title: Protease activity, secretion, cell entry, cytotoxicity, and cellular targets of secreted autotransporter toxin of uropathogenic Escherichia coli.
abstract: The secreted autotransporter toxin (Sat), found predominantly in uropathogenic Escherichia coli, is a member of the SPATE (serine protease autotransporters of Enterobacteriaceae) family and, as such, has serine protease activity and causes cytopathic effects on various cell types. To assess the contribution of the serine protease active site to the mechanism of action of Sat, mutations were made in the first (S256I), in the second (S258A), or in both (S256I/S258A) serine residues within the active site motif. Mutations in the first or both serines reduced protease activity to background levels (P<0.001); a single mutation in the second serine reduced activity by 60% compared to wild type (P<0.001). After reversion of the S256I mutation to wild type (I256S), we confirmed S256 as the catalytically active serine. None of these mutations affected secretion of the mature passenger domain or release into the supernatant. The S256I mutation, however, abrogated the cytotoxicity of Sat on human bladder (UM-UC-3) and kidney (HEK 293) epithelial cells, characterized by rounding and elongation, respectively, and a high level of cell detachment. Moreover, S256 is essential for Sat to mediate cytoskeletal contraction and actin loss in host cells as well as to degrade specific membrane/cytoskeletal (fodrin and leukocyte function-associated molecule 1) and nuclear [microtubule-associated proteins, LIM domain-only protein 7, Rap GTPase-activating protein, poly(ADP-ribose) polymerase] proteins in vitro. Lastly, Sat was internalized by host cells and localized to the cytoskeletal fraction where membrane/cytoskeletal target proteins reside.
authors.affiliation: Department of Microbiology and Immunology, University of Michigan Medical School, 5641 Medical Science Bldg II, 1150 West Medical Center Dr., Ann Arbor, MI 48109-0620, USA.
authors.forename: Nathalie M
authors.initials: NM
authors.lastname: Maroncle
authors.forename: Kelsey E
authors.initials: KE
authors.lastname: Sivick
authors.forename: Rebecca
authors.initials: R
authors.lastname: Brady
authors.forename: Faye-Ellen
authors.initials: FE
authors.lastname: Stokes
authors.forename: Harry L T
authors.initials: HL
authors.lastname: Mobley
mesh_terms: D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism / Q000633:toxicity*; D002460:Cell Line; D002478:Cells, Cultured; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity*; D004927:Escherichia coli Infections / Q000201:enzymology / Q000378:metabolism / Q000382:microbiology; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity*; D006801:Humans; D007668:Kidney / Q000166:cytology* / Q000201:enzymology / Q000382:microbiology; D016297:Mutagenesis, Site-Directed; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity*; D001743:Urinary Bladder / Q000166:cytology* / Q000201:enzymology / Q000382:microbiology; D019459:Urothelium / Q000166:cytology / Q000201:enzymology / Q000382:microbiology
chemical_list: D001427:Bacterial Toxins; D029968:Escherichia coli Proteins; C506813:secreted autotransporter toxin, E coli; D012697:Serine Endopeptidases