Subtilase cytotoxin produced by locus of enterocyte effacement‐negative Shiga‐toxigenic Escherichia coli induces stress granule formation

Abstract

Subtilase cytotoxin (SubAB) is mainly produced by locus of enterocyte effacement (LEE)-negative strains of Shiga-toxigenic Escherichia coli (STEC). SubAB cleaves an endoplasmic reticulum (ER) chaperone, BiP/Grp78, leading to induction of ER stress. This stress causes activation of ER stress sensor proteins and induction of caspase-dependent apoptosis. We found that SubAB induces stress granules (SG) in various cells. Aim of this study was to explore the mechanism by which SubAB induced SG formation. Here, we show that SubAB-induced SG formation is regulated by activation of double-stranded RNA-activated protein kinase (PKR)-like endoplasmic reticulum kinase (PERK). The culture supernatant of STEC O113:H21 dramatically induced SG in Caco2 cells, although subAB knockout STEC O113:H21 culture supernatant did not. Treatment with phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, and lysosomal inhibitors, NH4 Cl and chloroquine, suppressed SubAB-induced SG formation, which was enhanced by PKC and PKD inhibitors. SubAB attenuated the level of PKD1 phosphorylation. Depletion of PKCδ and PKD1 by siRNA promoted SG formation in response to SubAB. Furthermore, death-associated protein 1 (DAP1) knockdown increased basal phospho-PKD1(S916) and suppressed SG formation by SubAB. However, SG formation by an ER stress inducer, Thapsigargin, was not inhibited in PMA-treated cells. Our findings show that SubAB-induced SG formation is regulated by the PERK/DAP1 signalling pathway, which may be modulated by PKCδ/PKD1, and different from the signal transduction pathway that results in Thapsigargin-induced SG formation.

Diverse roles of endoplasmic reticulum stress sensors in bacterial infection

“…A recent study showed that SubAB induces stress granules in various cells in a PERK-dependent manner [ 22 ]. Furthermore, Halder et al reported that the Helicobacter pylori produced HP0175, a peptidyl-prolyl cis-trans isomerase, activates the PERK arm consequently leading to production of ATF4 and CHOP which both induce the expression of autophagy-related genes [ 23 ].…”

Preparation of Biodegradable PLGA-Nanoparticles Used for pH-Sensitive Intracellular Delivery of an Anti-inflammatory Bacterial Toxin to Macrophages

“…7,8) After translocation into ER via the trans-Golgi network (TGN), SubAB cleaves BiP, resulting in ER stressinduced cytotoxicity through activation of ER stress sensors, PERK, IRE1α and ATF6 signaling, which mediate a unique signaling pathway including phosphorylation of eIF2α and expression of C/EBP homologous protein (CHOP). 9,10) To date, several studies have demonstrated that SubAB induces cytotoxicity in vitro and lethal severe hemorrhagic inflammation in mice, inhibition of autophagy and stress granule formation. Our previous study demonstrated that SubAB inhibits LPSinduced nitric oxide (NO) production in macrophages through the suppression of nuclear factor-kappaB (NF-κB) activation and subsequent inducible NO synthase (iNOS) expression.…”

Mechanism of inhibition of Shiga-toxigenic Escherichia coli SubAB cytotoxicity by steroids and diacylglycerol analogues

“…After SubAB binds to its surface receptors 6 – 8 , the toxin translocates into cells through clathrin-mediated 9 or lipid rafts- and actin-dependent pathways 10 and then cleaves at a specific site on the chaperone protein BiP/Grp78 in the endoplasmic reticulum (ER) 4 . BiP cleavage by SubAB causes ER stress, followed by activation of ER-stress sensor proteins (e.g., IRE1, ATF6, PERK) 11 , 12 , which initiate cell damage pathways 11 , 12 and various cell responses including inhibition of iNOS synthesis 13 and stress granule formation 14 . In addition, administration of SubAB to mice causes a lethal severe hemorrhagic inflammation, injury to intestinal cells, extensive microvascular thrombosis, evidence of histological damage in kidneys, and liver, and dramatic splenic atrophy 15 – 17 .…”