title: Cortactin is essential for F-actin assembly in enteropathogenic Escherichia coli (EPEC)- and enterohaemorrhagic E. coli (EHEC)-induced pedestals and the alpha-helical region is involved in the localization of cortactin to bacterial attachment sites.
abstract: Enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) are important human pathogens. Upon attachment to host cells, EPEC and EHEC are able to induce actin polymerization, which accumulates, forming a pedestal-like structure beneath the attached bacteria. Using siRNA, we show here that EPEC- and EHEC-induced pedestals are dependent on cortactin, an F-actin-binding protein found in the mammalian cell cortex. Knock-down of cortactin by siRNA resulted in a dramatic reduction of the pedestal formation induced by both pathogens. We also show that disruption of the Src homology 3 (SH3) domain of cortactin, or its downregulation by specific point mutations, negatively affects pedestal formation, suggesting that this domain is important for regulation of F-actin assembly by EPEC and EHEC. Green fluorescent protein (GFP) fused with the SH3 domain (GFP-SH3), proline-rich region (GFP-PRR) or alpha-helical region of cortactin markedly reduced the amount of F-actin at the bacterial attachment sites. Interestingly, neither GFP-SH3 nor GFP-PRR was recruited to the vicinity of the bacterial adherence sites; however, GFP fused to the alpha-helical region was efficiently recruited and colocalized with the attached bacteria. These results demonstrate that cortactin is a requirement for pedestal formation and suggest a novel function for the predicted alpha-helical region of cortactin in actin assembly induced by EPEC and EHEC.
authors.affiliation: Research Institute for Microbial Diseases, Department of Infectious Diseases, Osaka University, Osaka, Japan. vcantarelli@weinmann.com.br
authors.forename: Vlademir V
authors.initials: VV
authors.lastname: Cantarelli
authors.forename: Toshio
authors.initials: T
authors.lastname: Kodama
authors.forename: Niels
authors.initials: N
authors.lastname: Nijstad
authors.forename: Said Kamal
authors.initials: SK
authors.lastname: Abolghait
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Iida
authors.forename: Takeshi
authors.initials: T
authors.lastname: Honda
mesh_terms: D000199:Actins / Q000378:metabolism*; D001422:Bacterial Adhesion; D051356:Cortactin / Q000235:genetics / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D004927:Escherichia coli Infections / Q000378:metabolism / Q000382:microbiology; D019453:Escherichia coli O157 / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D017433:Protein Structure, Secondary; D034741:RNA, Small Interfering / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D018909:src Homology Domains
chemical_list: D000199:Actins; D051356:Cortactin; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins