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Infection and Immunity, March 2003, p. 1255-1264, Vol. 71, No. 3
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.3.1255-1264.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Structural Characterization of the N Terminus of IpaC from Shigella flexneri

Amanda T. Harrington, Patricia D. Hearn, Wendy L. Picking, Jeffrey R. Barker, Andrew Wessel, and William D. Picking*

Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045

Received 28 August 2002/ Returned for modification 30 October 2002/ Accepted 4 December 2002

The primary effector for Shigella invasion of epithelial cells is IpaC, which is secreted via a type III secretion system. We recently reported that the IpaC N terminus is required for type III secretion and possibly other functions. In this study, mutagenesis was used to identify an N-terminal secretion signal and to determine the functional importance of the rest of the IpaC N terminus. The 15 N-terminal amino acids target IpaC for secretion by Shigella flexneri, and placing additional amino acids at the N terminus does not interfere with IpaC secretion. Furthermore, amino acid sequences with no relationship to the native IpaC secretion signal can also direct its secretion. Deletions introduced beyond amino acid 20 have no effect on secretion and do not adversely affect IpaC function in vivo until they extend beyond residue 50, at which point invasion function is completely eliminated. Deletions introduced at amino acid 100 and extending toward the N terminus reduce IpaC's invasion function but do not eliminate it until they extend to the N-terminal side of residue 80, indicating that a region from amino acid 50 to 80 is critical for IpaC invasion function. To explore this further, the ability of an IpaC N-terminal peptide to associate in vitro with its translocon partner IpaB and its chaperone IpgC was studied. The N-terminal peptide binds tightly to IpaB, but the IpaC central hydrophobic region also appears to participate in this binding. The N-terminal peptide also associates with the chaperone IpgC and IpaB is competitive for this interaction. Based on additional biophysical data, we propose that a region between amino acids 50 and 80 is required for chaperone binding, and that the IpaB binding domain is located downstream from, and possibly overlapping, this region. From these data, we propose that the secretion signal, chaperone binding region, and IpaB binding domain are located at the IpaC N terminus and are essential for presentation of IpaC to host cells during bacterial entry; however, IpaC effector activity may be located elsewhere.

* Corresponding author. Mailing address: Department of Molecular Biosciences, University of Kansas, 1200 Sunnyside Ave., Lawrence, KS 66045. Phone: (785) 864-3299. Fax: (785) 864-5294. E-mail: picking{at}ku.edu.

Editor: J. T. Barbieri

Infection and Immunity, March 2003, p. 1255-1264, Vol. 71, No. 3
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.3.1255-1264.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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