Deletion of Lipoprotein PG0717 in Porphyromonas gingivalis W83 Reduces Gingipain Activity and Alters Trafficking in and Response by Host Cells

Abstract

P. gingivalis (Pg), a causative agent of chronic generalized periodontitis, has been implicated in promoting cardiovascular disease. Expression of lipoprotein gene PG0717 of Pg strain W83 was found to be transiently upregulated during invasion of human coronary artery endothelial cells (HCAEC), suggesting this protein may be involved in virulence. We characterized the virulence phenotype of a PG0717 deletion mutant of pg W83. There were no differences in the ability of W83Δ717 to adhere and invade HCAEC. However, the increased proportion of internalized W83 at 24 hours post-inoculation was not observed with W83∆717. Deletion of PG0717 also impaired the ability of W83 to usurp the autophagic pathway in HCAEC and to induce autophagy in Saos-2 sarcoma cells. HCAEC infected with W83Δ717 also secreted significantly greater amounts of MCP-1, IL-8, IL-6, GM-CSF, and soluble ICAM-1, VCAM-1, and E-selectin when compared to W83. Further characterization of W83Δ717 revealed that neither capsule nor lipid A structure was affected by deletion of PG0717. Interestingly, the activity of both arginine (Rgp) and lysine (Kgp) gingipains was reduced in whole-cell extracts and culture supernatant of W83Δ717. RT-PCR revealed a corresponding decrease in transcription of rgpB but not rgpA or kgp. Quantitative proteome studies of the two strains revealed that both RgpA and RgpB, along with putative virulence factors peptidylarginine deiminase and Clp protease were significantly decreased in the W83Δ717. Our results suggest that PG0717 has pleiotropic effects on W83 that affect microbial induced manipulation of host responses important for microbial clearance and infection control.

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

“…An additional use of GFP-LC3 is to monitor colocalization with a target during autophagy-related processes such as organelle degradation or the sequestration of pathogenic microbes. [299][300][301][302] Preincubation of cells stably expressing GFP-LC3 with leupeptin can help stabilize the GFP-LC3 signal during fluorescence microscopy, especially under conditions of induced autophagic flux. Leupeptin is an inhibitor of lysosomal cysteine and serine proteases and will therefore inhibit degradation of membrane-conjugated GFP-LC3 that is present within autolysosomes.…”

Porphyromonas gingivalis induces autophagy in THP‐1‐derived macrophages

“…Porphyromonas gingivalis lipopolysaccharide (LPS) leads to reactive oxygen species-mediated autophagy (Bullon et al, 2012), and the lipoprotein gene PG0717 of P. gingivalis is required for the activation of autophagy in Saos-2 cell lines (Reyes et al, 2013). The P. gingivalis traffics to the autophagosome and subverts autophagy by delaying the fusion of the late autophagosome with the lysosome for survival (Dorn et al, 2001).…”

A Sialidase‐Deficient Porphyromonas gingivalis Mutant Strain Induces Less Interleukin‐1β and Tumor Necrosis Factor‐α in Epi4 Cells Than W83 Strain Through Regulation of c‐Jun N‐Terminal Kinase Pathway

“…Gingipains have diverse functions. They are indispensable for nutrient uptake 40 and are involved in interactions between P. gingivalis and host cells, including P. gingivalis adhesion, invasion, survival and host cell autophagy, along with microbial clearance and infection control 36 , 41 . In the present study, rgp gene expression and activities of Kgp and Rgp decreased in △PG0352, but there was no difference in kgp gene expression among the different P. gingivalis strains, suggesting that sialidase deficiency affected activities of Kgp and Rgp in different ways.…”