Essential Role for Verotoxin in Sustained Stress-Activated Protein Kinase and Nuclear Factor Kappa B Signaling, Stimulated by Escherichia coli O157:H7 in Vero Cells

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Abstract
The effects of Escherichia coli O157:H7 (strains E30480 and PM601) and the associated verotoxins (VTs), VT1 and VT2, on stress-activated protein kinase and nuclear factor kappa B (NF-κB) signaling were investigated with Vero cells, which are extremely sensitive to the cytotoxic effects of E. coli O157:H7 in vitro. Cell-free supernatants prepared from E30480 and PM601 cultures and purified VT1 and VT2 stimulated a strong and prolonged (>4-h) activation of both c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase. However, JNK activity stimulated in response to E30480 supernatants was substantially reduced following pretreatment with anti-VT1 and anti-VT2 antibodies, while a VT1 and VT2 gene knockout mutant of PM601 was unable to stimulate JNK activity. E30480 supernatants also caused a sustained activation of NF-κB DNA binding, degradation of inhibitory kappa B alpha (IκBα), and an increase in inhibitory kappa B kinase α activity, although PM601 supernatants and VT1 and VT2 had no effect. However, preincubation with VTs prolonged the transient activation of NF-κB and IκBα degradation stimulated by either tumor necrosis factor alpha or interleukin 1β, while preincubation with anti-VT antibodies prevented the prolonged loss of IκBα and partially reduced DNA binding in response to E30480 supernatants. These results strongly suggest that in Vero cells, VT plays an essential role in sustained JNK and NF-κB signaling in response to E. coli O157:H7 and that this action may underpin their cell-selective cytotoxic effects. These studies also suggest that another component released by strain E30480 contributes to the early activation of JNK and NF-κB.