Evidence that the A2 fragment of Shiga-like toxin type I is required for holotoxin integrity

P. R. Austin, P. E. Jablonski, G. A. Bohach, A. K. Dunker, C. J. Hovde

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Abstract

Escherichia coli Shiga-like toxin type I (SLT-I) is a potent cytotoxin consisting of an enzymatically active A subunit and a pentameric B subunit that mediates toxin binding to susceptible eukaryotic cells. Evidence that the carboxy-terminal 38 amino acids of the A subunit are involved in holotoxin 1A:5B association is presented. We compared the ability of purified recombinant SLT-I B subunit (Slt-IB) to combine in vitro with purified recombinant SLT-I A subunit (Slt-IA; full-length subunit A includes amino acids 1 to 293) and its ability to combine with purified recombinant SLT-I A1 subunit (Slt-IA1; truncated subunit A includes amino acids 1 to 255). Each mixture was analyzed for biological and physical evidence of toxin assembly. Although Slt-IA successfully combined with Slt-IB to form a molecular species similar to holotoxin that was detectable by nondenaturing polyacrylamide gel electrophoresis and immunoblotting and yielded a molecule which was cytotoxic to cultured Vero cells, Slt-IA1 did not have this ability. Slt-IA1 was 36-fold more active than Slt-IA in an in vitro protein synthesis inhibition assay. These findings suggest that the Slt-IA2 fragment is crucial for formation of SLT holotoxin and stabilizes the interaction between the A and B subunits.

Original languageEnglish (US)
Pages (from-to)1768-1775
Number of pages8
JournalInfection and immunity
Volume62
Issue number5
StatePublished - Jan 1 1994

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ASJC Scopus subject areas

  • Parasitology
  • Microbiology
  • Immunology
  • Infectious Diseases

Cite this

Austin, P. R., Jablonski, P. E., Bohach, G. A., Dunker, A. K., & Hovde, C. J. (1994). Evidence that the A2 fragment of Shiga-like toxin type I is required for holotoxin integrity. Infection and immunity, 62(5), 1768-1775.