This Article Full Text Full Text (PDF) Supplemental material Other Versions of this Article: IAI.01546-06v1 75/5/2189    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Shimizu, T. Articles by Noda, M. Search for Related Content PubMed PubMed Citation Articles by Shimizu, T. Articles by Noda, M.

The Serine 31 Residue of the B Subunit of Shiga Toxin 2 Is Essential for Secretion in Enterohemorrhagic Escherichia coli ^,

Takeshi Shimizu,^1* Satomi Kawakami,^2, Toshio Sato,³ Terumi Sasaki,⁴ Masato Higashide,⁴ Takashi Hamabata,³ Toshiko Ohta,² and Masatoshi Noda¹

Department of Molecular Infectiology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chiba 260-8670,¹ Department of Infection Biology, Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Ten-nohdai, Tsukuba 305-8575,² Department of Infectious Diseases, Research Institute, International Medical Center of Japan, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655,³ Kotobiken Medical Laboratories, 445-1 Kamiyokoba, Tsukuba 305-0854, Japan⁴

Received 26 September 2006/ Returned for modification 28 November 2006/ Accepted 12 February 2007

Shiga toxins produced by enterohemorrhagic Escherichia coli (EHEC) include Shiga toxin 1 (Stx1) as well as Shiga toxin 2 (Stx2). Stx1 is cell associated, whereas Stx2 is localized to the culture supernatant. We have analyzed the secretion of Stx2 by generating histidine-tagged StxB (StxB-H). Although neither StxB1-H nor StxB2-H was secreted in StxB-H-overexpressed EHEC, StxB2-H-overexpressed EHEC showed inhibited Stx2 secretion. On the other hand, StxB1-H-overexpressed EHEC showed no alteration of Stx2 secretion. B-subunit chimeras of Stx1 and Stx2 were used to identify the specific residue of StxB2 that the Stx2 secretory system recognizes. Alteration of the serine 31 residue to an asparagine residue (S31N) in StxB2-H enabled the recovery of Stx2 secretion. On the other hand, alteration of the asparagine 32 residue to a serine residue (N32S) in StxB1-H caused the partial secretion of a point-mutated histidine-tagged B subunit in EHEC. Based on the evidence, it appeared possible that this residue might contain secretion-related information for Stx2 secretion. To investigate this hypothesis, we constructed an isogenic mutant EHEC (Stx1B subunit, N32S) strain and an isogenic mutant EHEC (Stx2B subunit, S31N) strain. Although the mutant Stx2 was cell associated in isogenic mutant EHEC, mutant Stx1 was not extracellular. However, when we used plasmids for the expression of the mutant holotoxins, the overexpressed mutant Stx1 was found in the supernatant fraction, and the overexpressed mutant Stx2 was found in the cell-associated fraction in mutant holotoxin gene-transformed EHEC. These results indicate that the serine 31 residue of the B subunit of Stx2 contains secretion-related information.

Published ahead of print on 26 February 2007.

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

Editor: A. D. O'Brien

Present address: ICAM Co., Ltd., 3-28-14 Tokiwadai, Itabashi, Tokyo 174-0071, Japan.