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Journal of Bacteriology, November 2009, p. 6571-6583, Vol. 191, No. 21
0021-9193/09/$08.00+0     doi:10.1128/JB.00754-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Roles of Periplasmic Chaperone Proteins in the Biogenesis of Serine Protease Autotransporters of Enterobacteriaceae ^,

Fernando Ruiz-Perez,^1,2* Ian R. Henderson,⁵ Denisse L. Leyton,⁵ Amanda E. Rossiter,⁵ Yinghua Zhang,⁶ and James P. Nataro^1,2,3,4

Center for Vaccine Development,¹ Departments of Pediatrics,² Medicine,³ Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland 21201,⁴ School of Immunity and Infection, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom,⁵ The Biacore Facility, Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201⁶

Received 10 June 2009/ Accepted 25 August 2009

The serine protease autotransporters of Enterobacteriaceae (SPATEs) represent a large family of virulence factors. The prevailing model for autotransporter secretion comprises entry to the periplasm via the Sec apparatus, followed by an obscure series of steps in which the C terminus of the periplasmic species inserts into the outer membrane as a β-barrel protein, accompanied by translocation of the passenger domain to the bacterial cell surface. Little is known about the fate of the autotransporter proteins in the periplasm, including whether accessory periplasmic proteins are involved in translocation to the external milieu. Here we studied the role of the major periplasmic chaperones in the biogenesis of EspP, a prototype SPATE protein produced by Escherichia coli O157:H7. The yeast two-hybrid approach, secretion analysis of chaperone mutant strains, and surface plasmon resonance analysis (SPR) revealed direct protein-protein interactions between the periplasmic SurA and DegP chaperones and either the EspP-β or EspP passenger domains. The secretion of EspP was moderately reduced in the surA and skp mutant strains but severely impaired in the degP background. Site-directed mutagenesis of highly conserved aromatic amino acid residues in the SPATE family resulted in 80% reduction of EspP secretion. Synthetic peptides containing aromatic residues derived from the EspP passenger domain blocked DegP and SurA binding to the passenger domain. SPR suggested direct protein-protein interaction between periplasmic chaperones and the unfolded EspP passenger domain. Our data suggest that translocation of AT proteins may require accessory factors, calling into question the moniker "autotransporter."

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* Corresponding author. Mailing address: Room 480, Center for Vaccine Development, University of Maryland, 685 W. Baltimore St., Baltimore, MD 21201. Phone: (410) 706-5328. Fax: (410) 706-6205. E-mail: fruiz{at}medicine.umaryland.edu

Published ahead of print on 4 September 2009.

Supplemental material for this article may be found at http://jb.asm.org/.

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Journal of Bacteriology, November 2009, p. 6571-6583, Vol. 191, No. 21
0021-9193/09/$08.00+0     doi:10.1128/JB.00754-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.