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.
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.
School for Biomedical and Biomolecular Science, Belfield Campus, University College Dublin, Dublin, Ireland; Department of Microbiology, Otton von Guericke University, Magdeburg, Germany.
Manja
M
Boehm
Malgorzata
M
Krause-Gruszczynska
Manfred
M
Rohde
Nicole
N
Tegtmeyer
Seiichiro
S
Takahashi
Omar A
OA
Oyarzabal
Steffen
S
Backert
D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000502:physiology; D001483:Base Sequence; D002169:Campylobacter Infections / Q000209:etiology; D016123:Campylobacter jejuni / Q000235:genetics / Q000472:pathogenicity* / Q000648:ultrastructure; D002352:Carrier Proteins / Q000235:genetics / Q000502:physiology; D002460:Cell Line; D004789:Enzyme Activation; D004847:Epithelial Cells / Q000382:microbiology / Q000648:ultrastructure; D005353:Fibronectins / Q000172:deficiency / Q000235:genetics / Q000502:physiology; D051417:Focal Adhesion Kinase 1 / Q000172:deficiency / Q000235:genetics / Q000502:physiology; D005798:Genes, Bacterial; D020662:Guanine Nucleotide Exchange Factors / Q000502:physiology; D054884:Host-Pathogen Interactions / Q000235:genetics / Q000502:physiology*; D006801:Humans; D019012:Integrin beta1 / Q000235:genetics / Q000502:physiology; D051379:Mice; D018345:Mice, Knockout; D008855:Microscopy, Electron, Scanning; D008954:Models, Biological; D009154:Mutation; D009479:Neuropeptides / Q000502:physiology; D034741:RNA, Small Interfering / Q000235:genetics; D015398:Signal Transduction; D000075684:T-Lymphoma Invasion and Metastasis-inducing Protein 1; D014774:Virulence / Q000235:genetics / Q000502:physiology; D020744:rac GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology; D020830:rac1 GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology
D001425:Bacterial Outer Membrane Proteins; C107872:CadF protein, Campylobacter jejuni; D002352:Carrier Proteins; C099818:DOCK1 protein, human; C528110:Dock180 protein, mouse; D005353:Fibronectins; D020662:Guanine Nucleotide Exchange Factors; D019012:Integrin beta1; D009479:Neuropeptides; C107953:RAC1 protein, human; D034741:RNA, Small Interfering; C484602:Rac1 protein, mouse; D000075684:T-Lymphoma Invasion and Metastasis-inducing Protein 1; C513260:Tiam1 protein, mouse; D051417:Focal Adhesion Kinase 1; C496051:Ptk2 protein, mouse; D020744:rac GTP-Binding Proteins; D020830:rac1 GTP-Binding Protein