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J. Biol. Chem., Vol. 278, Issue 41, 39912-39920, October 10, 2003

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Structure-Function Analysis of the Enteroaggregative Escherichia coli Plasmid-encoded Toxin Autotransporter Using Scanning Linker Mutagenesis^*

Pinaki R. Dutta , Bao Quan Sui and James P. Nataro 

From the Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland 21201

The plasmid-encoded toxin (Pet) from enteroaggregative Escherichia coli is a cytopathic serine protease, which is prototypical of a large family of bacterial autotransporter toxins. To further elucidate the structure-function relationships of this toxin, we employed transposon-based scanning linker mutagenesis. A subset of insertions throughout the Pet mature toxin (passenger) domain reduced secretion to the extracellular space. Many of these mutants were undetectable, but secretion of a subset of mutants with insertions in the N-terminal half of the toxin could be restored to wild type secretion levels if cultured in the presence of 0.1% Triton X-100. Secretion of two mutants with insertions at the extreme C terminus was partially restored when co-expressed with a minimal clone of EspP, a related autotransporter protein. Several well secreted mutants with insertions in the N-terminal third of the molecule reduced protease activity over 20-fold, suggesting that the protease domain is located within this N-terminal region of Pet. We have also identified two insertional mutants in the middle of the passenger domain that were proteolytic but no longer cytopathic; these mutants displayed decreased binding and internalization upon incubation with HEp-2 cells. Our data suggest the existence of separate functional domains mediating Pet proteolysis, secretion, and cell interaction.

Received for publication, April 7, 2003 , and in revised form, July 17, 2003.

^* This work was supported by United States Public Health Service Grant AI 43615 (to J. P. N.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported in part by National Institutes of Health Training Grant 5 T32 AI07540-04.

To whom correspondence should be addressed: Center for Vaccine Development, HSF 480, 685 W. Baltimore St., Baltimore, MD 21201. Tel.: 410-706-8442; Fax: 410-706-6205; E-mail: jnataro{at}medicine.umaryland.edu.

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