RESUMEN
Enterohemorrhagic Escherichia coli (EHEC) has at least three enzymes, NorV, Hmp, and Hcp, that act independently to lower the toxicity of nitric oxide (NO), a potent antimicrobial molecule. This study aimed to reveal the cooperative roles of these defensive enzymes in EHEC against nitrosative stress. Under anaerobic conditions, combined deletion of all three enzymes significantly increased the NO sensitivity of EHEC determined by the growth at late stationary phase; however, the expression of norV restored the NO resistance of EHEC. On the other hand, the growth of Δhmp mutant EHEC was inhibited after early stationary phase, indicating that NorV and Hmp play a cooperative role in anaerobic growth. Under microaerobic conditions, the growth of Δhmp mutant EHEC was inhibited by NO, indicating that Hmp is the enzyme that protects cells from NO stress under microaerobic conditions. When EHEC cells were exposed to a lower concentration of NO, the NO level in bacterial cells of Δhcp mutant EHEC was higher than those of the other EHEC mutants, suggesting that Hcp is effective at regulating NO levels only at a low concentration. These findings of a low level of NO in bacterial cells with hcp indicate that the NO consumption activity of Hcp was suppressed by Hmp at a low range of NO concentrations. Taken together, these results show that the cooperative effects of NO-metabolizing enzymes are regulated by the range of NO concentrations to which the EHEC cells are exposed.
Asunto(s)
Escherichia coli Enterohemorrágica/enzimología , Proteínas de Escherichia coli/metabolismo , Óxido Nítrico/metabolismo , Estrés Nitrosativo/fisiología , Anaerobiosis , Escherichia coli Enterohemorrágica/metabolismo , Regulación Bacteriana de la Expresión Génica , Oxidorreductasas/metabolismoRESUMEN
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
The novel cytotoxic factor subtilase cytotoxin (SubAB) is produced mainly by non-O157 Shiga-toxigenic Escherichia coli (STEC). SubAB cleaves the molecular chaperone BiP/GRP78 in the endoplasmic reticulum (ER), leading to activation of RNA-dependent protein kinase (PKR)-like ER kinase (PERK), followed by caspase-dependent cell death. However, the SubAB uptake mechanism in HeLa cells is unknown. In this study, a variety of inhibitors and siRNAs were employed to characterize the SubAB uptake process. SubAB-induced BiP cleavage was inhibited by high concentrations of Dynasore, and methyl-ß-cyclodextrin (mßCD) and Filipin III, but not suppressed in clathrin-, dynamin I/II-, caveolin1- and caveolin2-knockdown cells. We observed that SubAB treatment led to dramatic actin rearrangements, e.g. formation of plasma membrane blebs, with a significant increase in fluid uptake. Confocal microscopy analysis showed that SubAB uptake required actin cytoskeleton remodelling and lipid raft cholesterol. Furthermore, internalized SubAB in cells was found in the detergent-resistant domain (DRM) structure. Interestingly, IPA-3, an inhibitor of serine/threonine kinase p21-activated kinase (PAK1), an important protein of macropinocytosis, directly inhibited SubAB-mediated BiP cleavage and SubAB internalization. Thus, our findings suggest that SubAB uses lipid raft- and actin-dependent, but not clathrin-, caveolin- and dynamin-dependent pathways as its major endocytic translocation route.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico/metabolismo , Microdominios de Membrana/metabolismo , Escherichia coli Shiga-Toxigénica/patogenicidad , Subtilisinas/metabolismo , Antivirales/farmacología , Transporte Biológico , Caveolinas/genética , Línea Celular , Colesterol/metabolismo , Clatrina/genética , Disulfuros/farmacología , Dinaminas/genética , Retículo Endoplásmico/metabolismo , Chaperón BiP del Retículo Endoplásmico , Activación Enzimática , Filipina/farmacología , Células HeLa , Humanos , Hidrazonas/farmacología , Naftoles/farmacología , Interferencia de ARN , ARN Interferente Pequeño , beta-Ciclodextrinas/farmacología , eIF-2 Quinasa/metabolismo , Quinasas p21 Activadas/antagonistas & inhibidoresRESUMEN
Autophagy and apoptosis play critical roles in cellular homeostasis and survival. Subtilase cytotoxin (SubAB), produced by non-O157 type Shiga-toxigenic Escherichia coli (STEC), is an important virulence factor in disease. SubAB, a protease, cleaves a specific site on the endoplasmic reticulum (ER) chaperone protein BiP/GRP78, leading to ER stress, and induces apoptosis. Here we report that in HeLa cells, activation of a PERK (RNA-dependent protein kinase [PKR]-like ER kinase)-eIF2α (α subunit of eukaryotic initiation factor 2)-dependent pathway by SubAB-mediated BiP cleavage negatively regulates autophagy and induces apoptosis through death-associated protein 1 (DAP1). We found that SubAB treatment decreased the amounts of autophagy markers LC3-II and p62 as well as those of mTOR (mammalian target of rapamycin) signaling proteins ULK1 and S6K. These proteins showed increased expression levels in PERK knockdown or DAP1 knockdown cells. In addition, depletion of DAP1 in HeLa cells dramatically inhibited the SubAB-stimulated apoptotic pathway: SubAB-induced Bax/Bak conformational changes, Bax/Bak oligomerization, cytochrome c release, activation of caspases, and poly(ADP-ribose) polymerase (PARP) cleavage. These results show that DAP1 is a key regulator, through PERK-eIF2α-dependent pathways, of the induction of apoptosis and reduction of autophagy by SubAB.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis/fisiología , Autofagia/fisiología , Proteínas de Escherichia coli/metabolismo , Regulación de la Expresión Génica/inmunología , Subtilisinas/metabolismo , Proteínas Reguladoras de la Apoptosis/genética , Chaperón BiP del Retículo Endoplásmico , Escherichia coli , Proteínas de Escherichia coli/genética , Células HeLa , Humanos , Subtilisinas/genética , Proteína Destructora del Antagonista Homólogo bcl-2/genética , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
In Helicobacter pylori infection, vacuolating cytotoxin (VacA)-induced mitochondrial damage leading to apoptosis is believed to be a major cause of cell death. It has also been proposed that VacA-induced autophagy serves as a host mechanism to limit toxin-induced cellular damage. Apoptosis and autophagy are two dynamic and opposing processes that must be balanced to regulate cell death and survival. Here we identify the low-density lipoprotein receptor-related protein-1 (LRP1) as the VacA receptor for toxin-induced autophagy in the gastric epithelial cell line AZ-521, and show that VacA internalization through binding to LRP1 regulates the autophagic process including generation of LC3-II from LC3-I, which is involved in formation of autophagosomes and autolysosomes. Knockdown of LRP1 and Atg5 inhibited generation of LC3-II as well as cleavage of PARP, a marker of apoptosis, in response to VacA, whereas caspase inhibitor, benzyloxycarbonyl-VAD-fluoromethylketone (Z-VAD-fmk), and necroptosis inhibitor, Necrostatin-1, did not inhibit VacA-induced autophagy, suggesting that VacA-induced autophagy via LRP1 binding precedes apoptosis. Other VacA receptors such as RPTPα, RPTPß, and fibronectin did not affect VacA-induced autophagy or apoptosis. Therefore, we propose that the cell surface receptor, LRP1, mediates VacA-induced autophagy and apoptosis.
Asunto(s)
Apoptosis , Autofagia , Proteínas Bacterianas/metabolismo , Infecciones por Helicobacter/metabolismo , Helicobacter pylori/metabolismo , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Clorometilcetonas de Aminoácidos/farmacología , Proteína 5 Relacionada con la Autofagia , Línea Celular , Inhibidores de Cisteína Proteinasa/farmacología , Epitelio/metabolismo , Epitelio/microbiología , Epitelio/patología , Fibronectinas/genética , Fibronectinas/metabolismo , Mucosa Gástrica/metabolismo , Infecciones por Helicobacter/genética , Infecciones por Helicobacter/patología , Helicobacter pylori/genética , Humanos , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas Tirosina Fosfatasas Clase 5 Similares a Receptores/genética , Proteínas Tirosina Fosfatasas Clase 5 Similares a Receptores/metabolismo , Estómago/microbiología , Estómago/patologíaRESUMEN
In enterohaemorrhagic Escherichia coli (EHEC) O157, there are two types of anaerobic nitric oxide (NO) reductase genes, an intact gene (norV) and a 204 bp deletion gene (norVs). Epidemiological analysis has revealed that norV-type EHEC are more virulent than norVs-type EHEC. Thus, to reveal the role of NO reductase during EHEC infection, we constructed isogenic norV-type and norVs-type EHEC mutant strains. Under anaerobic conditions, the norV-type EHEC was protected from NO-mediated growth inhibition, while the norVs-type EHEC mutant strain was not, suggesting that NorV of EHEC was effective in the anaerobic detoxification. We then investigated the role of NO reductase within macrophages. The norV-type EHEC produced a lower NO level within macrophages compared with the norVs-type EHEC. Moreover, the norV-type EHEC resulted in higher levels of Shiga toxin 2 (Stx2) within macrophages compared with the norVs-type EHEC. Finally, the norV-type EHEC showed a better level of survival than the norVs-type EHEC. These data suggest that the intact norV gene plays an important role for the survival of EHEC within macrophages, and is a direct virulence determinant of EHEC.
Asunto(s)
Escherichia coli O157/genética , Escherichia coli O157/patogenicidad , Macrófagos/microbiología , Oxidorreductasas/genética , Factores de Virulencia/genética , Anaerobiosis/genética , Escherichia coli O157/metabolismo , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica , Orden Génico , Macrófagos/metabolismo , Óxido Nítrico/metabolismo , Operón/genética , Oxidorreductasas/metabolismo , Toxina Shiga I/biosíntesis , Toxina Shiga I/genética , Factores de Virulencia/metabolismoRESUMEN
Cholix toxin (Cholix) is a novel ADP-ribosylating cytotoxin produced by Vibrio cholerae, which utilizes eukaryotic elongation factor 2 as a substrate and acts by a mechanism similar to that of diphtheria toxin and Pseudomonas exotoxin A. First it was found that Cholix-treated HeLa cells exhibited caspase-dependent apoptosis, whereas intestinal cells such as Caco-2, HCT116, and RKO did not. Here we investigated Cholix-induced cell death signaling pathways in HeLa cells. Cholix-induced cytochrome c release into cytosol was initiated by specific conformational changes of pro-apoptotic Bak associated with Bax. Silencing of bak/bax genes or bak gene alone using siRNA significantly suppressed cytochrome c release and caspase-7 activation, but not activation of caspases-3 and -9. Although pretreatment with a caspase-8 inhibitor (Z-IETD-FMK) reduced Cholix-induced cytochrome c release and activation of caspases-3, -7, and -9, cytotoxicity was not decreased. Pretreatment with Z-YVAD-FMK, which inhibits caspase-1, -4, and -5, suppressed not only cytochrome c release, activation of caspase-3, -7, -8, or -9, and PARP cleavage, but also cytotoxicity, indicating that caspase-1, -4, and -5 activation is initiated at an early stage of Cholix-induced apoptosis and promotes caspase-8 activation. These results show that the inflammatory caspases (caspase-1, -4, and -5) and caspase-8 are responsible for both mitochondrial signals and other caspase activation. In conclusion, we showed that Cholix-induced caspase activation plays an essential role in generation of apoptotic signals, which are mediated by both mitochondria-dependent and -independent pathways.
Asunto(s)
Factores de Ribosilacion-ADP/farmacología , Apoptosis/efectos de los fármacos , Toxinas Bacterianas/farmacología , Transducción de Señal/efectos de los fármacos , Vibrio cholerae/química , Factores de Ribosilacion-ADP/química , Apoptosis/genética , Toxinas Bacterianas/química , Inhibidores de Caspasas , Caspasas/genética , Caspasas/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Citocromos c/genética , Citocromos c/metabolismo , Activación Enzimática , Silenciador del Gen , Células HeLa , Humanos , Oligopéptidos/farmacología , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteolisis/efectos de los fármacos , ARN Interferente Pequeño/genética , Proteína Destructora del Antagonista Homólogo bcl-2/genética , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Shiga-toxigenic Escherichia coli (STEC) produces subtilase cytotoxin (SubAB), which cleaves the molecular chaperone BiP in the endoplasmic reticulum (ER), leading to an ER stress response and then activation of apoptotic signaling pathways. Here, we show that an early event in SubAB-induced apoptosis in HeLa cells is mediated by RNA-dependent protein kinase (PKR)-like ER kinase (PERK), not activating transcription factor 6 (ATF6) or inositol-requiring enzyme 1(Ire1), two other ER stress sensors. PERK knockdown suppressed SubAB-induced eIF2α phosphorylation, activating transcription factor 4 (ATF4) expression, caspase activation, and cytotoxicity. Knockdown of eIF2α by small interfering RNA (siRNA) or inhibition of eIF2α dephosphorylation by Sal003 enhanced SubAB-induced caspase activation. Treatment with proteasome inhibitors (i.e., MG132 and lactacystin), but not a general caspase inhibitor (Z-VAD) or a lysosome inhibitor (chloroquine), suppressed SubAB-induced caspase activation and poly(ADP-ribose) polymerase (PARP) cleavage, suggesting that the ubiquitin-proteasome system controls events leading to caspase activation, i.e., Bax/Bak conformational changes, followed by cytochrome c release from mitochondria. Levels of ubiquitinated proteins in HeLa cells were significantly decreased by SubAB treatment. Further, in an early event, some antiapoptotic proteins, which normally turn over rapidly, have their synthesis inhibited, and show enhanced degradation via the proteasome, resulting in apoptosis. In PERK knockdown cells, SubAB-induced loss of ubiquitinated proteins was inhibited. Thus, SubAB-induced ER stress is caused by BiP cleavage, leading to PERK activation, not by accumulation of ubiquitinated proteins, which undergo PERK-dependent degradation via the ubiquitin-proteasome system.
Asunto(s)
Apoptosis/fisiología , Proteínas de Escherichia coli/farmacología , Complejo de la Endopetidasa Proteasomal/metabolismo , Subtilisinas/farmacología , eIF-2 Quinasa/metabolismo , Factor de Transcripción Activador 4/genética , Factor de Transcripción Activador 4/metabolismo , Degradación Asociada con el Retículo Endoplásmico/fisiología , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Fosforilación , Complejo de la Endopetidasa Proteasomal/genética , Transducción de Señal , eIF-2 Quinasa/genéticaRESUMEN
Subtilase cytotoxin (SubAB), which is produced by certain strains of Shiga-toxigenic Escherichia coli (STEC), cleaves an endoplasmic reticulum (ER) chaperone, BiP/Grp78, leading to induction of ER stress and caspase-dependent apoptosis. SubAB alters the innate immune response. SubAB pretreatment of macrophages inhibited lipopolysaccharide (LPS)-induced production of both monocyte chemoattractant protein 1 (MCP-1) and tumor necrosis factor α (TNF-α). We investigated here the mechanism by which SubAB inhibits nitric oxide (NO) production by mouse macrophages. SubAB suppressed LPS-induced NO production through inhibition of inducible NO synthase (iNOS) mRNA and protein expression. Further, SubAB inhibited LPS-induced IκB-α phosphorylation and nuclear localization of the nuclear factor-κB (NF-κB) p65/p50 heterodimer. Reporter gene and chromatin immunoprecipitation (ChIP) assays revealed that SubAB reduced LPS-induced NF-κB p65/p50 heterodimer binding to an NF-κB binding site on the iNOS promoter. In contrast to the native toxin, a catalytically inactivated SubAB mutant slightly enhanced LPS-induced iNOS expression and binding of NF-κB subunits to the iNOS promoter. The SubAB effect on LPS-induced iNOS expression was significantly reduced in macrophages from NF-κB1 (p50)-deficient mice, which lacked a DNA-binding subunit of the p65/p50 heterodimer, suggesting that p50 was involved in SubAB-mediated inhibition of iNOS expression. Treatment of macrophages with an NOS inhibitor or expression of SubAB by E. coli increased E. coli survival in macrophages, suggesting that NO generated by macrophages resulted in efficient killing of the bacteria and SubAB contributed to E. coli survival in macrophages. Thus, we hypothesize that SubAB might represent a novel bacterial strategy to circumvent host defense during STEC infection.
Asunto(s)
Proteínas de Escherichia coli/farmacología , Escherichia coli/metabolismo , Macrófagos/efectos de los fármacos , FN-kappa B/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo II/genética , Subtilisinas/farmacología , Animales , Supervivencia Celular , Células Cultivadas , Chaperón BiP del Retículo Endoplásmico , Regulación de la Expresión Génica , Immunoblotting , Inmunoprecipitación , Lipopolisacáridos/farmacología , Macrófagos/metabolismo , Ratones , FN-kappa B/metabolismo , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa de Tipo II/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de SeñalRESUMEN
Subtilase cytotoxin (SubAB), which is produced by certain strains of Shiga-toxigenic Escherichia coli (STEC), causes the 78-kDa glucose-regulated protein (GRP78/BiP) cleavage, followed by induction of endoplasmic reticulum (ER) stress, leading to caspase-dependent apoptosis via mitochondrial membrane damage by Bax/Bak activation. The purpose of the present study was to identify SubAB receptors responsible for HeLa cell death. Four proteins, NG2, α2ß1 integrin (ITG), L1 cell adhesion molecule (L1CAM), and hepatocyte growth factor receptor (Met), were identified to be SubAB-binding proteins by immunoprecipitation and purification, followed by liquid chromatography-tandem mass spectrometry analysis. SubAB-induced Bax conformational change, Bax/Bak complex formation, caspase activation, and cell death were decreased in ß1 ITG, NG2, and L1CAM small interfering RNA-transfected cells, but unexpectedly, BiP cleavage was still observed. Pretreatment of cells with a function-blocking ß1 ITG antibody (monoclonal antibody [MAb] P5D2) enhanced SubAB-induced caspase activation; MAb P5D2 alone had no effect on caspase activation. Furthermore, we found that SubAB induced focal adhesion kinase fragmentation, which was mediated by a proteasome-dependent pathway, and caspase activation was suppressed in the presence of proteasome inhibitor. Thus, ß1 ITG serves as a SubAB-binding protein and may interact with SubAB-signaling pathways, leading to cell death. Our results raise the possibility that although BiP cleavage is necessary for SubAB-induced apoptotic cell death, signaling pathways associated with functional SubAB receptors may be required for activation of SubAB-dependent apoptotic pathways.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli Shiga-Toxigénica/metabolismo , Subtilisinas/metabolismo , Apoptosis , Chaperón BiP del Retículo Endoplásmico , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica/fisiología , Silenciador del Gen , Células HeLa , Humanos , Integrina alfa2beta1/metabolismo , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas c-met/metabolismo , Subtilisinas/genética , TapsigarginaRESUMEN
Subtilase cytotoxin (SubAB) is an AB(5) type toxin produced by a subset of Shiga-toxigenic Escherichia coli. The A subunit is a subtilase-like serine protease and cleaves an endoplasmic reticulum chaperone BiP. The B subunit binds to a receptor on the cell surface. Although SubAB is lethal for mice, the cause of death is not clear. In this study, we demonstrate in mice that SubAB induced small bowel hemorrhage and a coagulopathy characterized by thrombocytopenia, prolonged prothrombin time and activated partial thromboplastin time. SubAB also induced inflammatory changes in the small intestine as detected by ¹8F-fluoro-2-deoxy-d-glucose positron emission tomography imaging and histochemical analysis. Using RT-PCR and ELISA, SubAB was shown to increase interleukin-6 in a time-dependent manner. Thus, our results indicate that death in SubAB-treated mice may be associated with severe inflammatory response and hemorrhage of the small intestine, accompanied by coagulopathy and IL6 production.
Asunto(s)
Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/mortalidad , Proteínas de Escherichia coli/inmunología , Hemorragia/inmunología , Hemorragia/mortalidad , Escherichia coli Shiga-Toxigénica/patogenicidad , Subtilisinas/inmunología , Animales , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/genética , Hemorragia/microbiología , Humanos , Intestino Delgado/inmunología , Intestino Delgado/microbiología , Masculino , Ratones , Ratones Endogámicos BALB C , Escherichia coli Shiga-Toxigénica/genética , Escherichia coli Shiga-Toxigénica/inmunología , Subtilisinas/genéticaRESUMEN
Subtilase cytotoxin (SubAB) is an AB(5) cytotoxin produced by some strains of Shiga-toxigenic Escherichia coli. The A subunit is a subtilase-like serine protease and cleaves an endoplasmic reticulum (ER) chaperone, BiP, leading to transient inhibition of protein synthesis and cell cycle arrest at G(1) phase, and inducing caspase-dependent apoptosis via mitochondrial membrane damage in Vero cells. Here we investigated the mechanism of mitochondrial permeabilization in HeLa cells. SubAB-induced cytochrome c release into cytosol did not depend on mitochondrial permeability transition pore (PTP), since cyclosporine A did not suppress cytochrome c release. SubAB did not change the expression of anti-apoptotic Bcl-2 or Bcl-XL and pro-apoptotic Bax or Bak, but triggered Bax and Bak conformational changes and association of Bax with Bak. Silencing using siRNA of both bax and bak genes, but not bax, bak, or bim alone, resulted in reduction of cytochrome c release, caspase-3 activation, DNA ladder formation and cytotoxicity, indicating that Bax and Bak were involved in apoptosis. SubAB activated ER transmembrane transducers, Ire1alpha, and cJun N-terminal kinase (JNK), and induced C/EBF-homologue protein (CHOP). To investigate whether these signals were involved in cytochrome c release by Bax activation, we silenced ire1alpha, jnk or chop; however, silencing did not decrease SubAB-induced cytochrome c release, suggesting that these signals were not necessary for SubAB-induced mitochondrial permeabilization by Bax activation.
Asunto(s)
Apoptosis , Proteínas de Escherichia coli/toxicidad , Escherichia coli/patogenicidad , Membranas Mitocondriales/efectos de los fármacos , Permeabilidad/efectos de los fármacos , Subtilisinas/toxicidad , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Caspasa 3/metabolismo , Citocromos c/metabolismo , Citoplasma/química , Endorribonucleasas/metabolismo , Silenciador del Gen , Células HeLa , Humanos , MAP Quinasa Quinasa 4/metabolismo , Unión Proteica , Conformación Proteica/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Factor de Transcripción CHOP/metabolismo , Proteína Destructora del Antagonista Homólogo bcl-2/antagonistas & inhibidores , Proteína X Asociada a bcl-2/antagonistas & inhibidores , Proteína X Asociada a bcl-2/químicaRESUMEN
Shiga toxin 1 (Stx1) is located in the periplasmic fraction, while Stx2 is found in the extracellular fraction, suggesting that enterohemorrhagic Escherichia coli (EHEC) contains a specific Stx2 release system. Both stx(1) and stx(2) are found within the late operons of Stx-encoding phages. Stx2 production is greatly induced by mitomycin C, suggesting that stx(2) can transcribe from the late phage promoter of the Stx2-encoding phage. However, the Stx1 promoter adjacent to stx(1) is a dominant regulatory element in Stx1 production. With the deletion of phage lysis genes of the Stx2-encoding phage, Stx2 remains in the bacterial cells. Further, we demonstrate that the Stx2-encoding phage, but not the Stx1-encoding phage, is spontaneously induced at extremely low rates. These results indicate that spontaneously specific Stx2-encoding phage induction is involved in specific Stx2 release from bacterial cells. Furthermore, to examine whether another system for specific Stx2 release is present in EHEC, we analyze the stx-replaced mutants. As expected, Stx2 derived from the Stx1 promoter is located in both the extracellular and cell-associated fractions, while mutant Stx2 (B subunit, S31N) derived from the Stx1 promoter is found only in the cell-associated fraction. These results indicate that EHEC has another Stx2 release system that strictly recognizes the serine 31 residue of the B subunit. Overall, we present evidence that specific Stx2 release from bacterial cells is involved in both the Stx2-encoding phage induction system and another Stx2 release system.
Asunto(s)
Bacteriólisis , Escherichia coli Enterohemorrágica/metabolismo , Escherichia coli Enterohemorrágica/virología , Toxina Shiga II/metabolismo , Animales , Colifagos/crecimiento & desarrollo , Genes Virales , Profagos/crecimiento & desarrollo , Transporte de Proteínas , ConejosRESUMEN
Subtilase cytotoxin (SubAB) is an AB(5) cytotoxin produced by some strains of Shiga-toxigenic Escherichia coli. The A subunit is a subtilase-like serine protease and cleaves an endoplasmic reticulum chaperone, BiP, leading to transient inhibition of protein synthesis and cell cycle arrest at G(1) phase. Here we show that SubAB, but not the catalytically inactive mutant SubAB(S272A), induced apoptosis in Vero cells, as detected by DNA fragmentation and annexin V binding. SubAB induced activation of caspase-3, -7, and -8. Caspase-3 appeared earlier than caspase-8, and by use of specific caspase inhibitors, it was determined that caspase-3 may be upstream of caspase-8. A general caspase inhibitor blocked SubAB-induced apoptosis, detected by annexin V binding. SubAB also stimulated cytochrome c release from mitochondria, which was not suppressed by caspase inhibitors. In HeLa cells, Apaf-1 small interfering RNA inhibited caspase-3 activation, suggesting that cytochrome c might form an apoptosome, leading to activation of caspase-3. These data suggested that SubAB induced caspase-dependent apoptosis in Vero cells through mitochondrial membrane damage.
Asunto(s)
Apoptosis , Proteínas de Escherichia coli/toxicidad , Membranas Mitocondriales/efectos de los fármacos , Escherichia coli Shiga-Toxigénica/patogenicidad , Subtilisinas/toxicidad , Animales , Anexina A5/metabolismo , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Caspasa 8/metabolismo , Chlorocebus aethiops , Citocromos c/metabolismo , Fragmentación del ADN , Células HeLa , Humanos , Células VeroRESUMEN
Subtilase cytotoxin (SubAB) is a AB(5) type toxin produced by Shiga-toxigenic Escherichia coli, which exhibits cytotoxicity to Vero cells. SubAB B subunit binds to toxin receptors on the cell surface, whereas the A subunit is a subtilase-like serine protease that specifically cleaves chaperone BiP/Grp78. As noted previously, SubAB caused inhibition of protein synthesis. We now show that the inhibition of protein synthesis was transient and occurred as a result of ER stress induced by cleavage of BiP; it was closely associated with phosphorylation of double-stranded RNA-activated protein kinase-like ER kinase (PERK) and eukaryotic initiation factor-2alpha (eIF2alpha). The phosphorylation of PERK and eIF2alpha was maximal at 30-60 min and then returned to the control level. Protein synthesis after treatment of cells with SubAB was suppressed for 2 h and recovered, followed by induction of stress-inducible C/EBP-homologous protein (CHOP). BiP degradation continued, however, even after protein synthesis recovered. SubAB-treated cells showed cell cycle arrest in G1 phase, which may result from cyclin D1 downregulation caused by both SubAB-induced translational inhibition and continuous prolonged proteasomal degradation.
Asunto(s)
Ciclo Celular/efectos de los fármacos , Ciclina D1/metabolismo , Proteínas de Escherichia coli/farmacología , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Escherichia coli Shiga-Toxigénica/enzimología , Subtilisinas/farmacología , Animales , Western Blotting , Chlorocebus aethiops , Ciclina D1/genética , Chaperón BiP del Retículo Endoplásmico , Citometría de Flujo , Fase G1/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células VeroRESUMEN
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
Small GTPases of the ADP-ribosylation factor (ARF) family play a key role in membrane trafficking by regulating coated vesicle formation, and guanine nucleotide exchange is essential for the ARF function. Brefeldin A blocks the ARF-triggered coat assembly by inhibiting the guanine nucleotide exchange on ARFs and causes disintegration of the Golgi complex and tubulation of endosomal membranes. BIG2 is one of brefeldin A-inhibited guanine nucleotide exchange factors for the ARF GTPases and is associated mainly with the trans-Golgi network. In the present study, we have revealed that another population of BIG2 is associated with the recycling endosome and found that expression of a catalytically inactive BIG2 mutant, E738K, selectively induces membrane tubules from this compartment. We also have shown that BIG2 has an exchange activity toward class I ARFs (ARF1 and ARF3) in vivo and inactivation of either ARF exaggerates the BIG2(E738K)-induced tubulation of endosomal membranes. These observations together indicate that BIG2 is implicated in the structural integrity of the recycling endosome through activating class I ARFs.
Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Endosomas/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Factores de Ribosilacion-ADP/clasificación , Factores de Ribosilacion-ADP/genética , Membrana Celular/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Expresión Génica , Ácido Glutámico/genética , Ácido Glutámico/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Células HeLa , Humanos , Mutación/genética , Unión Proteica , Serina/genética , Serina/metabolismo , Toxina Shiga I/metabolismo , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab4/metabolismoRESUMEN
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
Cholix toxin (Cholix) from Vibrio cholerae is a potent virulence factor exhibiting ADP-ribosyltransferase activity on eukaryotic elongation factor 2 (eEF2) of host cells, resulting in the inhibition of protein synthesis. Administration of Cholix or its homologue Pseudomonas exotoxin A (PEA) to mice causes lethal hepatocyte damage. In this study, we demonstrate cytotoxicity of Cholix on human hepatocytes in the presence of tumor necrosis factor α (TNF-α), which has been reported to play a fatal role in PEA administered to mice. Compared with incubating HepG2 cells with Cholix alone, co-treatment with TNF-α and Cholix (TNF-α/Cholix) significantly enhanced the activation of caspases, cytochrome c release from mitochondria into cytoplasm, and poly-ADP-ribose polymerase (PARP) cleavage, while incubation with TNF-α alone or co-treatment with TNF-α/catalytically inactive Cholix did not. In the early stage of cell death, Cholix increased phosphorylation of mitogen-activated protein kinases (e.g., p38, ERK, JNK) and Akt, which was not affected by TNF-α alone. MAPK inhibitors (SP600125, SB20852, and U0126) suppressed PARP cleavage induced by TNF-α/Cholix. Protein kinase inhibitor Go6976 suppressed JNK phosphorylation and PARP cleavage by TNF-α/Cholix. In contrast, PKC activator PMA in the absence of TNF-α promoted Cholix-induced PARP cleavage. Reactive oxygen species (ROS) inhibitor, N-acetyl cysteine (NAC), suppressed TNF-α/Cholix-induced JNK and ERK phosphorylation, resulting in inhibition of PARP cleavage. These data suggest that ROS and JNK pathways are important mediators of TNF-α/Cholix-induced HepG2 cell death.