Evidence that the A2 fragment of Shiga-like toxin type I is required for holotoxin integrity.
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.
Austin
P R
PR
Department of Microbiology, Molecular Biology, and Biochemistry, University of Idaho, Moscow 83843.
Jablonski
P E
PE
Bohach
G A
GA
Dunker
A K
AK
Hovde
C J
CJ
D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry / Q000633:toxicity*; D002470:Cell Survival / Q000187:drug effects; D003603:Cytotoxins / Q000633:toxicity*; D022622:Shiga Toxin 1; D013329:Structure-Activity Relationship; D014709:Vero Cells
D001427:Bacterial Toxins; D003603:Cytotoxins; D022622:Shiga Toxin 1