Role of Tumor Necrosis Factor Alpha in Disease Using a Mouse Model of Shiga Toxin-Mediated Renal Damage

Mice have been extensively employed as an animal model of renal damage caused by Shiga toxins. In this study, we examined the role of the proinflammatory cytokine tumor necrosis factor alpha (TNF-α) in the development of toxin-mediated renal disease in mice. Mice pretreated with TNF-α and challenged with Shiga toxin type 1 (Stx1) showed increased survival compared to that of mice treated with Stx1 alone. Conversely, mice treated with Stx1 before TNF-α administration succumbed more quickly than mice given Stx1 alone. Increased lethality in mice treated with Stx1 followed by TNF-α was associated with evidence of glomerular damage and the loss of renal function. No differences in renal histopathology were noted between animals treated with Stx1 alone and the TNF-α pretreatment group, although we noted a sparing of renal function when TNF-α was administered before toxin. Compared to that of treatment with Stx1 alone, treatment with TNF-α after toxin altered the renal cytokine profile so that the expression of proinflammatory cytokines TNF-α and interleukin-1β (IL-1β) increased, and the expression of the anti-inflammatory cytokine IL-10 decreased. Increased lethality in mice treated with Stx1 followed by TNF-α was associated with higher numbers of dUTP-biotin nick end labeling-positive renal tubule cells, suggesting that increased lethality involved enhanced apoptosis. These data suggest that the early administration of TNF-α is a candidate interventional strategy blocking disease progression, while TNF-α production after intoxication exacerbates disease.Shiga toxins are a family of genetically and functionally related cytotoxic proteins expressed by the enteric pathogens Shigella dysenteriae serotype 1 and certain serotypes of Escherichia coli. Antigenic similarity to Shiga toxin expressed by S. dysenteriae serotype 1 is used to define Shiga toxin type 1 (Stx1) and type 2 (Stx2) expressed by Shiga toxin-producing E. coli (STEC) (44). Shiga toxins consist of a single A subunit in noncovalent association with a pentamer of B subunits. B subunits mediate binding to the neutral glycolipid receptor globotriaosylceramide (Gb₃), while the A subunit possesses an N-glycosidase activity (38). Following toxin internalization and routing to the endoplasmic reticulum (ER), a fragment of the toxin A subunit generated by furin or a furin-like protease is translocated across the ER membrane and mediates the cleavage of a single adenine residue (A4256 in the rat) from the 28S rRNA component of ribosomes (39). Stx-induced depurination leads to the disruption of elongation factor-dependent aminoacyl-tRNA binding to nascent polypeptides (30). Thus, Shiga toxins are potent protein synthesis inhibitors, with 50% cytotoxic doses measured in pg/ml amounts for many cell types in vitro. Shiga toxins also activate the ribotoxic and ER stress pathways, which are important in the activation of proinflammatory cytokine/chemokine production and apoptosis (6, 22, 41).The ingestion of small quantities of Stx-producing bacteria may lead to the development of bloody diarrhea with progression to acute renal failure, designated diarrhea-associated hemolytic uremic syndrome (D+HUS) (33). Epidemiologic studies have shown that the ingestion of STEC strains expressing Stx2 alone or Stx1 and Stx2 are more likely to progress to life-threatening extraintestinal complications (3, 17, 31). D+HUS is a leading cause of pediatric acute renal failure. D+HUS is characterized by rapid-onset oligouria or anuria, azotemia, microangiopathic hemolytic anemia with schistocytosis, and thrombocytopenia (33, 47). The histopathological examination of D+HUS renal tissues showed that glomerular microvascular endothelial cells were frequently swollen and detached from the basement membrane, and glomerular capillary lumina may be occluded with fibrin-rich microthrombi (21, 36).Numerous animal models have been employed to characterize the role of Stx1 and Stx2 in pathogenesis. Studies utilizing nonhuman primates showed that Shiga toxins are essential virulence determinants in the development of microangiopathic lesions. Fontaine et al. (9) showed that macaque monkeys fed toxigenic strains of S. dysenteriae developed colonic microvascular lesions, while baboons given purified intravenous Stx1 developed acute renal failure (48). The bolus intravenous administration of Stx1 or Stx2 into baboons revealed that the animals were more sensitive to Stx2, although the mean times to death were prolonged in Stx2-treated animals compared to that with Stx1 treatment. Both toxins mediated hematologic changes such as thrombocytopenia and schistocytosis, and both toxins produced renal pathology, but with different presentations. Renal damage caused by Stx1 was characterized by moderate congestion at the cortico-medullary junction, while Stx2-treated animals showed severe medullary congestion with cortical ischemia (42). Mice fed Stx2-producing E. coli or given a single bolus injection of purified Shiga toxins died without the development of glomerular thrombotic microangiopathy (50, 54). However, the administration of multiple low doses of Stx2 allowed the animals to survive initial toxin challenge and develop glomerular lesions characteristic of HUS in humans (40). In addition to the toxins, host response factors may contribute to D+HUS pathogenesis. Prodromal hemorrhagic colitis may alter normal colonic barrier function, and patients with D+HUS may be endotoxemic or show evidence of elevated antibody titers against lipopolysaccharides (LPS) expressed by Stx-producing E. coli (2, 10, 26). LPS elicit the expression of a broad array of pro- and anti-inflammatory cytokines and chemokines (45). In accordance with this, D+HUS patients frequently have increased serum or urinary proinflammatory cytokine and chemokine levels (15, 23). Studies using small-animal models support the hypothesis that additional bacterial and host response factors facilitate the development of renal disease. Keepers et al. (19) demonstrated that the coadministration of Stx2 and LPS to C57BL/6 mice did not produce major changes in lethality but resulted in pathophysiological changes more consistent with disease in humans: intraglomerular platelet and fibrin deposition, decreased renal function, neutrophilia, and lymphocytopenia. Barrett et al. (1) showed that the timing of toxin and LPS challenges were critical in disease outcome. LPS enhanced the lethal effects of purified Stx2 when administered to rabbits or mice after toxin challenge, whereas LPS protected the animals from Stx2 toxicity when administered before the toxin. Palermo et al. (32) showed that the LPS-induced modulation of Stx2 lethality was cytokine time and dose dependent. Mice given low doses of TNF-α or IL-1β 1 h before Stx2 treatment showed increased lethality when treated with Stx2, while mice given higher doses of IL-1β (sufficient to elicit corticosteroid production) were protected from Stx2 lethality.The proinflammatory cytokines TNF-α and IL-1β sensitize vascular endothelial cells to the cytotoxic action of Shiga toxins in vitro (24, 34, 53) through a mechanism involving the increased expression of genes involved in the biosynthesis of Gb₃ (43). Murine and human macrophages or macrophage-like cell lines express proinflammatory cytokines and chemokines when treated with purified Shiga toxins (12, 35, 51). Keepers et al. (18) showed a marked monocytic cell infiltrate into the kidneys of mice given Stx2 and LPS. Collectively, these data suggest that the innate immune response to Shiga toxins, in the presence or absence of LPS, alters the outcome of renal disease. In the present study, we have examined the role of a single proinflammatory cytokine, TNF-α, in pathogenesis using the mouse model of Stx-induced renal damage. Our data suggest that the timing of TNF-α production affects the outcome of disease in that the presence of elevated TNF-α levels prior to toxin challenge protects animals from disease, while high TNF-α levels occurring after toxin administration result in accelerated lethality.     

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.     

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.     

A DNA Vaccine Encoding the Enterohemorragic Escherichia coli Shiga-Like Toxin 2 A2 and B Subunits Confers Protective Immunity to Shiga Toxin Challenge in the Murine Model

Production of verocytotoxin or Shiga-like toxin (Stx), particularly Stx2, is the basis of hemolytic uremic syndrome, a frequently lethal outcome for subjects infected with Stx2-producing enterohemorrhagic Escherichia coli (EHEC) strains. The toxin is formed by a single A subunit, which promotes protein synthesis inhibition in eukaryotic cells, and five B subunits, which bind to globotriaosylceramide at the surface of host cells. Host enzymes cleave the A subunit into the A₁ peptide, endowed with N-glycosidase activity to the 28S rRNA, and the A₂ peptide, which confers stability to the B pentamer. We report the construction of a DNA vaccine (pStx2ΔAB) that expresses a nontoxic Stx2 mutated form consisting of the last 32 amino acids of the A₂ sequence and the complete B subunit as two nonfused polypeptides. Immunization trials carried out with the DNA vaccine in BALB/c mice, alone or in combination with another DNA vaccine encoding granulocyte-macrophage colony-stimulating factor, resulted in systemic Stx-specific antibody responses targeting both A and B subunits of the native Stx2. Moreover, anti-Stx2 antibodies raised in mice immunized with pStx2ΔAB showed toxin neutralization activity in vitro and, more importantly, conferred partial protection to Stx2 challenge in vivo. The present vector represents the second DNA vaccine so far reported to induce protective immunity to Stx2 and may contribute, either alone or in combination with other procedures, to the development of prophylactic or therapeutic interventions aiming to ameliorate EHEC infection-associated sequelae.Shiga toxin (Stx)-producing enterohemorrhagic Escherichia coli (EHEC) strains are important food-borne pathogens representing the major etiological agents of hemorrhagic colitis and hemolytic uremic syndrome (HUS), a life-threatening disease characterized by hemolytic anemia, thrombocytopenia, and renal failure (19). The infection correlates with ingestion of contaminated meat or vegetables but is also transmitted by water or even person-to-person contact (8, 14, 44). Sporadic or massive outbreaks have been reported in several developed countries but, in Argentina, HUS is endemic and represents a serious public health problem with high morbidity and mortality rates (29, 40). Production of verocytotoxin or Shiga-like toxin (Stx) is the basis of EHEC pathogenesis (18, 20). The toxin is formed by a single A subunit, which possesses N-glycosidase activity to the 28S rRNA and promotes protein synthesis inhibition in eukaryotic cells, and five B subunits, which bind to globotriaosylceramide at the surface of host cells (9, 28). Although two major types (Stx1 and Stx2) and several subtypes have been described, Stx2 and Stx2c are the most frequently found toxins in severe HUS cases among EHEC-infected subjects (12, 41). The degree of antigenic cross-reactivity between Stx2 and Stx1 is low, and several authors have reported that the two toxins do not provide heterologous protection, particularly concerning the B subunits (45, 47). On the other hand, Stx2c and Stx2d variants are readily neutralized by antibodies against Stx2 (27).Despite the magnitude of the social and economic impacts caused by EHEC infections, no licensed vaccine or effective therapy is presently available for human use. So far, attempts to develop vaccine formulations against EHEC-associated sequelae have relied mainly on induction of serum anti-Stx antibody responses. Several approaches have been pursed to generate immunogenic anti-Stx vaccine formulations and include the use of live attenuated bacterial strains (2, 32), protein-conjugated polysaccharides (21), purified B subunit (33, 48), B-subunit-derived synthetic peptides (15), and mutated Stx1 and Stx2 nontoxic derivatives (5, 6, 13, 16, 37, 39, 42, 45).In a previous report we described anti-Stx2 DNA vaccines encoding either the B subunit or a fusion protein between the B subunit and the first N-terminal amino acid of the A₁ subunit (8). The DNA vaccine encoding the hybrid protein elicited Stx-specific immune responses in mice and partial protection to Stx2 challenge (1, 33). Recent data have indicated that epitopes leading to generation of Stx-neutralizing antibodies are present on both the B as well as the A subunit (34, 45, 46). In addition, further evidence indicates that the A₂ subunit contains some of the most immunogenic epitopes of the Stx2 toxin (4). Thus, in line with such evidence, we attempted the construction of a new DNA vaccine encoding the last 32 amino acids from the A₂ subunit, in addition to the complete B subunit of Stx2, as separated polypeptides which could enhance the immunogenicity and protective effects of the vaccine formulation. In the present report, we describe the generation of a new DNA vaccine encoding both Stx2 A2 and B subunits as an approach to elicit protective antibody responses to Stx2. The results obtained demonstrate that immunization with this vaccine formulation results in systemic antibody responses to Stx2 A and B subunits and toxin neutralization activity both in vitro and in vivo.     

An Orally Applicable Shiga Toxin Neutralizer Functions in the Intestine To Inhibit the Intracellular Transport of the Toxin

Shiga toxin 2 (Stx2) is a major virulence factor in infections with Stx-producing Escherichia coli (STEC), which causes gastrointestinal diseases and sometimes fatal systemic complications. Recently, we developed an oral Stx2 inhibitor known as Ac-PPP-tet that exhibits remarkable therapeutic potency in an STEC infection model. However, the precise mechanism underlying the in vivo therapeutic effects of Ac-PPP-tet is unknown. Here, we found that Ac-PPP-tet completely inhibited fluid accumulation in the rabbit ileum caused by the direct injection of Stx2. Interestingly, Ac-PPP-tet accumulated in the ileal epithelial cells only through its formation of a complex with Stx2. The formation of Ac-PPP-tet-Stx2 complexes in cultured epithelial cells blocked the intracellular transport of Stx2 from the Golgi apparatus to the endoplasmic reticulum, a process that is essential for Stx2 cytotoxicity. Thus, Ac-PPP-tet is the first Stx neutralizer that functions in the intestine by altering the intracellular transport of Stx2 in epithelial cells.Infection with Shiga toxin (Stx)-producing Escherichia coli (STEC) in humans causes gastrointestinal diseases that are often followed by potentially fatal systemic complications such as acute encephalopathy and hemolytic-uremic syndrome (12, 22, 25, 26). Stx is produced in the gut, traverses the epithelium, and passes into the circulation. Circulating Stx then causes vascular damage in specific target tissues such as the brain and the kidney, resulting in systemic complications. For this reason, development of a neutralizer that specifically binds to and inhibits Stx in the gut and/or in the circulation would be a promising therapeutic approach.Stx is classified into two subgroups, Stx1 and Stx2. Stx2 is more closely related to the severity of STEC infections than Stx1 (6, 23, 31, 33). Stx consists of a catalytic A subunit and a pentameric B subunit. The former has 28S rRNA N-glycosidase activity and inhibits eukaryotic protein synthesis, while the latter is responsible for binding to the functional cell surface receptor Gb3 [Galα(1-4)-Galβ(1-4)-Glcβ1-ceramide] (11, 17, 25). The crystal structure of Stx reveals the presence of three distinctive binding sites (i.e., sites 1, 2, and 3) on each B subunit monomer for the trisaccharide moiety of Gb3 (7, 16). Highly selective and potent binding of Stx to Gb3 is attributed mainly to the multivalent interaction between the B subunit pentamer and the trisaccharide. This so-called clustering effect has formed the basis for the development of several synthetic Stx neutralizers that contain the trisaccharide in multiple configurations (3, 5, 14, 18, 19, 36). These neutralizers can strongly bind to Stx and inhibit its cytotoxic activity. Some are also effective in STEC infection models (18, 19, 36). However, the clinical application of these neutralizers has been substantially hampered by the synthetic complexity of the trisaccharide moiety.We have recently screened a library of novel tetravalent peptides that exert a clustering effect and have identified four peptide motifs that are superior to trisaccharide in binding Stx (20). Tetravalent forms of these peptides bind with high affinity to one trisaccharide-binding site (site 3) of Stx2 and effectively inhibit Stx2 cytotoxicity. This is particularly true of the neutralizer designated PPP-tet, which contains four Pro-Pro-Pro-Arg-Arg-Arg-Arg motifs. PPP-tet protects mice from a fatal dose of E. coli O157:H7, even when the peptide is orally administered after the establishment of infection (20). Moreover, the addition of acetyl groups to all the amino termini of PPP-tet (yielding Ac-PPP-tet) makes this compound resistant to proteolysis and markedly enhances its protective activity against STEC infection, indicating that Ac-PPP-tet holds promise as a therapeutic agent for STEC infections.After binding to Gb3, Stx is first transported to the Golgi apparatus in a retrograde manner and then transported to the endoplasmic reticulum (ER). On the other hand, the Stx catalytic A subunit is released into the cytoplasm, where it inhibits protein synthesis (27, 29). The retrograde transport of Stx is known to be essential for Stx cytotoxicity (2, 27, 28). In Vero cells, one of the cell types most sensitive to Stx, PPP-tet prevents Stx2 cytotoxicity by inducing the aberrant transport of Stx from the Golgi apparatus to an acidic compartment rather than to the ER, leading to the degradation of Stx (20). An advantage of PPP-tet is its ability to partially permeate cells, which allows it to inhibit the cytotoxicity of Stx2 already incorporated into cells (20). Nevertheless, the precise mechanism by which PPP-tet and Ac-PPP-tet function in vivo, as well as the identities of the organs or cells targeted by these compounds, is unknown.To understand how orally administered Ac-PPP-tet functions in vivo, we investigated the effect of Ac-PPP-tet on fluid accumulation in the rabbit ileum caused by the direct injection of Stx2. The rabbit ileal loop system is highly valid for evaluating the toxicity of Stx2 produced in the intestine after infection. We also examined the localization of the tetravalent peptide and Stx2 in the intact rabbit ileum, cultured ileal specimens, and Caco-2 intestinal epithelial cells. Our results reveal that Ac-PPP-tet functions as a potent Stx2 neutralizer in the intestine by altering the intracellular transport of Stx2 in epithelial cells.     

Salmonella enterica Serovar Typhimurium Vaccine Strains Expressing a Nontoxic Shiga-Like Toxin 2 Derivative Induce Partial Protective Immunity to the Toxin Expressed by Enterohemorrhagic Escherichia coli

Shiga-like toxin 2 (Stx2)-producing enterohemorrhagic Escherichia coli (referred to as EHEC or STEC) strains are the primary etiologic agents of hemolytic-uremic syndrome (HUS), which leads to renal failure and high mortality rates. Expression of Stx2 is the most relevant virulence-associated factor of EHEC strains, and toxin neutralization by antigen-specific serum antibodies represents the main target for both preventive and therapeutic anti-HUS approaches. In the present report, we describe two Salmonella enterica serovar Typhimurium aroA vaccine strains expressing a nontoxic plasmid-encoded derivative of Stx2 (Stx2ΔAB) containing the complete nontoxic A2 subunit and the receptor binding B subunit. The two S. Typhimurium strains differ in the expression of flagellin, the structural subunit of the flagellar shaft, which exerts strong adjuvant effects. The vaccine strains expressed Stx2ΔAB, either cell bound or secreted into the extracellular environment, and showed enhanced mouse gut colonization and high plasmid stability under both in vitro and in vivo conditions. Oral immunization of mice with three doses of the S. Typhimurium vaccine strains elicited serum anti-Stx2B (IgG) antibodies that neutralized the toxic effects of the native toxin under in vitro conditions (Vero cells) and conferred partial protection under in vivo conditions. No significant differences with respect to gut colonization or the induction of antigen-specific antibody responses were detected in mice vaccinated with flagellated versus nonflagellated bacterial strains. The present results indicate that expression of Stx2ΔAB by attenuated S. Typhimurium strains is an alternative vaccine approach for HUS control, but additional improvements in the immunogenicity of Stx2 toxoids are still required.Shiga-like toxins (Stx) play a crucial role in the pathogenesis of enterohemorrhagic Escherichia coli (EHEC) strains, which may lead to hemorrhagic colitis, central nervous system disturbances, and hemolytic-uremic syndrome (HUS) (27, 33). HUS involves acute renal failure, thrombocytopenia, and microangiopathic hemolytic anemia, with mortality rates ranging from 1% to 4% (45, 50). EHEC strains may express different serotypes, including the widely distributed O157:H7 serotype, and infection correlates with the ingestion of contaminated ground beef and cow manure-contaminated water, vegetables, juices, and other products (13, 18). The incidence of EHEC-associated HUS cases is particularly high in developed countries, and high incidence rates have been recorded in Argentina, where cultural and diverse epidemiological factors contribute to the widespread dissemination of the disease among children and teenagers (38).EHEC strains may express two different Stx types. Stx1 is virtually identical to Stx produced by Shigella dysenteriae, while Stx2 shows only 56% homology to Stx1 at the amino acid sequence level (14, 33, 51). Both toxin types are formed by one A subunit and five B subunits, which bind to glycosphingolipid receptors, such as globotriaosyl ceramide (Gb3), on host cell membranes and promote retrograde toxin transport through the Golgi complex and endoplasmic reticulum. In the cell cytoplasm, Stx2 subunit A is processed into two fragments; one of them (A1) is endowed with N-glycosidase activity, which depurinates a specific adenine residue of the eukaryotic 28S rRNA, inhibits protein synthesis, and induces apoptosis of the target cell (18, 51).After ingestion and gut colonization, Stx molecules are released by the bacterial cells and translocate across the gut epithelium to reach, via the bloodstream, capillary endothelial cells at renal glomeruli, where the most relevant tissue damage occurs (33, 45, 50). Epidemiological data indicate that individuals infected with Stx2-producing bacterial strains, and some closely related variants, have a high probability of developing HUS (45, 50). In addition, Stx2 expression has been shown to increase gut colonization by bacterial cells due to induction of increased receptor expression by enterocytes (39).So far, there is no effective prophylactic or therapeutic approach for the prevention of HUS development among EHEC-infected individuals. The treatments available involve platelet transfusion in cases of severe anemia, hemodialysis, and supportive care (7, 50). A more direct anti-Stx treatment under clinical or preclinical evaluation involves the use of synthetic Stx glycolipid receptor analogs and humanized anti-Stx monoclonal antibodies (44, 52).Attempts to develop prophylactic anti-HUS vaccines are focused on the generation of Stx-neutralizing antibodies or the blockade of gut colonization. The vaccine strategies based on Stx2 that have been tested under experimental conditions have included DNA vaccines (5, 12), protein-conjugated polysaccharides (28), purified recombinant B subunits (24, 25, 29, 30, 47, 53, 55, 58), and B-subunit-derived synthetic peptides (19, 20). Anti-EHEC vaccine approaches based on the blockade of gut colonization have employed intimin and type III secreted proteins, such as EspA and EspB (3, 37, 54).Live bivalent anti-Stx vaccines based on genetically modified, attenuated Vibrio cholerae or Salmonella enterica serovar Typhimurium strains have been reported to induce anti-StxB antibody responses following oral administration to mice or rabbits (1, 10, 49). Attenuated Salmonella strains, used as orally administered vaccine vectors for the expression of heterologous antigens, show several advantages over conventional parenterally delivered cellular or acellular vaccine formulations (15, 16). Attenuated Salmonella strains are safe, are easily administered by untrained personnel, and, more relevantly, may induce systemic and secreted antigen-specific antibody and cell-based immune responses against self and heterologous antigens. In addition, whole bacterial cells carry on their surfaces several molecular structures known to activate both innate and adaptive immune responses. These molecules, such as lipopolysaccharide and flagellin, act as strong adjuvants, both systemically and at mucosal surfaces.Flagellins, the structural subunit of flagellar filaments, contribute both to the virulence of bacterial pathogens and to the activation of inflammatory responses in mammalian hosts. Bacterial flagellins have been shown to bind both extracellular and intracellular receptors of antigen-presenting cells, leading to inflammation and increased adaptive immune responses, including the generation of antigen-specific antibodies and T cells (2, 26). The strong adjuvant effects of Salmonella flagellins, either when admixed with purified antigens or when used as hybrid proteins genetically fused to the target antigens, have been demonstrated recently (4, 8, 22, 23, 36). However, there is no clear evidence that the expression of flagellin affects the immunogenicity of heterologous antigens expressed by attenuated Salmonella vaccine strains.In the present study, we generated new experimental anti-HUS vaccine formulations based on two recombinant attenuated S. Typhimurium aroA vaccine strains differing in the expression of flagellin. The two strains were genetically modified in order to express a nontoxic Stx2 derivative consisting of the whole Stx2 B subunit and a partially deleted A subunit encompassing the first amino acid of the A1 subunit genetically fused to the whole A2 subunit (Stx2ΔAB). The Stx2ΔAB protein was previously tested in mice immunized with a DNA vaccine (5). The results of the present study show that the S. Typhimurium vaccine strains express and secrete the recombinant toxin and induce both systemic and mucosal anti-StxB antibodies with anti-Stx2 neutralization activity, conferring partial protection against intravenous (i.v.) challenge with Stx2.     

Monoclonal Antibody 11E10, Which Neutralizes Shiga Toxin Type 2 (Stx2), Recognizes Three Regions on the Stx2 A Subunit,Blocks the Enzymatic Action of the Toxin In Vitro,and Alters the Overall Cellular Distribution of the Toxin

Monoclonal antibody (MAb) 11E10 recognizes the Shiga toxin type 2 (Stx2) A₁ subunit. The binding of 11E10 to Stx2 neutralizes both the cytotoxic and lethal activities of Stx2, but the MAb does not bind to or neutralize Stx1 despite the 61% identity and 75% similarity in the amino acids of the A₁ fragments. In this study, we sought to identify the segment or segments on Stx2 that constitute the 11E10 epitope and to determine how recognition of that region by 11E10 leads to inactivation of the toxin. Toward those objectives, we generated a set of chimeric Stx1/Stx2 molecules and then evaluated the capacity of 11E10 to recognize those hybrid toxins by Western blot analyses and to neutralize them in Vero cell cytotoxicity assays. We also compared the amino acid sequences and crystal structures of Stx1 and Stx2 for stretches of dissimilarity that might predict a binding epitope on Stx2 for 11E10. Through these assessments, we concluded that the 11E10 epitope is comprised of three noncontiguous regions surrounding the Stx2 active site. To determine how 11E10 neutralizes Stx2, we examined the capacity of 11E10/Stx2 complexes to target ribosomes. We found that the binding of 11E10 to Stx2 prevented the toxin from inhibiting protein synthesis in an in vitro assay but also altered the overall cellular distribution of Stx2 in Vero cells. We propose that the binding of MAb 11E10 to Stx2 neutralizes the effects of the toxin by preventing the toxin from reaching and/or inactivating the ribosomes.Escherichia coli O157:H7 and other Shiga toxin (Stx)-producing E. coli (STEC) strains cause approximately 110,000 cases of infection and over 90 deaths each year in the United States according to the Centers for Disease Control and Prevention (16). Infections with STEC can lead to diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS). HUS occurs in about 6 to 15% of individuals after infection with E. coli O157:H7 (15)—but less frequently with other STEC strains (5)—and is characterized by hemolytic anemia, thrombotic thrombocytopenia, and renal failure. The development of this sequela is linked to the expression of Stxs by the bacteria (18).The Stx family comprises two serogroups, Stx/Stx1 and Stx2, and polyclonal antisera raised against either Stx1 or Stx2 do not cross-neutralize the other toxin (29, 30). Stx is produced by Shigella dysenteriae type 1 and differs by only 1 amino acid from the Stx1 made by the prototypic STEC O157:H7 strain, EDL933. A single isolate of STEC can express Stx1 (or one of its variants), Stx2 (or one of its variants), or both toxins. Variants of each toxin type are defined by either a biological or immunological difference from the prototypical toxin (31). Stx1 variants include Stx1c and Stx1d, while the variants of Stx2 are Stx2c, Stx2d, Stx2d-activatable (Stx2d_act), Stx2e, and Stx2f (reviewed in reference 18).Stxs are complex holotoxins with a stoichiometry of five identical binding (B) subunits and a single active (A) domain. These AB₅ molecules are potent cytotoxins with an N-glycosidase activity that stops protein synthesis by inactivation of the 60S ribosome (6); this activity eventually leads to eukaryotic cell death. The ∼32-kDa A subunit contains the enzymatic activity of the toxin with the active site glutamic acid residue at position 167. The A subunit is asymmetrically cleaved by trypsin or furin into an enzymatically active ∼28-kDa A₁ fragment and an ∼4-kDa A₂ peptide. The A₂ peptide remains linked to the large enzymatic domain through a disulfide bond and is encircled by the five identical B subunits of ∼7.7 kDa. The B subunits of the Stxs typically bind to the eukaryotic glycolipid receptor globotriaosylceramide (Gb₃), also known as CD77. The mature A and B subunits of Stx1 and Stx2 are approximately 68 and 73% similar at the amino acid level. The crystal structures of Stx and Stx2 have been resolved, and the two structures are remarkably similar (7, 8). Nevertheless, there are some features of these three-dimensional models that differ (summarized in reference 8).Currently, there are no Food and Drug Administration-approved therapies in the United States to treat STEC infections. However, our research group is one of several that investigate passive immunization strategies to neutralize the Stxs associated with STEC infections (3, 4, 10, 13, 19, 20). Our passive immunization strategy is based on murine monoclonal antibodies (MAbs) developed in this laboratory that specifically bind to and neutralize Stx/Stx1 or Stx2 (21, 28). The MAb 11E10 was generated by immunization of BALB/c mice with Stx2 turned into a toxoid (“toxoided”) by treatment with formaldehyde (21). MAb 11E10 specifically recognizes the A₁ fragment of Stx2 and neutralizes Stx2 for Vero cells and mice but does not bind to or neutralize Stx/Stx1 (21). The murine MAb 11E10 was modified to contain a human constant region to reduce the potential for an antibody recipient to generate an antimouse antibody response (4). This human-mouse chimeric antibody, called cαStx2, successfully underwent phase I clinical testing (3). In this report, we define the epitope on the A subunit of Stx2 recognized by the murine MAb 11E10 (and, therefore, also by cαStx2) and present evidence that the MAb blocks the enzymatic action of the toxin in vitro and also alters toxin trafficking in Vero cells.     

Human Intestinal Tissue and Cultured Colonic Cells Contain Globotriaosylceramide Synthase mRNA and the Alternate Shiga Toxin Receptor Globotetraosylceramide

Escherichia coli O157:H7 and other Shiga toxin (Stx)-producing E. coli (STEC) bacteria are not enteroinvasive but can cause hemorrhagic colitis. In some STEC-infected individuals, a life-threatening sequela of infection called the hemolytic uremic syndrome may develop that can lead to kidney failure. This syndrome is linked to the production of Stx by the infecting organism. For Stx to reach the kidney, the toxin must first penetrate the colonic epithelial barrier. However, the Stx receptor, globotriaosylceramide (Gb3), has been thought to be absent from human intestinal epithelial cells. Thus, the mechanisms by which the toxin associates with and traverses through the intestine en route to the kidneys have been puzzling aspects of STEC pathogenesis. In this study, we initially determined that both types of Stx made by STEC, Stx1 and Stx2, do in fact bind to colonic epithelia in fresh tissue sections and to a colonic epithelial cell line (HCT-8). We also discovered that globotetraosylceramide (Gb4), a lower-affinity toxin receptor derived from Gb3, is readily detectable on the surfaces of human colonic tissue sections and HCT-8 cells. Furthermore, we found that Gb3 is present on a fraction of HCT-8 cells, where it presumably functions to bind and internalize Stx1 and Stx2. In addition, we established by quantitative real-time PCR (qRT-PCR) that both fresh colonic epithelial sections and HCT-8 cells express Gb3 synthase mRNA. Taken together, our data suggest that Gb3 may be present in small quantities in human colonic epithelia, where it may compete for Stx binding with the more abundantly expressed glycosphingolipid Gb4.Shiga toxins (Stxs) are highly potent ribotoxic virulence factors associated with the worst pathological manifestations of infection by Escherichia coli serotype O157:H7 and other Stx-producing E. coli (STEC) bacteria. Two major types of Stxs are produced by STEC, Stx1 and Stx2, and an organism may produce one or both toxins. Stx1 and Stx2 share enzymatic and structural features but are immunologically distinct. More than 110,000 cases of STEC infection are estimated to occur each year in the United States, and about 75,000 of those cases are caused by E. coli O157:H7 infection. Many individuals infected with E. coli O157:H7 present with severe abdominal pain and bloody diarrhea, of which the latter may be caused by the action of Stxs on endothelial cells that line the small blood vessels (microvasculature) in the gastrointestinal tract (4, 26, 42, 44). In some patients, STEC infection leads to a serious sequela called the hemolytic uremic syndrome (HUS). The HUS is characterized by a triad of clinical features that include thrombocytopenia, hemolytic anemia, and acute kidney failure, and it occurs most frequently in children less than 10 years of age (2, 12). Of note, HUS associated with E. coli O157:H7 infection is a major cause of acute kidney failure in children in the United States and worldwide (6, 61). One hypothesis for how the renal injury occurs in HUS is that blood-borne Stx induces apoptosis in endothelial cells in the glomerular microvasculature (19). Thrombi then form in these damaged blood vessels, a characteristic pathological feature of the HUS. Death of renal tubular cells has also been linked to Stx production in humans and in mouse models of E. coli O157:H7 infection (7, 34, 56).How Stx moves from the lumen of the bowel to the blood vessels that lie below the surface of the gastrointestinal tract to reach the kidneys has not been determined. Presumably, the toxin breaches the epithelial barrier of the colon at or near the site of colonization by the noninvasive E. coli O157:H7. However, the colonic epithelium has been reported to lack globotriaosylceramide (Gb3), the established and preferred receptor for Stx1 and Stx2. The consensus in the literature that the Stx receptor, Gb3, is not present in the human colonic epithelial cells was originally based on conclusions that were drawn from analysis of the general glycolipid composition of the human large intestine (17, 52). In those studies, glycolipids from either mucosal scrapes or the entire mucosal layer were examined by thin-layer chromatography (TLC). Although these mucosal specimens were reported to contain small but measurable levels of Gb3, the samples included not only epithelial cells but also Gb3-enriched endothelial cells. Therefore, evidence of the presence of Gb3 on the cell surface of large bowel epithelial cells was inconclusive. In another investigation, trace amounts of Gb3 were found in epithelial cells isolated by sequential washes of colon tissue with buffer that contained EDTA and reducing agent to gently remove cells layer by layer; however, again, nonepithelial cell contamination could not be ruled out (16). Holgersson et al. ultimately concluded that large bowel epithelial cells do not express “glycolipid-borne Galα1-4 Gal sequence” (components of Gb3) based on the failure to detect Gb3 on the cell surface with Gb3-specific antibody (16). Nevertheless, some of these early studies do suggest that Gb3 may be present in trace amounts in colonic epithelia. These findings, however, appear to have been discounted by many in the field, who assert, based on the negative immunodetection by Holgersson et al. described above (16) and findings of subsequent studies (36, 48), that Gb3 is not present on the colonic epithelium (29).Given the presumed lack of colonic epithelial Gb3, a number of theories have been promulgated to explain how Stx is able to penetrate the epithelial barrier to reach the bloodstream (29). Two related hypotheses are as follows: (i) Stx follows a paracellular course and moves between cells during immune cell infiltration that follows infection (55), or (ii) Stx transits to the bloodstream from the bowel lumen through possible “holes” in the mucosa that result from attachment-and-effacement (A&E) lesions and Stx damage (33). A third hypothesis is that Stx moves by a transcellular route from the lumen to the vasculature in the lamina propria, such as through an alternative but undefined trafficking pathway that does not involve Gb3. In support of the latter conjecture, Stx was found to transcytose polarized tissue culture cells that appeared to lack Gb3 in the absence of cellular damage (1). However, in a human organ culture model, Stx-mediated intestinal epithelial damage was observed (48), a finding that seems to support the second hypothesis above. A fourth premise to explain how Stx crosses the mucosal barrier in the absence of Gb3 on enterocytes is that an alternative receptor exists (11). Such a putative receptor could either mediate Stx-induced cytotoxicity and thus cause a breach in the epithelial barrier or allow transcellular movement of the toxin across the epithelial cell. However, evidence in support of an alternative receptor to Gb3 is not strong. Rather, accumulated data suggest that Gb3 is the sole functional receptor for all Stxs (19, 22, 24, 27, 32, 34, 40, 43, 50, 51, 59), and they can be summarized by 4 lines of evidence. First, cellular cytotoxicity is directly proportional to cell surface Gb3 content (21, 46, 47). Second, Gb3 provided to resistant cells via liposomes induces Stx1 sensitivity in those cells, whereas Gb4 does not (58). Third, Gb3 expression appears to correlate with clinical manifestations of STEC disease. The highest Gb3 content is found in the microvascular glomeruli and proximal tubule cells of the kidney, consistent with renal pathology in HUS (41). Other Gb3-rich regions include the colonic microvascular endothelia, with its associated lamina propria (bloody colitis) (21), and endothelial vasculature of the cerebellum (neurological symptoms) (46). This link between high Gb3 content and Stx toxicity is also seen in animal models. In rabbits, intravenous administration of Stx1 or Stx2 produces vascular lesions in those tissues with high concentrations of Gb3, i.e., the intestine and the central nervous system (47). Furthermore, rabbit kidneys lack Gb3, and no tissue injury is observed there (7). Fourth, Gb3 synthase knockout mice are resistant to Stx effects, although in these mice, other glycosphingolipids, including Gb4, are eliminated as well (43).In this study, we asked whether Stxs could bind to sections of human colonic epithelia and if so by what receptor(s). Based on our initial observation that small amounts of Stx1 and Stx2 were capable of binding to the surface of colonic epithelia, we investigated the possibility that small amounts of Gb3 are present on colonic epithelial cells. Here we present the novel finding that Gb3 synthase mRNA can be detected in epithelial cells isolated by laser capture microdissection (LCM) from normal human colonic tissue sections and that Gb4, a glycosphingolipid derived from Gb3 (Fig. ​(Fig.1),1), is expressed in this tissue. Furthermore, we demonstrate that while both Stx1 and Stx2 can bind to the epithelial surfaces of those normal human colonic tissue sections, Stx1 binds at relatively higher levels. In addition, we demonstrate that the cytotoxicity of Stx1 and Stx2 for the human colonic cell line HCT-8 can be enhanced 20- to 60-fold by genetically manipulating the cells to increase the cell surface Gb3 content. Collectively, these data suggest that small amounts of Gb3 may mediate Stx uptake in normal colonic tissue, an event that leads to cell death, and that abundant Gb4 may compete with Gb3 for Stx binding.Open in a separate windowFIG. 1.Globotetraosylceramide (Gb4) synthesis pathway (38). The synthesis of Gb4 requires globotriaosylceramide (Gb3) as a substrate. Biosyntheses of Gb3 and Gb4 are accompanied by intracellular transport, presumably by a vesicle-bound exocytotic membrane flow, from the endoplasmic reticulum (ER) through the Golgi cisternae to the plasma membrane (57). Abbreviations: LacCer, lactose-ceramide; GlcCer, glucose-ceramide.     

Shiga Toxin,Cytolethal Distending Toxin,and Hemolysin Repertoires in Clinical Escherichia coli O91 Isolates

Shiga toxin (Stx)-producing Escherichia coli (STEC) strains of serogroup O91 are the most common human pathogenic eae-negative STEC strains. To facilitate diagnosis and subtyping of these pathogens, we genotypically and phenotypically characterized 100 clinical STEC O91 isolates. Motile strains expressed flagellar antigens H8 (1 strain), H10 (2 strains), H14 (52 strains), and H21 (20 strains) or were H nontypeable (Hnt) (10 strains); 15 strains were nonmotile. All nonmotile and Hnt strains possessed the fliC gene encoding the flagellin subunit of the H14 antigen (fliC_H14). Most STEC O91 strains possessed enterohemorrhagic E. coli hlyA and expressed an enterohemolytic phenotype. Among seven stx alleles identified, stx_2dact, encoding mucus- and elastase-activatable Stx2d, was present solely in STEC O91:H21, whereas most strains of the other serotypes possessed stx₁. Moreover, only STEC O91:H21 possessed the cdt-V cluster, encoding cytolethal distending toxin V; the toxin was regularly expressed and was lethal to human microvascular endothelial cells. Infection with STEC O91:H21 was associated with hemolytic-uremic syndrome (P = 0.0015), whereas strains of the other serotypes originated mostly in patients with nonbloody diarrhea. We conclude that STEC O91 clinical isolates belong to at least four lineages that differ by H antigens/fliC types, stx genotypes, and non-stx putative virulence factors, with accumulation of virulence determinants in the O91:H21 lineage. Isolation of STEC O91 from patients'' stools on enterohemolysin agar and the rapid initial subtyping of these isolates using fliC genotyping facilitate the identification of these emerging pathogens in clinical and epidemiological studies and enable prediction of the risk of a severe clinical outcome.Shiga toxin (Stx)-producing Escherichia coli (STEC) strains cause diarrhea and a life-threatening hemolytic-uremic syndrome (HUS) worldwide (23, 44). STEC strains isolated from patients usually possess, in addition to one or more stx genes, the eae gene, encoding adhesin intimin (7, 11, 16, 25, 26, 41, 49). However, a subset of STEC strains associated with human disease lack eae, and among these, strains of serogroup O91 are the most common (2, 7, 35, 37, 47, 48). In Germany during the last 5 years, serogroup O91 accounted for 6.4% to 11.0% of all STEC strains reported from human infections and was therefore the fourth-most-common STEC serogroup (after O157, O26, and O103) isolated (47, 48; http://www.rki.de). However, in contrast to eae-positive STEC strains of the three leading serogroups, which cause disease mostly in young children (47), STEC O91 is the most common serogroup isolated from adult patients (48).Despite their association with human diseases worldwide (7, 9, 11, 13, 14, 30, 35, 37, 38, 40, 47, 48), the spectrum of serotypes of STEC O91 isolates from patients and the pathogenic traits of such strains are poorly understood. Moreover, characteristics of STEC O91 strains which could assist with their isolation from human stools and further subtyping in clinical microbiological laboratories have not been systematically investigated or reported. To gain insight into the serotype composition and putative virulence factors of STEC O91 strains causing human disease and to identify characteristics which can facilitate laboratory diagnosis of these organisms, we determined the motility and flagellar phenotypes, fliC types, stx genotypes, non-stx putative virulence loci, and diagnostically useful phenotypes of 100 clinical STEC O91 isolates. Moreover, we investigated possible associations between bacterial characteristics and clinical infection phenotypes.     

Variation in Susceptibility of Bloodstream Isolates of Candida glabrata to Fluconazole According to Patient Age and Geographic Location in the United States in 2001 to 2007

We examined the susceptibilities to fluconazole of 642 bloodstream infection (BSI) isolates of Candida glabrata and grouped the isolates by patient age and geographic location within the United States. Susceptibility of C. glabrata to fluconazole was lowest in the northeast region (46%) and was highest in the west (76%). The frequencies of isolation and of fluconazole resistance among C. glabrata BSI isolates were higher in the present study (years 2001 to 2007) than in a previous study conducted from 1992 to 2001. Whereas the frequency of C. glabrata increased with patient age, the rate of fluconazole resistance declined. The oldest age group (≥80 years) had the highest proportion of BSI isolates that were C. glabrata (32%) and the lowest rate of fluconazole resistance (5%).Candidemia is without question the most important of the invasive mycoses (6, 33, 35, 61, 65, 68, 78, 86, 88). Treatment of candidemia over the past 20 years has been enhanced considerably by the introduction of fluconazole in 1990 (7, 10, 15, 28, 29, 31, 40, 56-58, 61, 86, 90). Because of its widespread usage, concern about the development of fluconazole resistance among Candida spp. abounds (2, 6, 14, 32, 47, 53, 55, 56, 59, 60, 62, 80, 86). Despite these concerns, fluconazole resistance is relatively uncommon among most species of Candida causing bloodstream infections (BSI) (5, 6, 22, 24, 33, 42, 54, 56, 65, 68, 71, 86). The exception to this statement is Candida glabrata, of which more than 10% of BSI isolates may be highly resistant (MIC ≥ 64 μg/ml) to fluconazole (6, 9, 15, 23, 30, 32, 36, 63-65, 71, 87, 91). Suboptimal fluconazole dosing practices (low dose [<400 mg/day] and poor indications) may lead to an increased frequency of isolation of C. glabrata as an etiological agent of candidemia in hospitalized patients (6, 17, 29, 32, 35, 41, 47, 55, 60, 68, 85) and to increased fluconazole (and other azole) resistance secondary to induction of CDR efflux pumps (2, 11, 13, 16, 43, 47, 50, 55, 69, 77, 83, 84) and may adversely affect the survival of treated patients (7, 10, 29, 40, 59, 90). Among the various Candida species, C. glabrata alone has increased as a cause of BSI in U.S. intensive care units since 1993 (89). Within the United States, the proportion of fungemias due to C. glabrata has been shown to vary from 11% to 37% across the different regions (west, midwest, northeast, and south) of the country (63, 65) and from <10% to >30% within single institutions over the course of several years (9, 48). It has been shown that the prevalence of C. glabrata as a cause of BSI is potentially related to many disparate factors in addition to fluconazole exposure, including geographic characteristics (3, 6, 63-65, 71, 88), patient age (5, 6, 25, 35, 41, 42, 48, 63, 82, 92), and other characteristics of the patient population studied (1, 32, 35, 51). Because C. glabrata is relatively resistant to fluconazole, the frequency with which it causes BSI has important implications for therapy (21, 29, 32, 40, 41, 45, 56, 57, 59, 80, 81, 86, 90).Previously, we examined the susceptibilities to fluconazole of 559 BSI isolates of C. glabrata and grouped the isolates by patient age and geographic location within the United States over the time period from 1992 to 2001 (63). In the present study we build upon this experience and report the fluconazole susceptibilities of 642 BSI isolates of C. glabrata collected from sentinel surveillance sites throughout the United States for the time period from 2001 through 2007 and stratify the results by geographic region and patient age. The activities of voriconazole and the echinocandins against this contemporary collection of C. glabrata isolates are also reported.     

Novel Subtilase Cytotoxin Produced by Shiga-Toxigenic Escherichia coli Induces Apoptosis in Vero Cells via Mitochondrial Membrane Damage

Subtilase cytotoxin (SubAB) is an AB₅ 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 G₁ 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.Shiga-toxigenic Escherichia coli (STEC) is an etiologic agent of hemorrhagic colitis. Gastrointestinal disease caused by STEC may progress to systemic complications, including hemolytic uremic syndrome (HUS), which is characterized by thrombocytopenia, microangiopathic hemolytic anemia, and renal failure (13, 23). Shiga toxin 1 (Stx1) and Stx2 are both produced by STEC. However, whether Shiga toxins are the only factors responsible for these devastating diseases is still not clear.A new member of the AB₅ toxin family, named subtilase cytotoxin (SubAB), was identified (22, 23) in E. coli O113:H21 strain 98NK2, which produced Stx2 and was responsible for an outbreak of HUS. SubAB consists of one A subunit and five B subunits, which form a pentamer, similar to the case for Stx. The SubAB A subunit, with a molecular size of 35 kDa, shares sequence homology with a subtilase-like serine protease of Bacillus anthracis, and the toxin was named “subtilase cytotoxin.” The A subunit cleaves at a specific single site of endoplasmic reticulum (ER) chaperone BiP (21). The B subunits bind to some N-glycosylated membrane proteins, and α2β1 integrin has been shown to one of the receptors for vacuolating activity of B subunits (18, 30). Recently, it was reported that B subunits specifically bound to glycans terminating in the sialic acid N-glycolylneuraminic acid (3). SubAB is lethal for mice, causing extensive microvascular thrombosis as well as necrosis in the brain, kidney, and liver and apoptosis in the spleen, kidney, and liver. These findings are similar to the histopathologic, biochemical, and hematologic changes seen in human HUS (22, 26).SubAB is cytotoxic to Vero cells. BiP cleavage by the A subunit is necessary for Vero cell death (17, 18, 21, 22). BiP is known as a master regulator of ER function and homeostasis (11). SubAB induces ER stress (17, 27), as shown by activation of double-stranded RNA-activated protein kinase-like ER kinase (PERK) and eukaryotic initiation factor 2α (eIF2α), leading to transient protein synthesis inhibition and stress-inducible C/EBP-homologous protein (CHOP) induction, with cell cycle arrest in G₁ phase as a result of downregulation of cyclin D1 (17).Apoptosis, or programmed cell death, is a physiological event important in a diverse array of biological processes ranging from embryo development to bacterial infection (7, 31, 33). Morphologically, cells undergoing apoptosis demonstrate nuclear/cytoplasmic condensation and membrane protrusions. Biochemically, apoptotic cells are characterized by reduction in the mitochondrial transmembrane potential, intracellular acidification, production of reactive oxygen species, externalization of phosphatidylserine residues in membrane bilayers, selective proteolysis of a subset of cellular proteins, and internucleosomal degradation of DNA, resulting in a typical fragmentation pattern (28). There are multiple potential participants described for ER stress-induced apoptosis; however, the precise mechanisms of ER stress-induced apoptosis have not been fully elucidated (29). Recently, SubAB-induced apoptosis was partially described (27). We report here that SubAB triggers apoptosis in Vero cells initiated via mitochondrial membrane damage, followed by activation of a caspase-dependent cell death pathway.     

Evaluation of Fab and F(ab′)2 Fragments and Isotype Variants of a Recombinant Human Monoclonal Antibody against Shiga Toxin 2

5C12 HuMAb is a human monoclonal antibody against the A subunit of Shiga toxin 2 (Stx2). We have previously shown that 5C12 HuMAb effectively neutralizes the cytotoxic effects of this toxin by redirecting its transport within the cell and also by neutralizing the toxin''s ability to inhibit protein synthesis. The 5C12 HuMAb and its recombinant IgG1 version protect mice at a dose of 0.6 μg against a lethal challenge of Stx2. The contribution of the Fc region to this observed neutralization activity of the 5C12 antibody against Stx2 was investigated in this study. Using recombinant DNA technology, 5C12 isotype variants (IgG1, IgG2, IgG3, and IgG4) and antibody fragments [Fab, F(ab′)₂] were expressed in Chinese hamster ovary cells and evaluated in vitro and in vivo. All four 5C12 isotype variants showed protection in vitro, with the IgG3 and IgG4 variants showing the highest protection in vivo. The Fab and F(ab′)₂ fragments also showed protection in vitro but no protection in the mouse toxicity model. Similar results were obtained for a second HuMAb (5H8) against the B subunit of Stx2. The data suggest the importance of the Fc region for neutralization activity, but it is not clear if this is related to the stability of the full-length antibody or if the Fc region is required for effective elimination of the toxin from the body.Approximately 20,000 cases of Shiga toxin (Stx)-producing Escherichia coli (STEC) infections, in which the O157:H7 serotype is the most prevalent serotype, are reported annually in the United States (for recent reviews, see references 6, 9, 10, 23, and 31). Transmission of E. coli O157:H7 is most frequently associated with the consumption of contaminated food (e.g., ground beef or spinach) or drinking unpasteurized dairy products. Infections can also be acquired through person-to-person contact. Infected individuals typically develop abdominal pain and bloody diarrhea 2 to 5 days following exposure. STEC infections are self-limiting and usually resolve in 7 to 10 days. However, in 10 to 15% of children under the age of 5 or in the elderly, E. coli O157:H7 infections can develop into diarrhea-associated hemolytic uremic syndrome (HUS), a serious, life-threatening complication (22, 26, 28, 31). HUS is associated with hemolytic anemia and thrombocytopenia as a result of the destruction of red blood cells and platelets, followed by acute renal failure. There are no effective therapies against HUS, and supportive therapies include dialysis and kidney transplantation. Thus, the best treatment for HUS is prevention or amelioration of the E. coli O157:H7 infection, as no protective therapies are presently available. Antibiotic therapy for treatment of E. coli O157:H7 infections does not shorten the infection period and, in fact, may increase the risk of developing HUS (34).The primary virulence factor for HUS is Shiga toxin 2 (Stx2), which is one of two antigenically distinct toxins produced by STEC. Stx2, like Stx1, consists of a single A subunit (32 kDa) linked to a ring of five B subunits (7 kDa) (18). The A subunit possesses RNA N-glycosidase activity, which cleaves a specific adenine residue in the 28S rRNA, resulting in the inhibition of protein synthesis. The B subunits are responsible for binding to the host cell receptor, globotriaosyl ceramide (Gb3; Galα[1-4]-Galβ[1-4]-Glcβ1-ceramide). The A subunit is cleaved as it is internalized through clathrin-dependent or independent endocytosis and translocated via the retrograde pathway to the cytosol where it inhibits protein synthesis (3, 4, 19).Therapies that inhibit cell binding, interfere with the intracellular transport of Stx2, or inhibit enzymatic activity are under development by several research groups. One of the most promising therapies is the use of Stx2-neutralizing human monoclonal antibodies, particularly those directed against the A subunit (13, 14). One of these antibodies, 5C12, was shown to protect mice and gnotobiotic piglets from the fatal complications of Stx2 (13, 24, 25). A recombinant 5C12 antibody also demonstrated similar protective activity in these two animal models (1). We have recently shown that 5C12 neutralizes the toxicity of Stx2 for HeLa cells by blocking the retrograde transport of the toxin to the cytosol where the A subunit inhibits protein synthesis (11). In the present study, we investigated the contribution of the Fc portion of 5C12 to its in vitro and in vivo toxin-neutralizing activity by evaluating the efficacies of the Fabs and F(ab′)₂ fragments of 5C12 in the HeLa cell and mouse toxicity assays. Smaller antibody fragments are advantageous for clinical use because of their lower immunogenicity and production costs. A comparison of a human monoclonal antibody against the B subunit of Stx2 (5H8) and its Fab fragment was performed to determine if similar results are obtained. We also investigated the contribution of the Fc functions by comparing the in vitro and in vivo neutralizing activities of the recombinant 5C12 isotype variants (e.g., IgG1, IgG2, IgG3, and IgG4).     

Hag Mediates Adherence of Moraxella catarrhalis to Ciliated Human Airway Cells

Moraxella catarrhalis is a human pathogen causing otitis media in infants and respiratory infections in adults, particularly patients with chronic obstructive pulmonary disease. The surface protein Hag (also designated MID) has previously been shown to be a key adherence factor for several epithelial cell lines relevant to pathogenesis by M. catarrhalis, including NCIH292 lung cells, middle ear cells, and A549 type II pneumocytes. In this study, we demonstrate that Hag mediates adherence to air-liquid interface cultures of normal human bronchial epithelium (NHBE) exhibiting mucociliary activity. Immunofluorescent staining and laser scanning confocal microscopy experiments demonstrated that the M. catarrhalis wild-type isolates O35E, O12E, TTA37, V1171, and McGHS1 bind principally to ciliated NHBE cells and that their corresponding hag mutant strains no longer associate with cilia. The hag gene product of M. catarrhalis isolate O35E was expressed in the heterologous genetic background of a nonadherent Haemophilus influenzae strain, and quantitative assays revealed that the adherence of these recombinant bacteria to NHBE cultures was increased 27-fold. These experiments conclusively demonstrate that the hag gene product is responsible for the previously unidentified tropism of M. catarrhalis for ciliated NHBE cells.Moraxella catarrhalis is a gram-negative pathogen of the middle ear and lower respiratory tract (29, 40, 51, 52, 69, 78). The organism is responsible for ∼15% of bacterial otitis media cases in children and up to 10% of infectious exacerbations in patients with chronic obstructive pulmonary disease (COPD). The cost of treating these ailments places a large financial burden on the health care system, adding up to well over $10 billion per annum in the United States alone (29, 40, 52, 95, 97). In recent years, M. catarrhalis has also been increasingly associated with infections such as bronchitis, conjunctivitis, sinusitis, bacteremia, pneumonia, meningitis, pericarditis, and endocarditis (3, 12, 13, 17-19, 24, 25, 27, 51, 67, 70, 72, 92, 99, 102-104). Therefore, the organism is emerging as an important health problem.M. catarrhalis infections are a matter of concern due to high carriage rates in children, the lack of a preventative vaccine, and the rapid emergence of antibiotic resistance in clinical isolates. Virtually all M. catarrhalis strains are resistant to β-lactams (34, 47, 48, 50, 53, 65, 81, 84). The genes specifying this resistance appear to be gram positive in origin (14, 15), suggesting that the organism could acquire genes conferring resistance to other antibiotics via horizontal transfer. Carriage rates as high as 81.6% have been reported for children (39, 104). In one study, Faden and colleagues analyzed the nasopharynx of 120 children over a 2-year period and showed that 77.5% of these patients became colonized by M. catarrhalis (35). These investigators also observed a direct relationship between the development of otitis media and the frequency of colonization. This high carriage rate, coupled with the emergence of antibiotic resistance, suggests that M. catarrhalis infections may become more prevalent and difficult to treat. This emphasizes the need to study pathogenesis by this bacterium in order to identify vaccine candidates and new targets for therapeutic approaches.One key aspect of pathogenesis by most infectious agents is adherence to mucosal surfaces, because it leads to colonization of the host (11, 16, 83, 93). Crucial to this process are surface proteins termed adhesins, which mediate the binding of microorganisms to human cells and are potential targets for vaccine development. M. catarrhalis has been shown to express several adhesins, namely UspA1 (20, 21, 59, 60, 77, 98), UspA2H (59, 75), Hag (also designated MID) (22, 23, 37, 42, 66), OMPCD (4, 41), McaP (61, 100), and a type 4 pilus (63, 64), as well as the filamentous hemagglutinin-like proteins MhaB1, MhaB2, MchA1, and MchA2 (7, 79). Each of these adhesins was characterized by demonstrating a decrease in the adherence of mutant strains to a variety of human-derived epithelial cell lines, including A549 type II pneumocytes and Chang conjunctival, NCIH292 lung mucoepidermoid, HEp2 laryngeal, and 16HBE14o-polarized bronchial cells. Although all of these cell types are relevant to the diseases caused by M. catarrhalis, they lack important aspects of the pathogen-targeted mucosa, such as the features of cilia and mucociliary activity. The ciliated cells of the respiratory tract and other mucosal membranes keep secretions moving out of the body so as to assist in preventing colonization by invading microbial pathogens (10, 26, 71, 91). Given this critical role in host defense, it is interesting to note that a few bacterial pathogens target ciliated cells for adherence, including Actinobacillus pleuropneumoniae (32), Pseudomonas aeruginosa (38, 108), Mycoplasma pneumoniae (58), Mycoplasma hyopneumoniae (44, 45), and Bordetella species (5, 62, 85, 101).In the present study, M. catarrhalis is shown to specifically bind to ciliated cells of a normal human bronchial epithelium (NHBE) culture exhibiting mucociliary activity. This tropism was found to be conserved among isolates, and analysis of mutants revealed a direct role for the adhesin Hag in binding to ciliated airway cells.     

Acanthamoeba culbertsoni Elicits Soluble Factors That Exert Anti-Microglial Cell Activity

Acanthamoeba culbertsoni is an opportunistic pathogen that causes granulomatous amoebic encephalitis (GAE), a chronic and often fatal disease of the central nervous system (CNS). A hallmark of GAE is the formation of granulomas around the amoebae. These cellular aggregates consist of microglia, macrophages, lymphocytes, and neutrophils, which produce a myriad of proinflammatory soluble factors. In the present study, it is demonstrated that A. culbertsoni secretes serine peptidases that degrade chemokines and cytokines produced by a mouse microglial cell line (BV-2 cells). Furthermore, soluble factors present in cocultures of A. culbertsoni and BV-2 cells, as well as in cocultures of A. culbertsoni and primary neonatal rat cerebral cortex microglia, induced apoptosis of these macrophage-like cells. Collectively, the results indicate that A. culbertsoni can apply a multiplicity of cell contact-independent modes to target macrophage-like cells that exert antiamoeba activities in the CNS.Acanthamoeba culbertsoni belongs to a group of free-living amoebae, such as Balamuthia mandrillaris, Naegleria fowleri, and Sappinia pedata, that can cause disease in humans (46, 56). Acanthamoeba spp. are found worldwide and have been isolated from a variety of environmental sources, including air, soil, dust, tap water, freshwater, seawater, swimming pools, air conditioning units, and contaminated contact lenses (30). Trophozoites feed on bacteria and algae and represent the infective form (47, 56). However, under unfavorable environmental conditions, such as extreme changes in temperature or pH, trophozoites transform into a double-walled, round cyst (22, 45).Acanthamoeba spp. cause an infection of the eye known as amoebic keratitis (AK), an infection of the skin referred to as cutaneous acanthamoebiasis, and a chronic and slowly progressing disease of the central nervous system (CNS) known as granulomatous amoebic encephalitis (GAE) (22, 23, 30, 56). GAE is most prevalent in humans who are immunocompromised (30, 33, 40) and has been reported to occur among individuals infected with the human immunodeficiency virus (HIV) (28). It has been proposed that Acanthamoeba trophozoites access the CNS by passage through the olfactory neuroepithelium (32) or by hematogenous spread from a primary nonneuronal site of infection (23, 24, 33, 53).In immune-competent individuals, GAE is characterized by the formation of granulomas. These cellular aggregates consist of microglia, macrophages, polymorphonuclear cells, T lymphocytes, and B lymphocytes (24, 30). The concerted action of these immune cells results in sequestration of amoebae and is instrumental in slowing the progression of GAE. This outcome is consistent with the observation that granulomas are rarely observed in immunocompromised individuals (34) and in mice with experimentally induced immune suppression following treatment with the cannabinoid delta-9-tetrahydrocannabinol (Δ⁹-THC) (8).Microglia are a resident population of macrophages in the CNS. These cells, along with CNS-invading peripheral macrophages, appear to play a critical early effector role in the control of Acanthamoeba spread during GAE (4, 5, 29, 31). In vitro, microglia have been shown to produce an array of chemokines and cytokines in response to Acanthamoeba (31, 51). However, these factors appear not to have a deleterious effect on these amoebae (29).Acanthamoeba spp. produce serine peptidases, cysteine peptidases, and metallopeptidases (1, 2, 9, 10, 14, 16, 18, 19, 21, 25, 26, 37, 38, 41, 42, 52). In the present study, it is demonstrated that serine peptidases secreted by A. culbertsoni degrade chemokines and cytokines that are produced by immortalized mouse BV-2 microglia-like cells. In addition, soluble factors present in cocultures of A. culbertsoni and BV-2 cells induced apoptosis of the BV-2 cells. Collectively, these results suggest a mode through which A. culbertsoni can evade immune responsiveness in the CNS.     

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.