Two distinct cytotoxic activities of subtilase cytotoxin produced by shiga-toxigenic Escherichia coli.
Subtilase cytotoxin (SubAB) is a recently identified AB5 subunit toxin produced by Shiga-toxigenic Escherichia coli. The A subunit is thought to be a subtilase-like, serine protease, whereas the B subunit binds to the toxin receptor on the cell surface. We cloned the genes from a clinical isolate; the toxin was produced as His-tagged proteins. SubAB induced vacuolation at concentrations greater than 1 microg/ml after 8 h, in addition to the reported cytotoxicity induced at a ng/ml level after 48 h. Vacuolation was induced with the B, but not the A, subunit and was dependent on V-type ATPase. The cytotoxicity of SubAB at low concentrations was associated with the inhibition of protein synthesis; the 50% inhibitory dose was approximately 1 ng/ml. The A subunit, containing serine 272, which is thought to be a part of the catalytic triad of a subtilase-like serine protease, plus the B subunit was necessary for this activity, both in vivo and in vitro. SubAB did not cleave azocasein, bovine serum albumin, ovalbumin, or synthetic peptides. These data suggest that SubAB is a unique AB toxin: first, the B subunit alone can induce vacuolation; second, the A subunit containing serine 272 plus the B subunit inhibited protein synthesis, both in vivo and in vitro; and third, the A subunit proteolytic activity may have a strict range of substrate specificity.
Morinaga
Naoko
N
Department of Molecular Infectiology, Graduate School of Medicine, Chiba University, 1-8-1, Inohana, Chuo-ku, Chiba 260-8670, Japan. nmorinaga@faculty.chiba-u.jp
Yahiro
Kinnosuke
K
Matsuura
Gen
G
Watanabe
Masaharu
M
Nomura
Fumio
F
Moss
Joel
J
Noda
Masatoshi
M
D000595:Amino Acid Sequence; D000818:Animals; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D003603:Cytotoxins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis / Q000502:physiology; D022603:Shiga Toxins / Q000235:genetics* / Q000378:metabolism*; D014617:Vacuoles; D014709:Vero Cells
D003603:Cytotoxins; D029968:Escherichia coli Proteins; D022603:Shiga Toxins