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Infection and Immunity, July 2005, p. 4327-4337, Vol. 73, No. 7
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.7.4327-4337.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

SepZ/EspZ Is Secreted and Translocated into HeLa Cells by the Enteropathogenic Escherichia coli Type III Secretion System

Kristen J. Kanack,1 J. Adam Crawford,1 Ichiro Tatsuno,1 Mohamed A. Karmali,2,3 and James B. Kaper1*

Center for Vaccine Development and Department of Microbiology and Immunology, University of Maryland School of Medicine, 685 W. Baltimore St., Baltimore, Maryland 21201,1 Laboratory for Foodborne Zoonoses, Population and Public Health Branch, Health Canada, 110 Stone Rd. W., Guelph, Ontario, Canada N1G 3W4,2 Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada3

Received 8 July 2004/ Returned for modification 20 August 2004/ Accepted 3 March 2005

Enteropathogenic Escherichia coli (EPEC) is a major bacterial cause of infantile diarrhea in developing countries and is the prototype for a group of gastrointestinal pathogens causing characteristic attaching and effacing (A/E) histopathology on intestinal epithelia. A/E pathogens utilize a type III secretion system (TTSS), encoded by the locus of enterocyte effacement (LEE) pathogenicity island, to deliver effector proteins into host cells. Here, we investigate sequence divergence of the LEE-encoded SepZ protein and identify it as a TTSS-secreted and -translocated molecule. SepZ is hypervariable among A/E pathogens, with sequences sharing between 60 to 81% amino acid identity with SepZ of EPEC. A SepZ-CyaA fusion was secreted and translocated into HeLa cells in a TTSS-dependent manner. Additionally, we determined that the first 20 amino acids of SepZ were sufficient to direct its translocation. In contrast to previous studies suggesting a role in invasion and the structure and/or regulation of the TTSS, we found that SepZ does not mediate uptake of EPEC into host cells or affect translocation and tyrosine phosphorylation of the translocated intimin receptor. Immunohistochemistry reveals that, after an extended HeLa cell infection, accumulated SepZ can be detected beneath the site of bacterial attachment in a subset of pedestal regions. To indicate its newly identified status as a translocated effector protein, we propose to rename SepZ as EspZ.

* Corresponding author. Mailing address: Center for Vaccine Development, University of Maryland School of Medicine, 685 W. Baltimore St., Baltimore, MD 21201. Phone: (410) 706-2493. Fax: (410) 706-0182. E-mail: jkaper{at}umaryland.edu.

Editor: J. B. Bliska

Infection and Immunity, July 2005, p. 4327-4337, Vol. 73, No. 7
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.7.4327-4337.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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