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Infection and Immunity, June 2003, p. 3310-3319, Vol. 71, No. 6
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.6.3310-3319.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Secretin of the Enteropathogenic Escherichia coli Type III Secretion System Requires Components of the Type III Apparatus for Assembly and Localization

Annick Gauthier,1 Jose Luis Puente,2 and B. Brett Finlay1*

Biotechnology Laboratory and Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3,1 Molecular Microbiology Department, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos 62250, Mexico2

Received 17 September 2002/ Returned for modification 16 January 2003/ Accepted 24 February 2003

At least 16 proteins are thought to be involved in forming the enteropathogenic Escherichia coli (EPEC) type III translocation apparatus which delivers virulence factors into host cells, yet their function and location have not been determined. A biochemical analysis was performed on three components: EscN, a predicted cytoplasmic ATPase; EscV, a predicted inner membrane protein; and EscC, a predicted outer membrane secretin. Wild-type EPEC and mutants constructed in these genes were fractionated by lysozyme treatment, ultracentrifugation, and selective detergent extraction. Fractionation revealed that the type III effectors Tir and EspB required a complete type III apparatus for any degree of export by EPEC, suggesting a continuous channel. Epitope-tagged EscC, EscV, and EscN were localized by fractionation, confirming computer modeling predictions for their location. Transcomplementation experiments revealed that localization of EscV and EscN was unaffected by mutations in other examined type III components. Remarkably, localization of EscC was altered in escV or escN mutants, where EscC accumulated in the periplasm. EscC was correctly localized in the escF needle component mutant, indicating that secretin localization is independent of needle formation. These data indicate that, contrary to previous indications, correct insertion and function of EscC secretin in the outer membrane depends not only on the sec-dependent secretion pathway but also on other type III apparatus components.

* Corresponding author. Mailing address: Biotechnology Laboratory, University of British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada. Phone: (604) 822-2210. Fax: (604) 822-9830. E-mail: bfinlay{at}interchange.ubc.ca.

Editor: V. J. DiRita

Infection and Immunity, June 2003, p. 3310-3319, Vol. 71, No. 6
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.6.3310-3319.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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