RESUMEN
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.
Asunto(s)
Gránulos Citoplasmáticos/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli Shiga-Toxigénica/metabolismo , Subtilisinas/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Células CACO-2/efectos de los fármacos , Células CACO-2/metabolismo , Células CACO-2/microbiología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Cloroquina/farmacología , Medios de Cultivo Condicionados/farmacología , ADN Helicasas , Chaperón BiP del Retículo Endoplásmico , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/farmacología , Técnicas de Inactivación de Genes , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Proteínas de Unión a Poli-ADP-Ribosa , Proteína Quinasa C-delta/metabolismo , ARN Helicasas , Proteínas con Motivos de Reconocimiento de ARN , Escherichia coli Shiga-Toxigénica/patogenicidad , Transducción de Señal/efectos de los fármacos , Estrés Fisiológico/efectos de los fármacos , Subtilisinas/genética , Subtilisinas/farmacología , eIF-2 Quinasa/metabolismoRESUMEN
Although the adhesion of enterohemorrhagic Escherichia coli (EHEC) is central to the EHEC-host interaction during infection, it remains unclear how such adhesion regulates virulence factors. Adhesion to abiotic surfaces by E. coli has been reported to be an outer membrane lipoprotein NlpE-dependent activation cue of the Cpx pathway. Therefore, we investigated the role of NlpE in EHEC on the adhesion-mediated expression of virulence genes. NlpE in EHEC contributed to upregulation of the locus of enterocyte effacement (LEE) genes encoded type III secretion system and to downregulated expression of the flagellin gene by activation of the Cpx pathway during adherence to hydrophobic glass beads and undifferentiated Caco-2 cells. Moreover, LysR homologue A (LrhA) in EHEC was involved in regulating the expression of the LEE genes and flagellin gene in response to adhesion. Gel mobility shift analysis revealed that response regulator CpxR bound to the lrhA promoter region and thereby regulated expressions of the LEE genes and flagellin gene via the transcriptional regulator LrhA in EHEC. Therefore, these results suggest that the sensing of adhesion signals via NlpE is important for regulation of the expression of the type III secretion system and flagella in EHEC during infection.
Asunto(s)
Adhesión Bacteriana , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/metabolismo , Escherichia coli Enterohemorrágica/genética , Escherichia coli Enterohemorrágica/fisiología , Proteínas de Escherichia coli/metabolismo , Flagelos/fisiología , Lipoproteínas/metabolismo , Sistemas de Secreción Tipo III/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/genética , Células CACO-2 , Escherichia coli Enterohemorrágica/patogenicidad , Proteínas de Escherichia coli/genética , Flagelos/metabolismo , Regulación Bacteriana de la Expresión Génica , Células HeLa , Humanos , Lipoproteínas/genética , Transducción de Señal/genética , Sistemas de Secreción Tipo III/genética , Factores de Virulencia/genéticaRESUMEN
Shiga toxigenic Escherichia coli (STEC) are responsible for a worldwide foodborne disease, which is characterized by severe bloody diarrhea and hemolytic uremic syndrome (HUS). Subtilase cytotoxin (SubAB) is a novel AB5 toxin, which is produced by Locus for Enterocyte Effacement (LEE)-negative STEC. Cleavage of the BiP protein by SubAB induces endoplasmic reticulum (ER) stress, followed by induction of cytotoxicity in vitro or lethal severe hemorrhagic inflammation in mice. Here we found that steroids and diacylglycerol (DAG) analogues (e.g., bryostatin 1, Ingenol-3-angelate) inhibited SubAB cytotoxicity. In addition, steroid-induced Bcl-xL expression was a key step in the inhibition of SubAB cytotoxicity. Bcl-xL knockdown increased SubAB-induced apoptosis in steroid-treated HeLa cells, whereas SubAB-induced cytotoxicity was suppressed in Bcl-xL overexpressing cells. In contrast, DAG analogues suppressed SubAB activity independent of Bcl-xL expression at early time points. Addition of Shiga toxin 2 (Stx2) with SubAB to cells enhanced cytotoxicity even in the presence of steroids. In contrast, DAG analogues suppressed cytotoxicity seen in the presence of both toxins. Here, we show the mechanism by which steroids and DAG analogues protect cells against SubAB toxin produced by LEE-negative STEC.
RESUMEN
Enterohemorrhagic Escherichia coli (EHEC) produces Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2). Nitric oxide (NO), which acts as an antimicrobial defense molecule, was found to enhance the production of Stx1 and Stx2 in EHEC under anaerobic conditions. Although EHEC O157 has two types of anaerobic NO reductase genes, an intact norV and a deleted norV, in the deleted norV-type EHEC, a high concentration of NO (12-29 µmol/L, maximum steady-state concentration) is required for enhanced Stx1 production and a low concentration of NO (~12 µmol/L, maximum steady-state concentration) is sufficient for enhanced Stx2 production under anaerobic conditions. These results suggested that different concentration thresholds of NO elicit a discrete set of Stx1 and Stx2 production pathways. Moreover, the enhancement of Shiga toxin production in the intact norV-type EHEC required treatment with a higher concentration of NO than was required for enhancement of Shiga toxin production in the deleted norV-type EHEC, suggesting that the specific NorV type plays an important role in the level of enhancement of Shiga toxin production in response to NO. Finally, Fur derepression and RecA activation in EHEC were shown to participate in the NO-enhanced Stx1 and Stx2 production, respectively.
Asunto(s)
Proteínas Bacterianas/metabolismo , Escherichia coli O157/efectos de los fármacos , Escherichia coli O157/metabolismo , Regulación Bacteriana de la Expresión Génica , Óxido Nítrico/metabolismo , Rec A Recombinasas/metabolismo , Proteínas Represoras/metabolismo , Toxina Shiga/biosíntesis , AnaerobiosisRESUMEN
A novel virulence gene, norV, that encodes nitric oxide (NO) reductase, was examined to investigate the emergence of enterohemorrhagic Escherichia coli (EHEC) O157 subgroup C clusters 2 and 3 from subgroup C cluster 1. Deletion of norV occurred at a point between cluster 1 and cluster 2 just after or at the same time that an stx2 bacteriophage, which retains Shiga toxin 2 gene, was inserted into wrbA, which encodes a novel multimeric flavodoxin-like protein, in EHEC O157. Sensitivity of NO to anaerobic growth was correlated with the deletion of norV in all EHEC O157 individuals tested. The C467A mutation of fimH, which encodes minor component of type 1 fimbriae, occurred within cluster 1, not as a transition from cluster 1 to cluster 2, indicating that there is a cluster 1 minority branch that leads to cluster 2. These data refine the evolutionary history of an emerging EHEC O157.