W83Δ717::717, was present within autophagosomes at 6 and 24 hours post-infection indicating that autophagy activation was restored.
Conclusions: Our results suggest a role for PG0717 in the regulation of gingipain activities and in the induction of autophagy during invasion of host cells. Future studies are required to understand how PG0717 functions and is regulated, especially with respect to its operon, a two-component system (PG0719 and PG0720).