title: The serine 31 residue of the B subunit of Shiga toxin 2 is essential for secretion in enterohemorrhagic Escherichia coli.
abstract: 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.
authors.affiliation: Department of Molecular Infectiology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chiba 260-8670, Japan. tshimizu@faculty.chiba-u.jp
authors.forename: Takeshi
authors.initials: T
authors.lastname: Shimizu
authors.forename: Satomi
authors.initials: S
authors.lastname: Kawakami
authors.forename: Toshio
authors.initials: T
authors.lastname: Sato
authors.forename: Terumi
authors.initials: T
authors.lastname: Sasaki
authors.forename: Masato
authors.initials: M
authors.lastname: Higashide
authors.forename: Takashi
authors.initials: T
authors.lastname: Hamabata
authors.forename: Toshiko
authors.initials: T
authors.lastname: Ohta
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Noda
mesh_terms: D000818:Animals; D004927:Escherichia coli Infections / Q000382:microbiology / Q000401:mortality; D019453:Escherichia coli O157 / Q000235:genetics / Q000254:growth & development / Q000378:metabolism* / Q000472:pathogenicity*; D015964:Gene Expression Regulation, Bacterial; D008297:Male; D051379:Mice; D008958:Models, Molecular; D009154:Mutation; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics; D012694:Serine / Q000737:chemistry*; D022641:Shiga Toxin 2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity
chemical_list: D021122:Protein Subunits; D022641:Shiga Toxin 2; D012694:Serine