Journal: PLoS Pathogens
Article Title: Pathogen-Mediated Proteolysis of the Cell Death Regulator RIPK1 and the Host Defense Modulator RIPK2 in Human Aortic Endothelial Cells
Figure Lengend Snippet: Model of P. gingivalis innate immune activation/invasion in endothelial cells. Outcome of innate immune responses is representative as a balance of functional, intact pathways (TLR-left panel) and dysregulated pathways (NLR-right panel). P. gingivalis represents a human pathogen that utilizes fimbriae for attachment and invasion of endothelial cells. Fimbriae are not only expressed on whole bacteria ( A ), but within outer membrane vesicles (OMV) that are released from the cell ( B ), as occurs with all Gram-negative bacteria identified to date. Fimbriae bind to TLR2 and MD2 to activate TLR2 and TLR4, resulting in the induction of NF-κB, leading to inflammation, and de novo protein synthesis ( C ) of cell adhesion molecules ( CAM ) and TLR ( TLR ) expressed at the cell surface ( D ). Many studies have demonstrated invasion of endothelial cells by P. gingivalis ( E ). However, recent studies have shown that OMV (containing fimbriae and active gingipain activity) gain entry into host cells rapidly in a gingipain-dependent manner ( E ), independent of whole organism. Upon entry, intracellular gingipain activity degrades RIPK1 and RIPK2 (abrogated by protease inhibitors) ( F ), resulting in a variety of possible consequences as a function of selective targeting of intracellular NOD1 or NOD2 (NOD1/2) signaling pathways and/or disruption of RIPK1 or RIPK2-mediated cell signaling. Alteration of immune signaling responses may result in decreased host cell death, decreased inflammatory mediator expression, and subsequent enhancement of intracellular bacterial cell survival, all of which contributes to an intracellular niche for P. gingivalis ( G ).
Article Snippet: P. gingivalis strain 381 was pretreated with 10 µM KYT-1, 10 µM KYT-36, 10 µM KYT-1 and 10 µM KYT-36, 1 mM TLCK, 100 µM zVAD-fmk, 100 µM BocD-fmk with or vehicle controls (HEPES, DMSO or acid water) for 45 min, then immediately co-cultured with 0.1 µg recombinant RIPK2 kinase (Invitrogen) for 1 h in a humidified chamber at 37°C with 5% CO2 .
Techniques: Activation Assay, Functional Assay, Chick Chorioallantoic Membrane Assay, Activity Assay, Expressing