Two Distinct Cytotoxic Activities of Subtilase Cytotoxin Produced by Shiga-Toxigenic Escherichia coli

Abstract

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 g/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 ϳ1 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.

Decreased ER-associated degradation of α-TCR induced by Grp78 depletion with the SubAB cytotoxin

“…Interestingly, such dramatic changes in the cytoplasm were not associated with induction of cytoplasmic chaperones. In contrast to Morinaga et al who associated the vacuolating activity with the B subunit of SubAB (Morinaga et al, 2007), we have observed only minimal changes after treatment with the mutant toxin, which can be explained by trace residual catalytic activity of SubA A272 B (Wolfson et al, 2008). Therefore, we must conclude that formation of the perinuclear vacuoles is downstream of BiP degradation and not an independent activity.…”

“…In accordance with other reports of the effects of SubAB on mammalian cells we observed vacuolization of HeLa cells after treatment with SubAB (Morinaga et al, 2007). Formation of intracellular vacuoles is a common response to some,but not all, stimuli causing ER stress in HeLa cells, such as RNAi of p97/VCP or extended inhibition of ERAD by proteasome inhibitors (Wojcik et al, 1996).…”

“…Since cytotoxic effects of SubAB were first investigated on Vero cells derived from African green monkey, we have also included COS7 cells (Morinaga et al, 2007;Paton et al, 2004;Paton et al, 2006).…”

“…Since SubAB is cytotoxic to Vero cells (Morinaga et al, 2007;Paton et al, 2004;Paton et al, 2006) and leads to increased apoptosis in many organs following intraperitoneal injection in mice (Wang et al, 2007), we therefore explored whether SubAB induces apoptotic features in human cells. In HeLa cells, SubAB induced activation of procaspase 3 as evidenced by cleavage of PARP and detection of trace amounts of cleaved caspase 3.…”

“…The A subunit of SubAB specifically cleaves BiP depleting cellular stores of this ER chaperone within 20-30 min upon addition to cell culture, an activity which is completely abolished by the Ser272Ala mutation of the A subunit (Paton et al, 2006). An independent group confirmed cytotoxic activity of SubAB, however it also has claimed an additional vacuolizing activity for the B subunit of SubAB (Morinaga et al, 2007).…”