M1167 GEF-H1 Mediates NOD1 Dependent NF-κB Activation Initiated By Shigella Flexneri Effectors

Abstract

Aim: To elucidate S. flexneri effectors and host mediators which signal the disruption of the apical junctional complex and initiate GEF-H1 mediated RhoA dependent activation of NF-kB in the epithelial cells. Methods: Membrane recruitment of S. flexneri effectors was assessed by live cell imaging of MDCK cells transfected with GFPand RFP-tagged GEFH1, IpgB2 and OspB1 expression vectors using the confocal microscopy. Shigella effectors found to target GEF-H1 in tight junctions were transfected into HEK293 cells along with a reporter construct containing NF-kB consensus binding motifs in the presence of control or specific Nod1 siRNA or GEF-H1 siRNA and stimulated with the NOD1 ligand γTriDAP or control peptides for 24 hours. NF-kB mediated transcriptional activation was assessed by dual-luciferase assays. Protein interactions were determined by immunoprecipitations. Results: IpgB2 localized to cellular junctions within epithelial monolayers, while OspB1 associated with an intracellular vesicular compartment close to cell membranes. Expression of IpgB2 and OspB1 in polarized epithelial cells resulted in the redistribution of GEF-H1 from tight junctions to basolateral membrane compartment and the cytosol. When expressed as GFP-fusion proteins IpgB2 and OspB1 were found to activate NF-kB activity 5.9±0.7 and 9.1±2.4 fold above background levels, respectively (p<0.05 vs basal). Remarkably, in the presence of γTriDAP, NF-kB activity in IpgB2 and OspB1 transfected cells increased up to 22.7±7.5 and 35.5±0.9 fold respectively (p<0.05 vs control). Depletion of Nod1 prevented the induction of NF-kB activation by IpgB2 and OspB1. Surprisingly, the induction of NFkB activity by IpgB2 and OspB1, alone or in the presence of γTriDAP, was completely abolished after depletion of GEF-H1 expression with specific siRNA. GEF-H1 directly interacted with Nod1 and both where found localized together at bacterial invasion sites. Exogenus GEF-H1 mediated RhoA activation greatly amplified Nod1 mediated NF-kB activation in intestinal epithelial cells. Conclusion: GEF-H1 is a critical component of epithelial defenses forming an intracellular sensing system with Nod1 for the detection of tight junction disruption and cell invasion by pathogens. GEF-H1 function is required for NF-κB activation by Nod1, raising the possibility that Nod family proteins detect modifications of the cytoskeleton by pathogens. The recognition of altered cell function by bacterial effectors may be important for the ability to distinguish between pathogenic and commensal microorganisms which share many ligands for pattern recognition receptors.