TLR激动剂抑制IL-27诱导的THP-1细胞HLA-DR的表达

目的研究白介素27(IL-27)对THP-1单核细胞系Ⅱ类转录活化子(classⅡtransactivator,CⅡTA)和Ⅱ类主要组织相容性复合物(major histocom patibility complex classⅡ,MHCⅡ)分子的表达的影响及Toll样受体(Toll-likereceptor,TLR)激动剂LPS和Pam3CSK4的干预作用。方法RT-PCR检测CⅡTAⅠ、Ⅲ和Ⅳ以及人白细胞抗原(human leukocyte antigen,HLA)-DRA和干扰素调节因子-1(interferon regulatory factor-1,IRF-1)mRNA的表达。半定量PCR检测HLA-DRA、DRB、DPA、DPB、DQA、DQB、IRF-1、CⅡTA mRNA的表达。流式细胞术检测细胞表面HLA-DR的表达。结果IL-27刺激24h后可分别上调THP-1细胞CⅡTAⅢ、CⅡTAⅣ和IRF-1mRNA表达水平。IL-27刺激24h和48h可升高MHCⅡ类分子mRNA的表达,并能诱导HLA-DR在28%和53%的THP-1细胞表面表达。在THP-1细胞和PMA诱导的THP-1巨噬细胞,LPS和Pam3CSK4均可抑制IL-27诱导的CⅡTA和HLA-DR的表达。结论IL-27可上调THP-1细胞CⅡTA和MHCⅡ类分子的表达,这种作用可被LPS和Pam3CSK4抑制。     

Protection of Human Podocytes from Shiga Toxin 2-Induced Phosphorylation of Mitogen-Activated Protein Kinases and Apoptosis by Human Serum Amyloid P Component

Hemolytic uremic syndrome (HUS) is mainly induced by Shiga toxin 2 (Stx2)-producing Escherichia coli. Proteinuria can occur in the early phase of the disease, and its persistence determines the renal prognosis. Stx2 may injure podocytes and induce proteinuria. Human serum amyloid P component (SAP), a member of the pentraxin family, has been shown to protect against Stx2-induced lethality in mice in vivo, presumably by specific binding to the toxin. We therefore tested the hypothesis that SAP can protect against Stx2-induced injury of human podocytes. To elucidate the mechanisms underlying podocyte injury in HUS-associated proteinuria, we assessed Stx2-induced activation of mitogen-activated protein kinases (MAPKs) and apoptosis in immortalized human podocytes and evaluated the impact of SAP on Stx2-induced damage. Human podocytes express Stx2-binding globotriaosylceramide 3. Stx2 applied to cultured podocytes was internalized and then activated p38α MAPK and c-Jun N-terminal kinase (JNK), important signaling steps in cell differentiation and apoptosis. Stx2 also activated caspase 3, resulting in an increased level of apoptosis. Coincubation of podocytes with SAP and Stx2 mitigated the effects of Stx2 and induced upregulation of antiapoptotic Bcl2. These data suggest that podocytes are a target of Stx2 and that SAP protects podocytes against Stx2-induced injury. SAP may therefore be a useful therapeutic option.     

Shiga Toxin 1-Induced Inflammatory Response in Lipopolysaccharide-Sensitized Astrocytes Is Mediated by Endogenous Tumor Necrosis Factor Alpha

Hemolytic-uremic syndrome (HUS) is generally caused by Shiga toxin (Stx)-producing Escherichia coli. Endothelial dysfunction mediated by Stx is a central aspect in HUS development. However, inflammatory mediators such as bacterial lipopolysaccharide (LPS) and polymorphonuclear neutrophils (PMN) contribute to HUS pathophysiology by potentiating Stx effects. Acute renal failure is the main feature of HUS, but in severe cases, patients can develop neurological complications, which are usually associated with death. Although the mechanisms of neurological damage remain uncertain, alterations of the blood-brain barrier associated with brain endothelial injury is clear. Astrocytes (ASTs) are the most abundant inflammatory cells of the brain that modulate the normal function of brain endothelium and neurons. The aim of this study was to evaluate the effects of Stx type 1 (Stx1) alone or in combination with LPS in ASTs. Although Stx1 induced a weak inflammatory response, pretreatment with LPS sensitized ASTs to Stx1-mediated effects. Moreover, LPS increased the level of expression of the Stx receptor and its internalization. An early inflammatory response, characterized by the release of tumor necrosis factor alpha (TNF-α) and nitric oxide and PMN-chemoattractant activity, was induced by Stx1 in LPS-sensitized ASTs, whereas activation, evidenced by higher levels of glial fibrillary acid protein and cell death, was induced later. Furthermore, increased adhesion and PMN-mediated cytotoxicity were observed after Stx1 treatment in LPS-sensitized ASTs. These effects were dependent on NF-κB activation or AST-derived TNF-α. Our results suggest that TNF-α is a pivotal effector molecule that amplifies Stx1 effects on LPS-sensitized ASTs, contributing to brain inflammation and leading to endothelial and neuronal injury.The epidemic form of hemolytic-uremic syndrome (HUS) has been associated with enterohemorrhagic infections caused by Shiga toxin (Stx)-producing Escherichia coli (STEC) organisms (33). HUS is the most common cause of acute renal failure in children and is related to the endothelial damage of glomeruli and/or arterioles of the kidney and epithelial cell damage induced by Stx through the interaction with its globotriaosylceramide (Gb₃) receptor (35). Although Stx is the main pathogenic factor and is necessary for epidemic HUS development, clinical and experimental evidence suggests that the inflammatory response is able to potentiate Stx toxicity. In fact, both bacterial lipopolysaccharide (LPS) and polymorphonuclear neutrophils (PMN) play a key role in the full development of HUS (15). Moreover, PMN leukocytosis in patients correlates with a poor prognosis (17).Endothelial cell damage is not limited to the kidney but extends to other organs; in severe cases, the brain can be affected. In fact, central nervous system (CNS) complications indicate severe HUS, and brain damage involvement is the most common cause of death (14).However, the pathogenesis of CNS impairment is not yet fully understood. Although it has been demonstrated that human brain endothelial cells (BECs) are relatively resistant to Stx, inflammatory mediators, such as tumor necrosis factor alpha (TNF-α), markedly increase human BEC sensitivity to Stx cytotoxicity (11).BECs are part of the blood-brain barrier (BBB), which protects the brain from potentially harmful substances and leukocytes present in the bloodstream. Thus, the integrity of BBB function is theorized to be a key component in CNS-associated pathologies, and BEC damage is thought to be one of the possible mechanisms involved in the disruption of the BBB in HUS. In fact, LPS from bacterial infections leads to the release of TNF-α, interleukin-1β (IL-1β), and reactive oxygen species (ROS), all of which have the ability to open the BBB.Several in vivo studies demonstrated previously that Stx is able to impair BBB function, increasing its permeability (21). Moreover, Stx itself is able to cross the endothelial barrier and enter into the CNS, since Stx activity in cerebrospinal fluid was previously observed (19, 23), and Stx was previously immunodetected in many brain cells including astrocytes (ASTs) and neurons (44).ASTs, which are inflammatory cells found throughout the CNS, are in close contact with BECs by end-foot processes (24), and their interaction with the cerebral endothelium determines BBB function (2, 4). In addition, ASTs interact with neurons through gap junctions and release neurotrophins that are essential for neuronal survival (6). However, in response to brain injury, ASTs become activated and release inflammatory mediators such as nitric oxide (NO) and TNF-α, altering the permeability of the BBB and affecting neuronal survival and tissue integrity (1, 9). In addition, AST-derived cytokines and chemokines can stimulate the peripheral immune system and attract peripheral inflammatory leukocytes to the site of injury (46).ASTs are therefore in a critical position to influence neuronal viability and BEC integrity once Stx and factors associated with the STEC infection reach the brain parenchyma. We hypothesize that the effects of LPS and Stx on ASTs may be involved in the brain damage observed with severe cases of HUS. Thus, the aim of this study was to evaluate whether Stx type 1 (Stx1) alone or in combination with LPS is capable of inducing an inflammatory response in ASTs.     

Signaling through C/EBP Homologous Protein and Death Receptor 5 and Calpain Activation Differentially Regulate THP-1 Cell Maturation-Dependent Apoptosis Induced by Shiga Toxin Type 1

Shiga toxins (Stxs) induce apoptosis via activation of the intrinsic and extrinsic pathways in many cell types. Toxin-mediated activation of the endoplasmic reticulum (ER) stress response was shown to be instrumental in initiating apoptosis in THP-1 myeloid leukemia cells. THP-1 cells responded to Shiga toxin type 1 (Stx1) in a cell maturation-dependent manner, undergoing rapid apoptosis in the undifferentiated state but reduced and delayed apoptosis in differentiated cells. The onset of apoptosis was associated with calpain activation and changes in expression of C/EBP homologous protein (CHOP), Bcl-2 family members, and death receptor 5 (DR5). Ligation of DR5 by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) activates the extrinsic pathway of apoptosis. We show here that expression of TRAIL and DR5 is increased by Stx1 treatment. Addition of exogenous TRAIL enhances, and anti-TRAIL antibodies inhibit, Stx1-induced apoptosis of THP-1 cells. Silencing of CHOP or DR5 expression selectively prevented caspase activation, loss of mitochondrial membrane potential, and Stx1-induced apoptosis of macrophage-like THP-1 cells. In contrast, the rapid kinetics of apoptosis induction in monocytic THP-1 cells correlated with rates of calpain cleavage. The results suggest that CHOP-DR5 signaling and calpain activation differentially contribute to cell maturation-dependent Stx1-induced apoptosis. Inhibition of these signaling pathways may protect cells from Stx cytotoxicity.Shiga toxins (Stxs) are major virulence factors expressed by the enteric pathogens Shigella dysenteriae serotype 1 and certain Escherichia coli serotypes referred to as Shiga toxin-producing E. coli (STEC). Infections with Stx-producing bacteria are associated with watery diarrhea that may progress to bloody diarrhea, acute renal failure, and central nervous system complications such as lethargy, seizures, and paralysis (60). STEC is a particular public health concern in developed nations, with approximately 73,000 cases annually of hemorrhagic colitis caused by E. coli O157:H7 and 37,000 annual cases caused by STEC non-O157 serotypes in the United States (42). The histopathological hallmark of disease caused by Stxs is damage to endothelial cells lining colonic capillaries, renal glomeruli and arterioles, and central nervous system (CNS) blood vessels (46). The essential role of Stxs in pathogenesis has been confirmed using animal models in which the infusion of the toxins causes extensive microvascular thromboses in the kidney and CNS and, in some cases, ataxia and limb paralysis (43, 61). S. dysenteriae serotype 1 produces Shiga toxin, while STEC may express one or more toxin variants categorized as Shiga toxin type 1 (Stx1) or Shiga toxin type 2 (Stx2) based on their antigenic similarity to Shiga toxin (56). All Stxs possess an AB₅ structure composed of a monomeric A subunit in noncovalent association with a pentamer of B subunits (17). The B subunits mediate toxin binding by interaction with the membrane neutral glycolipid globotriaosylceramide (Gb₃) (38). The toxins are then internalized and undergo a complex series of intracellular routing events, collectively termed retrograde transport, which ultimately deliver the toxins to the endoplasmic reticulum (ER) lumen (50). In the ER, the A subunit is proteolytically processed, and a fragment of the A subunit retrotranslocates into the cytosol. The N-glycosidase activity associated with the processed A subunit catalyzes the inactivation of eukaryotic ribosomes and inhibits protein synthesis (12, 51).In addition to the capacity to inhibit protein synthesis, Stxs have been shown to induce apoptosis, or programmed cell death, in many cell types (5). The toxins appear to activate apoptotic signaling through an extrinsic (death receptor-mediated signaling) or an intrinsic (mitochondrion-mediated signaling) pathway. For example, the toxins have been shown to be capable of directly activating initiator and executioner caspase cascades but also to generate truncated BID (tBID) which translocates to mitochondrial membranes, leading to increased mitochondrial membrane permeability, release of cytochrome c, and formation of the apoptosome (6, 18, 34). As a result of signaling through the intrinsic or extrinsic pathway, intoxicated cells display characteristics of apoptosis such as DNA fragmentation, cell shrinkage, membrane blebbing, and chromatin condensation.We previously showed that Stx1 induced apoptosis in the human myelogenous leukemia cell line THP-1 in a cell maturation-dependent manner. Undifferentiated, nonadherent monocytic THP-1 cells underwent rapid apoptosis when treated with Stx1, while differentiation to the adherent, macrophage-like state was associated with increased resistance to the cytotoxic action of the toxins, with only approximately 30% of cells undergoing delayed apoptosis (22). The induction of apoptosis by Stx1 involved the activation of the ER stress response in both monocytic and macrophage-like THP-1 cells (33, 36). Stx1 induced the expression of the ER stress effectors C/EBP homologous protein (CHOP), tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), and death receptor 5 (DR5) in monocytic THP-1 cells. Delivery of functional Stx1 into the cytosol of monocytic THP-1 cells led to downregulated expression of the prosurvival factor Bcl-2, while the delayed-apoptosis phenotype in macrophage-like cells was associated with increased Bcl-2 expression, phosphorylation, and mitochondrial translocation.Increased expression of the apoptosis-inducing factor TRAIL and its death-inducing receptor, DR5, enhances cell death signals triggered during a prolonged ER stress response (23, 68). TRAIL may be membrane associated or may be cleaved from the cell surface by proteases to generate a soluble ligand (26, 40). Engagement of TRAIL with its cognate receptor DR5 activates the extrinsic pathway of apoptosis through DR5 aggregation, the recruitment of the Fas-associated death domain (FADD), and the formation of the death-inducing signaling complex (DISC) (31, 53). The observation that expression of TRAIL and DR5 was upregulated by Stx1 treatment of monocytic THP-1 cells suggested that this receptor-ligand pair may contribute to rapid apoptosis induced by the toxin in these cells. However, we also showed that calpains were rapidly activated by Stx1 in monocytic THP-1 cells, and calpains may directly cleave caspase-3 (36). The studies reported here were designed to characterize the roles of TRAIL/DR5 and calpains in the rapid apoptosis response of monocytic cells and in delayed apoptosis in macrophage-like cells. We show that Stx1-induced apoptotic signaling is amplified by the addition of soluble TRAIL (sTRAIL) and inhibited by exposure of cells to neutralizing anti-TRAIL antibodies prior to intoxication. A reduction in CHOP or DR5 expression using RNA interference (RNAi) techniques markedly protected cells from apoptosis induced by Stx1, linking activation of the ER stress response with apoptosis in this system. Signaling through CHOP and DR5 led to activation of the initiator caspase, caspase-8, and the executioner caspase, caspase-3, in macrophage-like THP-1 cells, but the effect of CHOP and DR5 knockdown on caspase activation and apoptosis of monocytic cells was minimal. In contrast, the rate of calpain activation (cleavage) was directly correlated with the rapid onset of apoptosis in monocytic THP-1 cells.     

The A1 Subunit of Shiga Toxin 2 Has Higher Affinity for Ribosomes and Higher Catalytic Activity than the A1 Subunit of Shiga Toxin 1

Shiga toxin (Stx)-producing Escherichia coli (STEC) infections can lead to life-threatening complications, including hemorrhagic colitis (HC) and hemolytic-uremic syndrome (HUS), which is the most common cause of acute renal failure in children in the United States. Stx1 and Stx2 are AB5 toxins consisting of an enzymatically active A subunit associated with a pentamer of receptor binding B subunits. Epidemiological evidence suggests that Stx2-producing E. coli strains are more frequently associated with HUS than Stx1-producing strains. Several studies suggest that the B subunit plays a role in mediating toxicity. However, the role of the A subunits in the increased potency of Stx2 has not been fully investigated. Here, using purified A1 subunits, we show that Stx2A1 has a higher affinity for yeast and mammalian ribosomes than Stx1A1. Biacore analysis indicated that Stx2A1 has faster association and dissociation with ribosomes than Stx1A1. Analysis of ribosome depurination kinetics demonstrated that Stx2A1 depurinates yeast and mammalian ribosomes and an RNA stem-loop mimic of the sarcin/ricin loop (SRL) at a higher catalytic rate and is a more efficient enzyme than Stx1A1. Stx2A1 depurinated ribosomes at a higher level in vivo and was more cytotoxic than Stx1A1 in Saccharomyces cerevisiae. Stx2A1 depurinated ribosomes and inhibited translation at a significantly higher level than Stx1A1 in human cells. These results provide the first direct evidence that the higher affinity for ribosomes in combination with higher catalytic activity toward the SRL allows Stx2A1 to depurinate ribosomes, inhibit translation, and exhibit cytotoxicity at a significantly higher level than Stx1A1.     

柯萨奇病毒B3通过caspase依赖途径诱导HeLa细胞凋亡

目的 评价柯萨奇病毒B3（CVB3）致HeLa细胞死亡的方式及分子机制。方法 用CVB3作用于HeLa细胞,在不同时间收集细胞,通过相差显微镜、电子显微镜、流式细胞仪以及分子生物学手段,HeLa细胞的病变及caspase-3基因mRNA和蛋白质的表达。结果 CVB3作用于HeLa细胞后,细胞很快发生变性和坏死,24h后较多细胞凋亡;caspase基因在病毒作用早期即被活化,表现在caspase-3 mRNA在病毒作用后6h内,迅速增高达峰值。在24h内,又降至接近病毒作用前的水平。caspase-3蛋白表达在42h内逐渐增高。结论CVB3可诱导HeLa细胞发生坏死和凋亡两种反应,坏死早于凋亡,细胞凋亡与caspase-3的表达密切相关。     

Toxicity of Shiga Toxin 1 in the Central Nervous System of Rabbits

The action of Shiga toxin (Stx) on the central nervous system was examined in rabbits. Intravenous Stx1 was 44 times more lethal than Stx2 and acted more rapidly than Stx2. However, Stx1 accumulated more slowly in the cerebrospinal fluid than did Stx2. Magnetic resonance imaging demonstrated a predominance of Stx1-dependent lesions in the spinal cord. Pretreatment of the animals with anti-Stx1 antiserum intravenously completely protected against both development of brain lesions and mortality.     

Shiga Toxin 2 Targets the Murine Renal Collecting Duct Epithelium

Hemolytic-uremic syndrome (HUS) caused by Shiga toxin-producing Escherichia coli infection is a leading cause of pediatric acute renal failure. Bacterial toxins produced in the gut enter the circulation and cause a systemic toxemia and targeted cell damage. It had been previously shown that injection of Shiga toxin 2 (Stx2) and lipopolysaccharide (LPS) caused signs and symptoms of HUS in mice, but the mechanism leading to renal failure remained uncharacterized. The current study elucidated that murine cells of the glomerular filtration barrier were unresponsive to Stx2 because they lacked the receptor glycosphingolipid globotriaosylceramide (Gb₃) in vitro and in vivo. In contrast to the analogous human cells, Stx2 did not alter inflammatory kinase activity, cytokine release, or cell viability of the murine glomerular cells. However, murine renal cortical and medullary tubular cells expressed Gb₃ and responded to Stx2 by undergoing apoptosis. Stx2-induced loss of functioning collecting ducts in vivo caused production of increased dilute urine, resulted in dehydration, and contributed to renal failure. Stx2-mediated renal dysfunction was ameliorated by administration of the nonselective caspase inhibitor Q-VD-OPH in vivo. Stx2 therefore targets the murine collecting duct, and this Stx2-induced injury can be blocked by inhibitors of apoptosis in vivo.Shiga toxin-producing Escherichia coli (STEC) is the principal etiologic agent of diarrhea-associated hemolytic-uremic syndrome (HUS) (42, 60, 66). Renal disease is thought to be due to the combined action of Shiga toxins (Shiga toxin 1 [Stx1] and Stx2), the primary virulence factors of STEC, and bacterial lipopolysaccharide (LPS) on the renal glomeruli and tubules (6, 42, 60, 66). Of these, Stx2 is most frequently associated with the development of HUS (45). Shiga toxin enters susceptible cell types after binding to the cell surface receptor glycosphingolipid globotriaosylceramide (Gb₃) and specifically depurinates the 28S rRNA, thereby inhibiting protein synthesis (42, 60, 66). The damage initiates a ribotoxic stress response consisting of mitogen-activated protein (MAP) kinase activation, and this response can be associated with cytokine release and cell death (21, 22, 25-27, 61, 69, 73). This cell death is often caspase-dependent apoptosis (18, 61). Gb₃ is expressed by human glomerular endothelial cells, podocytes, and multiple tubular epithelial cell types, and damage markers for these cells can be detected in urine samples from HUS patients (10-12, 15, 49, 73). Shiga toxin binds to these cells in renal sections from HUS patients, and along with the typical fibrin-rich glomerular microangiopathy, biopsy sections demonstrate apoptosis of both glomerular and tubular cell types (9, 29, 31).Concomitant development of the most prominent features of HUS: anemia, thrombocytopenia, and renal failure, requires both Shiga toxin and LPS in the murine model (30, 33). Nevertheless, our previous work demonstrated that renal failure is mediated exclusively by Stx2 (33). While it is established that Gb₃ is the unique Shiga toxin receptor (46), the current literature regarding the mechanism by which Shiga toxin causes renal dysfunction in mice is inconsistent. Even though Gb₃ has been localized to some murine renal tubules and tubular damage has been observed (19, 23, 46, 53, 65, 68, 72, 74), the specific types of tubules affected have been incompletely characterized. Although multiple groups have been unable to locate the Shiga toxin receptor Gb₃ in glomeruli in murine renal sections (19, 53), one group has reported that murine glomerular podocytes possess Gb₃ and respond to Stx2 in vitro (40), and another group has reported that renal tubular capillaries express the Gb₃ receptor (46). Furthermore, murine glomerular abnormalities, including platelet and fibrin deposition, occur in some murine HUS models (28, 30, 33, 46, 59, 63). We demonstrate here that murine glomerular endothelial cells and podocytes are unresponsive to Stx2 because they do not produce the glycosphingolipid receptor Gb₃ in vitro or in vivo. Further, murine renal tubules, including collecting ducts, express Gb₃ and undergo Stx2-induced apoptosis, resulting in dysfunctional urine production and dehydration.     

Clostridium difficile Toxin B Induces Apoptosis in Intestinal Cultured Cells

Toxigenic strains of the anaerobic bacterium Clostridium difficile produce at least two large, single-chain protein exotoxins involved in the pathogenesis of antibiotic-associated diarrhea and colitis. Toxin A (CdA) is a cytotoxic enterotoxin, while toxin B (CdB) is a more potent cytotoxin lacking enterotoxic activity. This study dealt with CdB, providing the first evidence that intestinal cells exposed to this toxin exhibit typical features of apoptosis in that a significant proportion of the treated cells displayed nuclear fragmentation and chromatin condensation. In keeping with ultrastructural data, CdB-treated cells showed the typical flow cytometric hallmark of apoptosis consisting of a distinct sub-G₁ peak. The CdB-induced apoptotic response was dose and time dependent and not simply due to the actin-disrupting effect of the toxin or to the subsequent impairment of cell anchorage. Rather, the inhibition of proteins belonging to the Rho family due to CdB seems to play a role in the induction of apoptosis in intestinal cells. The origin of cells and the growth rate may also be cofactors relevant to such a response.     

Disruption of an Internal Membrane-Spanning Region in Shiga Toxin 1 Reduces Cytotoxicity

Shiga toxin type 1 (Stx1) belongs to the Shiga family of bipartite AB toxins that inactivate eukaryotic 60S ribosomes. The A subunit of Stxs are N-glycosidases that share structural and functional features in their catalytic center and in an internal hydrophobic region that shows strong transmembrane propensity. Both features are conserved in ricin and other ribosomal inactivating proteins. During eukaryotic cell intoxication, holotoxin likely moves retrograde from the Golgi apparatus to the endoplasmic reticulum. The hydrophobic region, spanning residues I224 through N241 in the Stx1 A subunit (Stx1A), was hypothesized to participate in toxin translocation across internal target cell membranes. The TMpred computer program was used to design a series of site-specific mutations in this hydrophobic region that disrupt transmembrane propensity to various degrees. Mutations were synthesized by PCR overlap extension and confirmed by DNA sequencing. Mutants StxAF226Y, A231D, G234E, and A231D-G234E and wild-type Stx1A were expressed in Escherichia coli SY327 and purified by dye-ligand affinity chromatography. All of the mutant toxins were similar to wild-type Stx1A in enzymatic activity, as determined by inhibition of cell-free protein synthesis, and in susceptibility to trypsin digestion. Purified mutant or wild-type Stx1A combined with Stx1B subunits in vitro to form a holotoxin, as determined by native polyacrylamide gel electrophoresis immunoblotting. StxA mutant A231D-G234E, predicted to abolish transmembrane propensity, was 225-fold less cytotoxic to cultured Vero cells than were the wild-type toxin and the other mutant toxins which retained some transmembrane potential. Furthermore, compared to wild-type Stx1A, A231D-G234E Stx1A was less able to interact with synthetic lipid vesicles, as determined by analysis of tryptophan fluorescence for each toxin in the presence of increasing concentrations of lipid membrane vesicles. These results provide evidence that this conserved internal hydrophobic motif contributes to Stx1 translocation in eukaryotic cells.     

Purification and Biological Characterization of Shiga Toxin from Shigella dysenteriae 1

Shiga toxin has been purified in milligram quantities to near homogeneity from cell lysates of Shigella dysenteriae 1 strain 3818-0. Purification involved an initial ultracentrifugation, ammonium sulfate fractionation, chromatography on DEAE-cellulose and carboxymethyl cellulose, gel filtration, and preparative isoelectric focusing in sucrose gradients. The purified toxin was resolved by discontinuous polyacrylamide gel electrophoresis into a major cytotoxic protein band and a closely migrating, cytotoxic protease-nicked minor band. Antiserum generated by immunization with glutaraldehyde-inactivated toxin was shown to be monospecific against S. dysenteriae cell lysates. This highly purified toxin was cytotoxic to HeLa cells, enterotoxic in rabbit ileal loops, and lethal to mice. Monospecific antiserum to the toxin neutralized completely these toxin activities in both purified toxin preparations and crude shigella cell lysates.     

Antiapoptotic Proteins Bcl-2 and Bcl-XL Inhibit Clostridium difficile Toxin A-Induced Cell Death in Human Epithelial Cells

It has been well established that Clostridium difficile toxin A (TcdA) induces cell death in human epithelial cells. However, the mechanism of TcdA-induced cell death remains to be fully characterized. Here, we show that TcdA induces dose-dependent cell death in ovarian carcinoma and colonic carcinoma cell lines. TcdA-mediated cell death, as well as caspase 8 and caspase 3 activation, were specifically abrogated by anti-toxin antibodies. Although caspase 8 and caspase 3 were activated by TcdA in OVCAR3 ovarian carcinoma and T84 colonic cancer cells, pancaspase and caspase 8, 3, and 9 inhibitors did not block TcdA-induced cell death. In contrast, tumor necrosis factor-related apoptosis-inducing ligand-induced cell death was nearly completely blocked by caspase inhibitors in OVCAR3 cells. In these cells, TcdA induces the mitochondrial pathway of apoptosis, as demonstrated by changes in mitochondrial outer membrane permeabilization (MOMP). Furthermore, overexpression of the antiapoptotic proteins Bcl-2 and Bcl-X_L significantly inhibited TcdA-induced cell death, as well as TcdA-induced MOMP. Conversely, small interfering RNA-mediated inhibition of Bcl-X_L in TcdA-resistant SKOV3ip1 cells enhanced TcdA-induced cell death. Overexpression of the antiapoptotic proteins Bcl-2 and Bcl-X_L in T84 cells also inhibited TcdA-induced cell death. Altogether, our data demonstrate that TcdA induces cell death in both ovarian and colonic cancer cells preferentially via the mitochondrial pathway of apoptosis by a death receptor-independent and a caspase-independent mechanism. This process is regulated by antiapoptotic members of the Bcl-2 family.Apoptosis can be mediated by a variety of stimuli, including binding of ligands to death receptors, DNA-damaging agents, and growth factor withdrawal. Depending on the signal, apoptosis is initiated either by the death receptor pathway or by a mitochondrion-dependent pathway (31-33). In both pathways, however, effector caspases (caspases 3, 6, and 7) are activated and cleavage of cellular substrates occurs, leading to the morphological changes observed in apoptosis. In the mitochondrion-dependent pathway of apoptosis, effector caspase activation is triggered by an increase in mitochondrial outer membrane permeabilization (MOMP), resulting in the release of cytochrome c and the formation of the apoptosome (31, 33). Changes in MOMP are regulated by a balance between pro- and antiapoptotic members of the Bcl-2 family (31). The proapoptotic family members Bax and Bak form channels into the outer membrane of the mitochondria that allow the release of cytochrome c and other mitochondrial intermembrane proteins. Insertion of Bax and Bak into the outer mitochondrial membrane is regulated by antiapoptotic members of the Bcl-2 family. Antiapoptotic members, such as Bcl-2 and Bcl-X_L, bind and neutralize Bax and/or Bak. Stimulation of death receptors by death ligands, such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), results in activation of initiator caspase 8. Upon binding to TRAIL, activated TRAIL receptors recruit the Fas-associated death domain (3). Via its death effector domain, the Fas-associated death domain recruits caspase 8 and assembles into a death-inducing signaling complex (16, 27). When recruited to the death-inducing signaling complex, pro-caspase 8 is activated and subsequently cleaves downstream effector caspases, leading to apoptosis. This process is efficiently blocked by the inhibition of caspases. An interconnection between cell surface death receptors and mitochondrion-initiated pathways of apoptosis has been found in many cellular systems. In this context, apoptosis can be inhibited by Bcl-X_L or Bcl-2 (2, 10, 13). In contrast to the death receptor pathway, which is highly dependent on caspase activation, the inhibition of caspases fails to prevent apoptosis in caspase-independent cell death (32). Furthermore, as caspase-independent cell death often requires MOMP, this process can be blocked by Bcl-2 overexpression (2, 13).C. difficile is the leading cause of hospital-acquired diarrhea and the etiological agent of pseudomembranous colitis. In humans, the intestinal damage is produced by the actions of toxin A (TcdA) and toxin B (TcdB), which are the major virulence determinants of C. difficile. The emergence in 2000, first in the United States and Canada and more recently in Western Europe, of a hypervirulent strain of C. difficile (NAP1/BI/027) has led to an increase in the incidence and the case-fatality ratio of hospital-acquired diarrhea, resulting, on average, in 10.7 additional days in the hospital (14, 23, 26, 28, 29, 35). This epidemic NAP1/B1/027 strain produces higher levels of TcdA and TcdB (35). TcdA is primarily responsible for the mucosal damage and the inflammatory response in animal models (24). TcdA was shown to induce apoptosis in many human cell types in vitro, including endothelial cells (11), monocytes (34), HeLa cells (30), and intestinal epithelial cells (4, 5, 9). The mechanisms by which TcdA induces apoptosis in the cells remain to be fully characterized. Brito et al. demonstrated that TcdA-induced intestinal cell death involves caspase 8, 3, and 9 activation, but the inhibition of these caspases only partially blocked TcdA-induced DNA laddering (4). Carneiro et al. have shown that TcdA induces caspase 6, 8, 9, and 3 and Bid (a proapoptotic member of the Bcl-2 family) cleavage, resulting in cell death in human intestinal epithelial cells (5). Bid cleavage, however, occurred by a caspase-independent mechanism. Gerhard et al. reported activation of caspases 3, 8, and 9 in colonic crypt cells treated with TcdA (9). Finally, Qa''Dan et al. showed that pancaspase inhibitor slowed but did not inhibit TcdB-induced cell death in HeLa cells, suggesting that cell death occurs by both caspase-dependent and caspase-independent mechanisms (30). In contrast to previous studies, apoptosis was completely blocked by the pancaspase inhibitor z-VAD-fmk. The role of caspase activation in TcdA-induced cell death thus remains controversial. In addition, the degree of mitochondrial contribution to TcdA-induced cell death has not been investigated.In this study, we examined the mechanism of TcdA-induced cell death using two cell culture models involving human colonic carcinoma and ovarian carcinoma cell lines. The advantage of using the ovarian carcinoma cell model was our ability to compare the effect of caspase inhibitors on TcdA-induced cell death to that of a well-established caspase-dependent stimulus (TRAIL), which cannot be assessed in colonic cell lines because of their inherent resistance to TRAIL. In this way, we show that TcdA induces primarily caspase-independent cell death in both ovarian and colonic carcinoma cells. We further demonstrate that TcdA-induced cell death is death receptor pathway independent but strongly dependent on the activation of the mitochondrial pathway. Thus, our findings define a novel mechanism in which TcdA-induced cell death involves a mitochondrion-dependent, caspase- and death receptor-independent signaling pathway that contrasts with previous data.     

鲨鱼软骨制剂通过下调Bcl-2诱导K562细胞凋亡

目的探讨鲨鱼软骨制剂(SCP)诱导人红白血病细胞系(K562)凋亡的作用机制。方法以不同浓度SCP加入体外培养的K562细胞中,用MTT比色法检测细胞存活率;Hoechst33342/PI荧光染色,荧光显微镜分析凋亡细胞百分率;流式细胞术进行细胞凋亡定量;免疫细胞化学染色法检测Bcl-2蛋白的表达。结果SCP明显抑制K562细胞生长,IC50值为1mg/ml以下;荧光显微镜下可见50%以上细胞为凋亡细胞的形态学改变;免疫细胞化学检测显示SCP诱导细胞凋亡过程中Bcl-2表达明显降低。结论SCP诱导K562细胞凋亡,可能与下调Bcl-2表达有关。     

Identification and Characterization of a Newly Isolated Shiga Toxin 2-Converting Phage from Shiga Toxin-Producing Escherichia coli

Shiga toxins 1 (Stx1) and 2 (Stx2) are encoded by toxin-converting bacteriophages of Stx-producing Escherichia coli (STEC), and so far two Stx1- and one Stx2-converting phages have been isolated from two STEC strains (A. D. O’Brien, J. W. Newlands, S. F. Miller, R. K. Holmes, H. W. Smith, and S. B. Formal, Science 226:694–696, 1984). In this study, we isolated two Stx2-converting phages, designated Stx2Φ-I and Stx2Φ-II, from two clinical strains of STEC associated with the outbreaks in Japan in 1996 and found that Stx2Φ-I resembled 933W, the previously reported Stx2-converting phage, in its infective properties for E. coli K-12 strain C600 while Stx2Φ-II was distinct from them. The sizes of the plaques of Stx2Φ-I and Stx2Φ-II in C600 were different; the former was larger than the latter. The restriction maps of Stx2Φ-I and Stx2Φ-II were not identical; rather, Stx2Φ-II DNA was approximately 3 kb larger than Stx2Φ-I DNA. Furthermore, Stx2Φ-I and Stx2Φ-II showed different phage immunity, with Stx2Φ-I and 933W belonging to the same group. Infection of C600 by Stx2Φ-I or 933W was affected by environmental osmolarity differently from that by Stx2Φ-II. When C600 was grown under conditions of high osmolarity, the infectivity of Stx2Φ-I and 933W was greatly decreased compared with that of Stx2Φ-II. Examination of the plating efficiency of the three phages for the defined mutations in C600 revealed that the efficiency of Stx2Φ-I and 933W for the fadL mutant decreased to less than 10⁻⁷ compared with that for C600 whereas the efficiency of Stx2Φ-II decreased to 0.1% of that for C600. In contrast, while the plating efficiency of Stx2Φ-II for the lamB mutant decreased to a low level (0.05% of that for C600), the efficiencies of Stx2Φ-I and 933W were not changed. This was confirmed by the phage neutralization experiments with isolated outer membrane fractions from C600, fadL mutant, or lamB mutant or the purified His₆-tagged FadL and LamB proteins. Based on the data, we concluded that FadL acts as the receptor for Stx2Φ-I and Stx2Φ-II whereas LamB acts as the receptor only for Stx2Φ-II.