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Activation of Shiga toxin type 2d (Stx2d) by elastase involves cleavage of the C‐terminal two amino acids of the A 2 peptide in the context of the appropriate B pentamer
Author(s) -
MeltonCelsa Angela R.,
KokaiKun John F.,
O’Brien Alison D.
Publication year - 2002
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.2002.02733.x
Subject(s) - shiga toxin , biology , shigella dysenteriae , escherichia coli , diphtheria toxin , vero cell , biochemistry , peptide sequence , furin , toxin , microbiology and biotechnology , shiga like toxin , amino acid , enzyme , gene , in vitro
Summary Shiga toxins (Stx) are potent ribosome‐inactivating toxins that are produced by Shigella dysenteriae type 1 or certain strains of Escherichia coli. These toxins are composed of one A subunit that can be nicked and reduced to an enzymatically active A 1 ( ≈ 27 kDa) and an A 2 peptide ( ≈ 4 kDa) as well as a pentamer of B subunits ( ≈ 7 kDa/monomer) that binds the eukaryotic cell. Purified Shiga toxin type 2d is activated 10‐ to 1000‐fold for Vero cell toxicity by preincubation with mouse or human intestinal mucus or purified mouse elastase, whereas Stx2, Stx2c, Stx2e and Stx1 are not activatable. E. coli strains that produce the activatable Stx2d are more virulent in a streptomycin (str)‐treated mouse model of infection [lethal dose 50% (LD 50 ) = 10 1 ] than are E. coli strains that produce any other type of Stx (LD 50 = 10 10 ). To identify the element(s) of Stx2d that are required for mucus‐mediated activation, toxin genes were constructed such that the expressed mutant toxins consisted of hybrids of Stx2d and Stx1, Stx2 or Stx2e, contained deletions of up to six amino acids from the C‐terminus of the A 2 of Stx2d or were altered in one or both of the two amino acids of the A 2 of Stx2d that represent the only amino acid differences between the activatable Stx2d and the non‐activatable Stx2c. Analysis of these mutant toxins revealed that the A 2 portion of Stx2d is required for toxin activation and that activation is abrogated if the Stx1 or Stx2e B subunit is substituted for the Stx2d B polypeptide. Furthermore, mass spectrometry performed on buffer‐ or elastase‐treated Stx2d indicated that the A 2 peptide of the activated Stx2d was two amino acids smaller than the A 2 peptide from buffer‐treated Stx2d. This finding, together with the toxin hybrid results, suggests that activation involves B pentamer‐dependent cleavage by elastase of the C‐terminal two amino acids from the Stx2d A 2 peptide.