Microbial Degradation of Cellular Kinases Impairs Innate Immune Signaling and Paracrine TNFα Responses.

Kenneth Barth,Caroline Attardo Genco

doi:10.1038/srep34656

Abstract

The NFκB and MAPK signaling pathways are critical components of innate immunity that orchestrate appropriate immune responses to control and eradicate pathogens. Their activation results in the induction of proinflammatory mediators, such as TNFα a potent bioactive molecule commonly secreted by recruited inflammatory cells, allowing for paracrine signaling at the site of an infection. In this study we identified a novel mechanism by which the opportunistic pathogen Porphyromonas gingivalis dampens innate immune responses by disruption of kinase signaling and degradation of inflammatory mediators. The intracellular immune kinases RIPK1, TAK1, and AKT were selectively degraded by the P. gingivalis lysine-specific gingipain (Kgp) in human endothelial cells, which correlated with dysregulated innate immune signaling. Kgp was also observed to attenuate endothelial responsiveness to TNFα, resulting in a reduction in signal flux through AKT, ERK and NFκB pathways, as well as a decrease in downstream proinflammatory mRNA induction of cytokines, chemokines and adhesion molecules. A deficiency in Kgp activity negated decreases to host cell kinase protein levels and responsiveness to TNFα. Given the essential role of kinase signaling in immune responses, these findings highlight a unique mechanism of pathogen-induced immune dysregulation through inhibition of cell activation, paracrine signaling, and dampened cellular proinflammatory responses.

Highlights

We previously demonstrated that P. gingivalis degrades both RIPK1 and RIPK2 in endothelial cells and macrophages in a dose and fimbriae dependent manner[22]
We previously reported that P. gingivalis mediated degradation of RIPK1 in human endothelial cells occurred early after stimulation with consistent generation of lower molecular weight fragments that were not products of new protein synthesis[22]
human umbilical vein endothelial cells (HUVEC) were immediately cocultured with medium or pretreated preparations of P. gingivalis for 2 hr. (f) HUVEC were cocultured with wild-type strains 381, 33277, or gingipain mutant strains (ΔRgpA, ΔRgpA/B, ΔKgp) at an MOI 100 for 2 hr. (a–f) Whole cell lysates were analyzed for RIPK1, TAK1, cellular inhibitor of apoptosis 2 (cIAP2), IKKβ, P- IKKβ, IκBα, P-IκBα, AKT, ubiquitin, ERK, or GAPDH via Western blot analysis

Summary

Introduction

Antibacterial peptides, complement, cell adhesion molecules, coreceptors or MAPK pathways leads to dysregulation of cytokine networks, cell activation/recruitment, opsonization/phagocytosis and neutrophil function, effectively subverting protective proinflammatory host responses[16,17,18,19,20,21]. These studies highlight a significant role for gingipains on disruption of innate immunity and suggest dysregulation of host cell signaling may be a critical factor in many of these evasion strategies. Given the essential role of kinase signaling in immune responses, these findings highlight a novel pathogen mechanism of immune dysregulation via impairment of innate immune signaling and paracrine TNFαresponses

Methods

Results

Conclusion