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Infection and Immunity, November 2006, p. 6124-6134, Vol. 74, No. 11
0019-9567/06/$08.00+0     doi:10.1128/IAI.01086-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Protease Activity, Secretion, Cell Entry, Cytotoxicity, and Cellular Targets of Secreted Autotransporter Toxin of Uropathogenic Escherichia coli

Department of Microbiology and Immunology, University of Michigan School of Medicine, Ann Arbor, Michigan 48104,¹ Department of Biomedical Sciences, University of Maryland Dental School, Baltimore, Maryland 21201,² Society for General Microbiology, Reading, United Kingdom³

Received 11 July 2006/ Returned for modification 10 August 2006/ Accepted 25 August 2006

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.

Published ahead of print on 5 September 2006.

Editor: D. L. Burns

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