title: Enterohemorrhagic E. coli requires N-WASP for efficient type III translocation but not for EspFU-mediated actin pedestal formation.
abstract: Upon infection of mammalian cells, enterohemorrhagic E. coli (EHEC) O157:H7 utilizes a type III secretion system to translocate the effectors Tir and EspF(U) (aka TccP) that trigger the formation of F-actin-rich 'pedestals' beneath bound bacteria. EspF(U) is localized to the plasma membrane by Tir and binds the nucleation-promoting factor N-WASP, which in turn activates the Arp2/3 actin assembly complex. Although N-WASP has been shown to be required for EHEC pedestal formation, the precise steps in the process that it influences have not been determined. We found that N-WASP and actin assembly promote EHEC-mediated translocation of Tir and EspF(U) into mammalian host cells. When we utilized the related pathogen enteropathogenic E. coli to enhance type III translocation of EHEC Tir and EspF(U), we found surprisingly that actin pedestals were generated on N-WASP-deficient cells. Similar to pedestal formation on wild type cells, Tir and EspF(U) were the only bacterial effectors required for pedestal formation, and the EspF(U) sequences required to interact with N-WASP were found to also be essential to stimulate this alternate actin assembly pathway. In the absence of N-WASP, the Arp2/3 complex was both recruited to sites of bacterial attachment and required for actin assembly. Our results indicate that actin assembly facilitates type III translocation, and reveal that EspF(U), presumably by recruiting an alternate host factor that can signal to the Arp2/3 complex, exhibits remarkable versatility in its strategies for stimulating actin polymerization.
authors.affiliation: Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.
authors.forename: Didier
authors.initials: D
authors.lastname: Vingadassalom
authors.forename: Kenneth G
authors.initials: KG
authors.lastname: Campellone
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Brady
authors.forename: Brian
authors.initials: B
authors.lastname: Skehan
authors.forename: Scott E
authors.initials: SE
authors.lastname: Battle
authors.forename: Douglas
authors.initials: D
authors.lastname: Robbins
authors.forename: Archana
authors.initials: A
authors.lastname: Kapoor
authors.forename: Gail
authors.initials: G
authors.lastname: Hecht
authors.forename: Scott B
authors.initials: SB
authors.lastname: Snapper
authors.forename: John M
authors.initials: JM
authors.lastname: Leong
mesh_terms: D000199:Actins / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D054324:Enterohemorrhagic Escherichia coli / Q000378:metabolism* / Q000472:pathogenicity; D004927:Escherichia coli Infections / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism; D055786:Gene Knockout Techniques; D006801:Humans; D015151:Immunoblotting; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D008856:Microscopy, Fluorescence; D021381:Protein Transport / Q000502:physiology; D011956:Receptors, Cell Surface / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal / Q000378:metabolism*
chemical_list: D000199:Actins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C489109:EspFU protein, E coli; D047908:Intracellular Signaling Peptides and Proteins; D011956:Receptors, Cell Surface; C109287:Tir protein, E coli; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal
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