Characterization of non-toxic mutant toxins of Vero toxin 1 that were constructed by replacing amino acids in the A subunit

Shiga Toxin 1 Induces Apoptosis in the Human Myelogenous Leukemia Cell Line THP-1 by a Caspase-8-Dependent, Tumor Necrosis Factor Receptor-Independent Mechanism

“…Purified Stx1 pentameric B-subunits were the kind gift of Cheleste Thorpe, Tufts University School of Medicine, Boston, MA. Purified Stx1A Ϫ (E167Q-R170L), an enzymatic mutant in which glutamate at position 167 and arginine at position 170 were replaced by glutamine and leucine, respectively, by oligonucleotide-directed site-specific mutagenesis (32), was the kind gift of Yoshifumi Takeda, Jissen Women's University, Tokyo, Japan.…”

“…To determine whether Stx1 binding to Gb 3 and/or toxin enzymatic activity is required for the induction of THP-1 cell apoptosis, we treated cells with equal amounts of purified Stx1, Stx1 pentameric B-subunits, or Stx1A Ϫ (E167Q-R170L), a Stx1 holotoxin containing mutations in the A-subunit active site that reduce Vero cell cytotoxicity approximately 3 ϫ 10 5 -fold (32). As shown in Fig.…”

Vero Toxins (Shiga‐Like Toxins) Produced by Enterohemorrhagic Escherichia coli (Verocytotoxin‐Producing E. coli)

“…It was found that, among these 22 mutant toxins, two mutants that had the replacement of Glu 167 by glutamine (E167Q) and of Arg 170 by leucine (R 170L) showed significantly reduced toxicity both in cytotoxicity to Vero cells and in inhibitory activity of protein synthesis in rabbit reticulocyte lysate (Table 3) (99) . Ohmura et al (70) further constructed a mutant gene of VT1 that encodes a mutant toxin with the replacement of two amino acids, Glu 167 and Arg 170, by glutamine and leucine, respectively (E167Q-R170L) . Similar mutant SLT-IIv (or VT2vp1) , E167Q, E167D, and E167D-R170K with less toxic activities were prepared by Gordon et al (24) .…”

“…However, the decrease of these activities in the purified R17OL was less than that of the other two mutant VT1s. Since Ohmura et al (70) showed that the mutant VT1 possessed similar antigenicities to that of wildtype, these mutant toxins may be used as a candidate toxoid to protect VT1-mediated diseases as suggested by Gordon et al (24).…”

Protective Immunization of Mice with an Active-Site Mutant of Subtilase Cytotoxin of Shiga Toxin-Producing Escherichia coli

“…Thus, parenteral immunization with chemical or genetic Shiga toxin toxoids is likely to be highly protective against the life-threatening complications of STEC disease. Indeed, several studies have shown that A subunit active-site mutants of Stx1 or Stx2 are capable of eliciting protective immune responses in animals (2,4,6,7,13). However, the recent discovery (15) that some STEC strains produce an additional potent AB 5 cytotoxin (SubAB) that has the potential to significantly augment clinical manifestations, or indeed to cause disease in its own right, raises the issue of whether immunity to Shiga toxin alone will be sufficient to prevent disease caused by such strains.…”