title: 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.
authors.affiliation: Department of Microbiology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan.
authors.forename: Hiroyasu
authors.initials: H
authors.lastname: Tsutsuki
authors.affiliation: Department of Molecular Infectiology, Graduate School of Medicine, Chiba University, Chiba, Japan.
authors.forename: Kinnosuke
authors.initials: K
authors.lastname: Yahiro
authors.affiliation: Pathogenic Microbe Laboratory, Research Institute, National Centre for Global Health and Medicine, Tokyo, Japan.
authors.forename: Kohei
authors.initials: K
authors.lastname: Ogura
authors.affiliation: Department of Molecular Infectiology, Graduate School of Medicine, Chiba University, Chiba, Japan.
authors.forename: Kimitoshi
authors.initials: K
authors.lastname: Ichimura
authors.affiliation: Department of Bacteriology I, National Institute of Infectious Diseases, Tokyo, Japan.
authors.forename: Sunao
authors.initials: S
authors.lastname: Iyoda
authors.affiliation: Department of Bacteriology I, National Institute of Infectious Diseases, Tokyo, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Ohnishi
authors.affiliation: Department of Legal Medicine, Graduate School of Medicine, Chiba University, Chiba, Japan.
authors.forename: Sayaka
authors.initials: S
authors.lastname: Nagasawa
authors.affiliation: Division of Bacteriology, Osaka Prefectural Institute of Public Health, Osaka, Japan.
authors.forename: Kazuko
authors.initials: K
authors.lastname: Seto
authors.affiliation: Cardiovascular and Pulmonary Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA.
authors.forename: Joel
authors.initials: J
authors.lastname: Moss
authors.affiliation: Department of Molecular Infectiology, Graduate School of Medicine, Chiba University, Chiba, Japan.
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Noda
mesh_terms: D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D018938:Caco-2 Cells / Q000187:drug effects / Q000378:metabolism / Q000382:microbiology; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002738:Chloroquine / Q000494:pharmacology; D017077:Culture Media, Conditioned / Q000494:pharmacology; D003594:Cytoplasmic Granules / Q000378:metabolism*; D004265:DNA Helicases; D000091342:Endoplasmic Reticulum Chaperone BiP; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D055786:Gene Knockout Techniques; D006367:HeLa Cells; D054884:Host-Pathogen Interactions; D006801:Humans; D000075223:Poly-ADP-Ribose Binding Proteins; D051745:Protein Kinase C-delta / Q000378:metabolism; D020365:RNA Helicases; D000067776:RNA Recognition Motif Proteins; D054323:Shiga-Toxigenic Escherichia coli / Q000378:metabolism* / Q000472:pathogenicity; D015398:Signal Transduction / Q000187:drug effects; D013312:Stress, Physiological / Q000187:drug effects; D013381:Subtilisins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D019892:eIF-2 Kinase / Q000378:metabolism
chemical_list: D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins; D017077:Culture Media, Conditioned; C091572:DAP protein, human; D000091342:Endoplasmic Reticulum Chaperone BiP; D029968:Escherichia coli Proteins; C000717247:HSPA5 protein, human; D000075223:Poly-ADP-Ribose Binding Proteins; D000067776:RNA Recognition Motif Proteins; D002738:Chloroquine; C460640:EIF2AK3 protein, human; D019892:eIF-2 Kinase; D051745:Protein Kinase C-delta; D013381:Subtilisins; C527234:subtilase cytotoxin, E coli; D004265:DNA Helicases; C116070:G3BP1 protein, human; D020365:RNA Helicases
keywords: ER stress; protein kinases; stress granule; subtilase cytotoxin