Mapping the minimal contiguous gene segment that encodes functionally active Shiga-like toxin II.
Shiga-like toxin type II (SLT-II) is one of two antigenically distinct cytotoxins produced by enterohemorrhagic Escherichia coli that are believed to play a central role in the pathogenesis of enterohemorrhagic E. coli-induced disease. SLT-II is a bipartite toxin with an enzymatically active A subunit that inhibits protein synthesis and an oligomeric B subunit that binds to the glycolipid globotriaosylceramide on eukaryotic cells. In this study, functional boundaries of the slt-II operon were mapped. Mutant proteins lacking the last four amino acids from the carboxy terminus of the 70-amino-acid mature SLT-II B polypeptide had no cytotoxic activity. However, when only two amino acids were removed from the carboxy terminus of the B subunit, the cytotoxic activity of the holotoxin was not altered drastically. Furthermore, a 21-amino-acid extension to the carboxy terminus of the SLT-II B polypeptide was tolerated with a minimum reduction in cytotoxic activity of the holotoxin. Deletion of the region coding for amino acids 3 through 18 of the 296-amino-acid mature SLT-II A polypeptide resulted in complete ablation of the cytotoxic activity of the holotoxin as well as abolition of the enzymatic activity of the A subunit. Thus, it appears that both 5'- and 3'-terminal coding sequences are essential for function of the slt-II operon.
Perera
L P
LP
Department of Microbiology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799.
Samuel
J E
JE
Holmes
R K
RK
O'Brien
A D
AD
D000595:Amino Acid Sequence; D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics*; D001483:Base Sequence; D002855:Chromatography, Thin Layer; D002872:Chromosome Deletion; D002874:Chromosome Mapping; D004269:DNA, Bacterial; D004706:Endodeoxyribonucleases; D005798:Genes, Bacterial / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D009876:Operon / Q000235:genetics; D010957:Plasmids; D014176:Protein Biosynthesis; D022641:Shiga Toxin 2; D013329:Structure-Activity Relationship; D014709:Vero Cells
D001427:Bacterial Toxins; D004269:DNA, Bacterial; D015345:Oligonucleotide Probes; D022641:Shiga Toxin 2; D004706:Endodeoxyribonucleases; C038407:exonuclease Bal 31