Novel subtilase cytotoxin produced by Shiga-toxigenic Escherichia coli induces apoptosis in vero cells via mitochondrial membrane damage.
Subtilase cytotoxin (SubAB) is an AB(5) cytotoxin produced by some strains of Shiga-toxigenic Escherichia coli. The A subunit is a subtilase-like serine protease and cleaves an endoplasmic reticulum chaperone, BiP, leading to transient inhibition of protein synthesis and cell cycle arrest at G(1) phase. Here we show that SubAB, but not the catalytically inactive mutant SubAB(S272A), induced apoptosis in Vero cells, as detected by DNA fragmentation and annexin V binding. SubAB induced activation of caspase-3, -7, and -8. Caspase-3 appeared earlier than caspase-8, and by use of specific caspase inhibitors, it was determined that caspase-3 may be upstream of caspase-8. A general caspase inhibitor blocked SubAB-induced apoptosis, detected by annexin V binding. SubAB also stimulated cytochrome c release from mitochondria, which was not suppressed by caspase inhibitors. In HeLa cells, Apaf-1 small interfering RNA inhibited caspase-3 activation, suggesting that cytochrome c might form an apoptosome, leading to activation of caspase-3. These data suggested that SubAB induced caspase-dependent apoptosis in Vero cells through mitochondrial membrane damage.
Matsuura
Gen
G
Department of Molecular Infectiology, Graduate School of Medicine, Chiba University, Chiba, Japan.
Morinaga
Naoko
N
Yahiro
Kinnosuke
K
Komine
Reiko
R
Moss
Joel
J
Yoshida
Hideo
H
Noda
Masatoshi
M
D000818:Animals; D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis; D053148:Caspase 3 / Q000378:metabolism; D053179:Caspase 7 / Q000378:metabolism; D053181:Caspase 8 / Q000378:metabolism; D002522:Chlorocebus aethiops; D045304:Cytochromes c / Q000378:metabolism; D053938:DNA Fragmentation; D029968:Escherichia coli Proteins / Q000633:toxicity*; D006367:HeLa Cells; D006801:Humans; D051336:Mitochondrial Membranes / Q000187:drug effects*; D054323:Shiga-Toxigenic Escherichia coli / Q000472:pathogenicity*; D013381:Subtilisins / Q000633:toxicity*; D014709:Vero Cells
D017304:Annexin A5; D029968:Escherichia coli Proteins; D045304:Cytochromes c; D013381:Subtilisins; C527234:subtilase cytotoxin, E coli; D053148:Caspase 3; D053179:Caspase 7; D053181:Caspase 8