Kohei Ogura

Subtilase Cytotoxin Enhances Escherichia coli Survival in Macrophages by Suppression of Nitric Oxide Production through the Inhibition of NF-κB Activation

ABSTRACT 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.

Identification of Subtilase Cytotoxin (SubAB) Receptors Whose Signaling, in Association with SubAB-Induced BiP Cleavage, Is Responsible for Apoptosis in HeLa Cells

ABSTRACT 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, α2β1 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 β1 ITG, NG2, and L1CAM small interfering RNA-transfected cells, but unexpectedly, BiP cleavage was still observed. Pretreatment of cells with a function-blocking β1 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, β1 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.

Characterization of Cholix Toxin-induced Apoptosis in HeLa Cells

Background: Cholix toxin (Cholix) is a novel ADP-ribosyl transferase cytotoxin produced by Vibrio cholerae. Results: Cholix-induced apoptosis is dependent on caspase activation, which is regulated by both mitochondria-dependent and -independent pathways. Conclusion: Inflammatory caspases and caspase-8 are responsible for both mitochondrial signals and other caspase activation. Significance: The cell death pathway induced by eEF2-ADP-ribosylation might differ in various cell types. 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.

Uptake of Shiga-toxigenic Escherichia coli SubAB by HeLa cells requires an actin- and lipid raft-dependent pathway

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‐β‐cyclodextrin (mβCD) 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.

Regulation of subtilase cytotoxin-induced cell death by an RNA-dependent protein kinase-like endoplasmic reticulum kinase-dependent proteasome pathway in HeLa cells.

ABSTRACT 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.

Severe invasive streptococcal infection by Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis.

Streptococcus pyogenes, a group A Streptococcus (GAS), has been recognized as the causative pathogen in patients with severe invasive streptococcal infection with or without necrotizing fasciitis. In recent epidemiological studies, Streptococcus dysgalactiae subsp. equisimilis (SDSE) has been isolated from severe invasive streptococcal infection. Complete genome sequence showed that SDSE is the closest bacterial species to GAS, with approximately 70% of genome coverage. SDSE, however, lacks several key virulence factors present in GAS, such as SPE‐B, the hyaluronan synthesis operon and active superantigen against human immune cells. A key event in the ability of GAS to cause severe invasive streptococcal infection was shown to be the acquisition of novel genetic traits such as phages. Strikingly, however, during severe invasive infection, GAS destroys its own covRS two‐component system, which negatively regulates many virulence factor genes, resulting in a hyper‐virulent phenotype. In contrast, this phenomenon has not been observed in SDSE. The present review describes the epidemiology of severe invasive streptococcal infection and the detailed pathogenic mechanisms of GAS and SDSE, emphasizing findings from their genome sequences and analyses of gene expression.

DAP1, a negative regulator of autophagy, controls SubAB-mediated apoptosis and autophagy

ABSTRACT 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.

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

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, NH4Cl 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.

Vibrio cholerae Cholix Toxin-Induced HepG2 Cell Death is Enhanced by Tumor Necrosis Factor-Alpha Through ROS and Intracellular Signal-Regulated Kinases

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.

Pathogenicity Induced by Invasive Infection of Streptococcus dysgalactiae subsp. equisimilis in a Mouse Model of Diabetes

Streptococcus dysgalactiae subsp. equisimilis (SDSE) causes severe invasive diseases such as streptococcal toxic shock syndrome, similar to that caused by S. pyogenes (GAS). Invasive SDSE infections are increasing, particularly among patients with diabetes mellitus. Here we investigate the association between the pathogenicity of SDSE and diabetes mellitus in a mouse model, using GAS infection for comparison. Intraperitoneal injection of highly hemolytic SDSE-167 into C57BL6/J mice induced a rapid rise in blood glucose concentrations within 4 h, which was otherwise seen only in mice injected with high doses of hypervirulent GAS mutants. The survival rates of mice injected with SDSE-167 were significantly lower in mice (db/db) with type 2 diabetes than in nondiabetic mice. Injection of db/db mice with SDSE-167 increased the concentrations of cytokines and chemokines, particularly those of interleukin 6 and monocyte chemotactic protein-1. Microarray data indicate that multiple pathways are involved in the pathogenicity of SDSE-167 in db/db mice. These data reveal that the mechanisms underlying streptococcal infection differ between SDSE and GAS.