title: Actin pedestal formation by enterohemorrhagic Escherichia coli enhances bacterial host cell attachment and concomitant type III translocation.
abstract: Attachment of enterohemorrhagic Escherichia coli (EHEC) to intestinal epithelial cells is critical for colonization and is associated with localized actin assembly beneath bound bacteria. The formation of these actin "pedestals" is dependent on the translocation of effectors into mammalian cells via a type III secretion system (T3SS). Tir, an effector required for pedestal formation, localizes in the host cell plasma membrane and promotes attachment of bacteria to mammalian cells by binding to the EHEC outer surface protein Intimin. Actin pedestal formation has been shown to foster intestinal colonization by EHEC in some animal models, but the mechanisms responsible for this remain undefined. Investigation of the role of Tir-mediated actin assembly promoting host cell binding is complicated by other, potentially redundant EHEC-encoded binding pathways, so we utilized cell binding assays that specifically detect binding mediated by Tir-Intimin interaction. We also assessed the role of Tir-mediated actin assembly in two-step assays that temporally segregated initial translocation of Tir from subsequent Tir-Intimin interaction, thereby permitting the distinction of effects on translocation from effects on cell attachment. In these experimental systems, we compromised Tir-mediated actin assembly by chemically inhibiting actin assembly or by infecting mammalian cells with EHEC mutants that translocate Tir but are specifically defective in Tir-mediated pedestal formation. We found that an inability of Tir to promote actin assembly resulted in a significant and striking decrease in bacterial binding mediated by Tir and Intimin. Bacterial mutants defective for pedestal formation translocated type III effectors to mammalian cells with reduced efficiency, but the decrease in translocation could be entirely accounted for by the decrease in host cell attachment.
authors: Departments of Medicine and Microbiology/Immunology, Division of Gastroenterology and Nutrition, Loyola University Chicago, Maywood, Illinois, USA.
authors: Scott E
authors: SE
authors: Battle
authors: Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts, USA.
authors: Michael J
authors: MJ
authors: Brady
authors: Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts, USA.
authors: Sivapriya Kailasan
authors: SK
authors: Vanaja
authors: Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts, USA.
authors: John M
authors: JM
authors: Leong
authors: Departments of Medicine and Microbiology/Immunology, Division of Gastroenterology and Nutrition, Loyola University Chicago, Maywood, Illinois, USA Hines VA Medical Center, Hines, Illinois, USA ghecht@lumc.edu.
authors: Gail A
authors: GA
authors: Hecht
mesh_terms: D000199:Actins / Q000378:metabolism*; D018829:Adhesins, Bacterial / Q000378:metabolism; D001422:Bacterial Adhesion / Q000502:physiology*; D018988:Bacterial Translocation / Q000502:physiology*; D002352:Carrier Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D054324:Enterohemorrhagic Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D011485:Protein Binding / Q000502:physiology
chemical_list: D000199:Actins; D018829:Adhesins, Bacterial; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C489109:EspFU protein, E coli; D047908:Intracellular Signaling Peptides and Proteins; C073819:eaeA protein, E coli