Major Host Factors Involved in Epithelial Cell Invasion of Campylobacter jejuni: Role of Fibronectin, Integrin Beta1, FAK, Tiam-1, and DOCK180 in Activating Rho GTPase Rac1

Abstract

Host cell entry by the food-borne pathogen Campylobacter jejuni has been reported as one of the primary reasons of tissue damage in infected humans, however, molecular invasion mechanisms and cellular factors involved in this process are widely unclear. Here we used knockout cell lines derived from fibronectin−/−, integrin beta1−/−, and focal adhesion kinase (FAK)−/− deficient mice and corresponding wild-type (WT) controls, to study C. jejuni-induced signaling cascades involved in the bacterial invasion process. Using high resolution scanning electron microscopy, GTPase pull-downs, G-LISA, and gentamicin protection assays we found that each of these host cell factors is indeed required for activation of the small Rho GTPase member Rac1 and maximal host cell invasion of this pathogen. Interestingly, membrane ruffling, tight engulfment of bacteria and invasion were only seen during infection of WT control cells, but not in fibronectin−/−, integrin beta1−/−, and FAK−/− knockout cell lines. We also demonstrate that C. jejuni activates FAK autophosphorylation activity at Y-397 and phosphorylation of Y-925, which is required for stimulating two downstream guanine exchange factors, DOCK180 and Tiam-1, which are upstream of Rac1. Small interfering (si) RNA studies further show that DOCK180 and Tiam-1 act cooperatively to trigger Rac1 activation and C. jejuni invasion. Moreover, mutagenesis data indicate that the bacterial fibronectin-binding protein CadF and the intact flagellum are involved in Rho GTPase activation and host cell invasion. Collectively, our results suggest that C. jejuni infection of host epithelial target cells hijacks a major fibronectin → integrin beta1 → FAK → DOCK180/Tiam-1 signaling cascade, which has a crucial role for Rac1 GTPase activity and bacterial entry into host target cells.

Campylobacter jejuniserine protease HtrA plays an important role in heat tolerance, oxygen resistance, host cell adhesion, invasion, and transmigration

“…Both studies concluded that particularly the chaperone activity of HtrA may have a signifi cant impact on the interaction of C. jejuni and host cells. Lack of HtrA reduced bacterial binding to epithelial cells 5-10 times more [22], as compared to any other known adhesin [16,25,[58][59][60], suggesting a pleiotropic effect. Impaired adherence and invasion of the htrA mutant cannot be explained by reduced C. jejuni motility, since the htrA mutation does not affect these parameters [26].…”

Rho GTPases as pathogen targets: Focus on curable sexually transmitted infections

“…119 For instance, the bacterial fibronectin-binding protein of Campylobacter jejuni, CadF, and the intact flagellum are involved in eukaryotic Rac1 GTPase activation and host cell invasion. 116,120 It has been postulated that CadF binding to fibronectin results in integrin clustering and activation. These events lead to the activation of signaling cascades that involves epidermal growth factor receptor (EGFR) and focal adhesion kinase (FAK), resulting in association of c-Src and phosphorylation of paxillin.…”

“…Finally, the recruitment of Dock180, a Rac1-specific guanine nucleotide exchange factor, provokes the activation of Rac1, leading to a local restructuring of the actin cytoskeleton and the engulfment of the bacteria. 116,120 Similarly, in infections caused by T. pallidum, the bacterial adhesins might target fibronectin and laminin not only to enhance the binding to host cells [108][109][110] but also to trigger intracellular signaling pathways that might promote Rac1 activation and consequently actin remodelling at the attachment site, facilitating the invasion of host tissues. Nevertheless, the role of Rho GTPases and the actin cytoskeleton in treponemal infections remains almost unexplored.…”

Extracellular secretion of protease HtrA fromCampylobacter jejuniis highly efficient and independent of its protease activity and flagellum

“…The detailed signaling pathways leading to the activation of Rac1 and Cdc42 involve CadF targeting of fi bronectin and require certain receptors (integrins, EGF and PDGF receptors), kinases (FAK, Src, and PI3-kinase) and guanine exchange factors (DOCK180, Vav2, and Tiam-1). Studies using knockout cell lines, scanning electron microscopy, GTPase, and other signaling assays have shown that C. jejuni triggers the CadF → fi bronectin → integrin-β1 → FAK/Src → EGFR/PDGFR → PI3-kinase → Vav2 and CadF → fi bronectin → integrin-β1 → FAK → DOCK180/Tiam-1 signaling cascades to activate Cdc42 and Rac1, respectively, stimulating cytoskeletal rearrangements and bacterial uptake [18,19].…”