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. 1994 May;62(5):1768–1775. doi: 10.1128/iai.62.5.1768-1775.1994

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

P R Austin 1, P E Jablonski 1, G A Bohach 1, A K Dunker 1, C J Hovde 1
PMCID: PMC186405  PMID: 8168939

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.

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