Protection of Mice against Shiga Toxin 2 (Stx2)-Associated Damage by Maternal Immunization with a Brucella Lumazine Synthase-Stx2 B Subunit Chimera

Hemolytic-uremic syndrome (HUS) is defined as the triad of anemia, thrombocytopenia, and acute kidney injury. Enterohemorrhagic Shiga toxin (Stx)-producing Escherichia coli (EHEC), which causes a prodromal hemorrhagic enteritis, remains the most common etiology of the typical or epidemic form of HUS. Because no licensed vaccine or effective therapy is presently available for human use, we recently developed a novel immunogen based on the B subunit of Shiga toxin 2 (Stx2B) and the enzyme lumazine synthase from Brucella spp. (BLS) (BLS-Stx2B). The aim of this study was to analyze maternal immunization with BLS-Stx2B as a possible approach for transferring anti-Stx2 protection to the offspring. BALB/c female mice were immunized with BLS-Stx2B before mating. Both dams and pups presented comparable titers of anti-Stx2B antibodies in sera and fecal extracts. Moreover, pups were totally protected against a lethal dose of systemic Stx2 injection up to 2 to 3 months postpartum. In addition, pups were resistant to an oral challenge with an Stx2-producing EHEC strain at weaning and did not develop any symptomatology associated with Stx2 toxicity. Fostering experiments demonstrated that anti-Stx2B neutralizing IgG antibodies were transmitted through breast-feeding. Pups that survived the EHEC infection due to maternally transferred immunity prolonged an active and specific immune response that protected them against a subsequent challenge with intravenous Stx2. Our study shows that maternal immunization with BLS-Stx2B was very effective at promoting the transfer of specific antibodies, and suggests that preexposure of adult females to this immunogen could protect their offspring during the early phase of life.     

Protection against Shiga toxin-producing Escherichia coli infection by transcutaneous immunization with Shiga toxin subunit B

Enterohemorrhagic Escherichia coli (EHEC) strains are important human food-borne pathogens. EHEC strains elaborate potent Shiga toxins (Stx1, and/or Stx2) implicated in the development of hemorrhagic colitis (HC) or hemolytic-uremic syndrome (HUS). In this report, we evaluated the immunogenicity and protective efficacy of Stx1 subunit B (StxB1) administered by transcutaneous immunization (TCI). Three groups of Dutch Belted rabbits received patches containing StxB1, StxB1 in combination with Escherichia coli heat-labile enterotoxin (LT), or LT alone. An additional group of naïve rabbits served as controls. The protective efficacy following TCI with StxB1 was assessed by challenging rabbits with a virulent Stx1-producing strain, RDEC-H19A, capable of inducing HC and HUS in rabbits. Antibodies specific to StxB1 from serum and bile samples were determined by enzyme-linked immunosorbent assay and toxin neutralization test. Rabbits immunized with StxB1 demonstrated improved weight gain and reduced Stx-induced histopathology. Rabbits receiving StxB or StxB1/LT showed a significant increase in serum immunoglobulin G titers specific to StxB1 as well as toxin neutralization titers. These data demonstrated that the StxB delivered by TCI could induce significant systemic immune responses. Thus, Stx subunit B vaccine delivered by a patch for a high-risk population may be a practical approach to prevent (and/or reduce) Stx-induced pathology.     

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.     

Oral Intoxication of Mice with Shiga Toxin Type 2a (Stx2a) and Protection by Anti-Stx2a Monoclonal Antibody 11E10

Shiga toxin (Stx)-producing Escherichia coli (STEC) strains cause food-borne outbreaks of hemorrhagic colitis and, less commonly, a serious kidney-damaging sequela called the hemolytic uremic syndrome (HUS). Stx, the primary virulence factor expressed by STEC, is an AB₅ toxin with two antigenically distinct forms, Stx1a and Stx2a. Although both toxins have similar biological activities, Stx2a is more frequently produced by STEC strains that cause HUS than is Stx1a. Here we asked whether Stx1a and Stx2a act differently when delivered orally by gavage. We found that Stx2a had a 50% lethal dose (LD₅₀) of 2.9 μg, but no morbidity occurred after oral intoxication with up to 157 μg of Stx1a. We also compared several biochemical and histological parameters in mice intoxicated orally versus intraperitoneally with Stx2a. We discovered that both intoxication routes caused similar increases in serum creatinine and blood urea nitrogen, indicative of kidney damage, as well as electrolyte imbalances and weight loss in the animals. Furthermore, kidney sections from Stx2a-intoxicated mice revealed multifocal, acute tubular necrosis (ATN). Of particular note, we detected Stx2a in kidney sections from orally intoxicated mice in the same region as the epithelial cell type in which ATN was detected. Lastly, we showed reduced renal damage, as determined by renal biomarkers and histopathology, and full protection of orally intoxicated mice with monoclonal antibody (MAb) 11E10 directed against the toxin A subunit; conversely, an irrelevant MAb had no therapeutic effect. Orally intoxicated mice could be rescued by MAb 11E10 6 h but not 24 h after Stx2a delivery.     

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.     

Effects of Shiga Toxin Type 2 on a Bioengineered Three-Dimensional Model of Human Renal Tissue

Shiga toxins (Stx) are a family of cytotoxic proteins that can cause hemolytic-uremic syndrome (HUS), a thrombotic microangiopathy, following infections by Shiga toxin-producing Escherichia coli (STEC). Renal failure is a key feature of HUS and a major cause of childhood renal failure worldwide. There are currently no specific therapies for STEC-associated HUS, and the mechanism of Stx-induced renal injury is not well understood primarily due to a lack of fully representative animal models and an inability to monitor disease progression on a molecular or cellular level in humans at early stages. Three-dimensional (3D) tissue models have been shown to be more in vivo-like in their phenotype and physiology than 2D cultures for numerous disease models, including cancer and polycystic kidney disease. It is unknown whether exposure of a 3D renal tissue model to Stx will yield a more in vivo-like response than 2D cell culture. In this study, we characterized Stx2-mediated cytotoxicity in a bioengineered 3D human renal tissue model previously shown to be a predictor of drug-induced nephrotoxicity and compared its response to Stx2 exposure in 2D cell culture. Our results demonstrate that although many mechanistic aspects of cytotoxicity were similar between 3D and 2D, treatment of the 3D tissues with Stx resulted in an elevated secretion of the kidney injury marker 1 (Kim-1) and the cytokine interleukin-8 compared to the 2D cell cultures. This study represents the first application of 3D tissues for the study of Stx-mediated kidney injury.     

Syntheses and Immunologic Properties of Escherichia coli O157 O-Specific Polysaccharide and Shiga Toxin 1 B Subunit Conjugates in Mice

Escherichia coli O157 is the major cause of diarrhea-associated hemolytic uremic syndrome (HUS). Strains causing HUS contain either Shiga toxin 1 (Stx1) or Stx2, or both. In adult volunteers, conjugate vaccines of detoxified lipopolysaccharide (LPS) elicited bactericidal antibodies to E. coli O157. Here, the detoxified LPS was conjugated with improved schemes to the nontoxic B subunit of Stx1. Mice injected with these bivalent conjugates elicited both bactericidal antibodies to E. coli O157 and neutralization antibodies to Stx1.     

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.     

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.     

Human monocytes stimulated by Shiga toxin 1a via globotriaosylceramide release proinflammatory molecules associated with hemolytic uremic syndrome

The life-threatening sequela of hemorrhagic colitis induced by Shiga toxins (Stx)-producing Escherichia coli (STEC) infections in humans is hemolytic uremic syndrome (HUS), the main cause of acute renal failure in early childhood. The key step in the pathogenesis of HUS is the appearance of Stx in the blood of infected patients because these powerful virulence factors are capable of inducing severe microangiopathic lesions in the kidney. During precocious toxemia, which occurs in patients before the onset of HUS during the intestinal phase, Stx bind to several different circulating cells. An early response of these cells might include the release of proinflammatory mediators associated with the development of HUS. Here, we show that primary human monocytes stimulated with Shiga toxin 1a (Stx1a) through the glycolipid receptor globotriaosylceramide released larger amounts of proinflammatory molecules (IL-1β, TNFα, IL-6, G-CSF, CXCL8, CCL2, CCL4) than Stx1a-treated neutrophils. The mediators (except IL-1β) are among the top six proinflammatory mediators found in the sera from patients with HUS in different studies. The molecules appear to be involved in different pathogenetic steps of HUS, i.e. sensitization of renal endothelial cells to the toxin actions (IL-1β, TNFα), activation of circulating monocytes and neutrophils (CXCL8, CCL2, CCL4) and increase in neutrophil counts in patients with poor prognosis (G-CSF). Hence, a role of circulating monocytes in the very early phases of the pathogenetic process culminating with HUS can be envisaged. Impairment of the events of precocious toxemia would prevent or reduce the risk of HUS in STEC-infected children.     

In Vivo Transduction with Shiga Toxin 1-Encoding Phage

To facilitate the study of intestinal transmission of the Shiga toxin 1 (Stx1)-converting phage H-19B, Tn10d-bla mutagenesis of an Escherichia coli H-19B lysogen was undertaken. Two mutants containing insertions in the gene encoding the A subunit of Stx1 were isolated. The resultant ampicillin-resistant E. coli strains lysogenic for these phages produced infectious H-19B particles but not active toxin. These lysogens were capable of transducing an E. coli recipient strain in the murine gastrointestinal tract, thereby demonstrating that lysogens of Shiga toxin-converting phages give rise to infectious virions within the host gastrointestinal tract.     

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.     

Mouse model of hemolytic-uremic syndrome caused by endotoxin-free Shiga toxin 2 (Stx2) and protection from lethal outcome by anti-Stx2 antibody

Hemolytic-uremic syndrome (HUS) results from infection by Shiga toxin (Stx)-producing Escherichia coli and is the most common cause of acute renal failure in children. We have developed a mouse model of HUS by administering endotoxin-free Stx2 in multiple doses over 7 to 8 days. At sacrifice, moribund animals demonstrated signs of HUS: increased blood urea nitrogen and serum creatinine levels, proteinuria, deposition of fibrin(ogen), glomerular endothelial damage, hemolysis, leukocytopenia, and neutrophilia. Increased expression of proinflammatory chemokines and cytokines in the sera of Stx2-treated mice indicated a systemic inflammatory response. Currently, specific therapeutics for HUS are lacking, and therapy for patients is primarily supportive. Mice that received 11E10, a monoclonal anti-Stx2 antibody, 4 days after starting injections of Stx2 recovered fully, displaying normal renal function and normal levels of neutrophils and lymphocytes. In addition, these mice showed decreased fibrin(ogen) deposition and expression of proinflammatory mediators compared to those of Stx2-treated mice in the absence of antibody. These results indicate that, when performed during progression of HUS, passive immunization of mice with anti-Stx2 antibody prevented the lethal effects of Stx2.     

Distinct Physiologic and Inflammatory Responses Elicited in Baboons after Challenge with Shiga Toxin Type 1 or 2 from Enterohemorrhagic Escherichia coli

Shiga toxin-producing Escherichia coli is a principal source of regional outbreaks of bloody diarrhea and hemolytic-uremic syndrome in the United States and worldwide. Primary bacterial virulence factors are Shiga toxin types 1 and 2 (Stx1 and Stx2), and we performed parallel analyses of the pathophysiologies elicited by the toxins in nonhuman primate models to identify shared and unique consequences of the toxemias. After a single intravenous challenge with purified Stx1 or Stx2, baboons (Papio) developed thrombocytopenia, anemia, and acute renal failure with loss of glomerular function, in a dose-dependent manner. Differences in the timing and magnitude of physiologic responses were observed between the toxins. The animals were more sensitive to Stx2, with mortality at lower doses, but Stx2-induced renal injury and mortality were delayed 2 to 3 days compared to those after Stx1 challenge. Multiplex analyses of plasma inflammatory cytokines revealed similarities (macrophage chemoattractant protein 1 [MCP-1] and tumor necrosis factor alpha [TNF-α]) and differences (interleukin-6 [IL-6] and granulocyte colony-stimulating factor [G-CSF]) elicited by the toxins with respect to the mediator induced and timing of the responses. Neither toxin induced detectable levels of plasma TNF-α. To our knowledge, this is the first time that the in vivo consequences of the toxins have been compared in a parallel and reproducible manner in nonhuman primates, and the data show similarities to patient observations. The availability of experimental nonhuman primate models for Stx toxemias provides a reproducible platform for testing antitoxin compounds and immunotherapeutics with outcome criteria that have clinical meaning.Infection with Shiga toxin-producing Escherichia coli (STEC) results in intestinal cramps and bloody diarrhea, followed 5 to 12 days later in some patients by the development of hemolytic-uremic syndrome (HUS) (16, 18). HUS is characterized clinically by the triad of thrombocytopenia, hemolytic microangiopathy, and renal injury and is the leading cause of acute renal failure in otherwise healthy children in the United States. An antibiotic regimen is not recommended, and treatment options are limited to critical care support (47). Patients with diarrhea-associated HUS can have long-term renal impairment of varying severity, and approximately one-fourth of patients have neurologic sequelae, including seizures, coma/stupor, cortical blindness, ataxia, and paraplegia (10, 14).The natural infection route is gastrointestinal, via contaminated food or water. The bacteria colonize the intestinal lumen, with most strains forming characteristic attaching-and-effacing lesions, and the organisms may synthesize and release one or more toxins that are primary virulence factors contributing to the clinical manifestations of HUS (19). The toxins are AB₅ holotoxins, referred to as Shiga toxins due to their functional and structural similarities to Shiga toxin expressed by Shigella dysenteriae serotype 1 (4). Shiga toxin type 1 (Stx1) is essentially identical to the Shigella toxin (4), differing by one amino acid, but shares only 58% amino acid identity with Shiga toxin type 2 (Stx2). Stx1 and Stx2 have distinct spatial conformations (8) and dissociation rates from receptor-lipid surfaces (24). STEC strains may secrete one or both toxins and several toxin variants, and clinical studies have demonstrated that HUS is most often associated with the expression of Stx2 (3), particularly following infection with E. coli O157:H7 strains (12, 20). All Shiga toxins share a cellular intoxication mechanism in which B subunits oligomerize into pentamers for interaction with a cell surface globotriaosylceramide Gb₃ (CD77) receptor. Following binding, holotoxins are internalized via clathrin-dependent or clathrin-independent mechanisms and undergo retrograde transport through the trans-Golgi network and Golgi apparatus to reach the endoplasmic reticulum (33, 46). During transport, the A subunit undergoes limited proteolysis, and once in the endoplasmic reticulum, a fragment of the A subunit translocates into the cytoplasm, where its N-glycosidase activity inactivates the 28S rRNA component of eukaryotic ribosomes to inhibit protein synthesis and cause cell death (25, 43).While Stx1 and Stx2 share many characteristics, they are not identical and there is evidence that toxin-specific activities may be clinically relevant. Both toxins are internalized after binding to Gb₃, but the mechanisms of their intracellular trafficking through polarized intestinal epithelial cells to reach the intestinal endothelium are very different (15). Also, endothelial sensitivities to Stx1 and Stx2 differ depending on the vascular bed, with intestinal endothelium being more sensitive to the Shiga toxins than saphenous vein endothelium (12), and glomerular endothelial cells are about 1,000 times more sensitive to Stx2 than human umbilical vein endothelial cells (17). The mechanisms for these differences are not completely understood but may be related to receptor density, toxin effects on endoplasmic reticulum stress responses and apoptosis (22, 41), or local availability of sensitizing cytokines (5, 7, 11).Most animal models show greater sensitivity to Stx2, including murine, rabbit, and gnotobiotic piglet models, although renal and neurologic micropathologies differ from those in humans and between animal species (6, 9, 45). Earlier studies with the baboon (Papio) model showed that a bolus infusion of purified Stx1 induced intestinal injury, kidney glomerular injury, microangiopathic anemia, thrombocytopenia, and neurologic abnormalities similar to those in humans, suggesting that the baboon represents a promising preclinical animal model (44). A systemic inflammatory response was minimal after Stx1 challenge, but urinary tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) levels were consistent with local kidney inflammatory responses. Baboons were also more sensitive to Stx2 (38), but a direct comparison of the pathophysiologies elicited by the two toxins was difficult because of differing experimental designs. We sought to expand these earlier studies of baboons to identify similarities and differences elicited by Stx1 and Stx2 under reproducible experimental conditions. Given the clinical relevance of Stx2 production during STEC infection in patients, we were particularly interested in responses after Stx2 challenge, for which few data are available from the baboon model. We present the metabolic, physiologic, and inflammatory responses in baboons after intravenous challenge with Stx1 or Stx2. The observed differences in pathophysiology elicited by the two toxins may contribute to a better understanding of the differences in clinical manifestations produced by the toxins.     

Geographically Distinct Escherichia coli O157 Isolates Differ by Lineage,Shiga Toxin Genotype,and Total Shiga Toxin Production

While the differential association of Escherichia coli O157 genotypes with animal and human hosts has recently been well documented, little is known about their distribution between countries and how this might affect regional disease rates. Here, we used a 48-plex single nucleotide polymorphism (SNP) assay to segregate 148 E. coli O157 isolates from Australia, Argentina, and the United States into 11 SNP lineages. We also investigated the relationship between SNP lineages, Shiga toxin (Stx) gene profiles, and total Stx production. E. coli O157 isolates clearly segregated into SNP lineages that were differentially associated with each country. Of the 11 SNP lineages, seven were detected among isolates from a single country, two were detected among isolates from all three countries, and another two were detected only among U.S. and Argentinean isolates. A number of Australian (30%) and Argentinean (14%) isolates were associated with novel, previously undescribed SNP lineages that were unique to each country. Isolates within SNP lineages that were strongly associated with the carriage of stx_2a produced comparatively more Stx on average than did those lacking the stx_2a subtype. Furthermore, the proportion of isolates in stx_2a-associated SNP lineages was significantly higher in Argentina and the United States than Australia (P < 0.05). This study provides evidence for the geographic divergence of E. coli O157 and for a prominent role of stx_2a in total Stx production. These results also highlight the need for more comprehensive studies of the global distribution of E. coli O157 lineages and the impacts of regionally predominant E. coli O157 lineages on the prevalence and severity of disease.     

Enhanced Virulence of the Escherichia coli O157:H7 Spinach-Associated Outbreak Strain in Two Animal Models Is Associated with Higher Levels of Stx2 Production after Induction with Ciprofloxacin

Shiga toxin (Stx)-producing Escherichia coli (STEC) causes hemorrhagic colitis and the hemolytic-uremic syndrome (HUS). STEC strains may produce Stx1a and/or Stx2a or variants of either toxin. A 2006 spinach-associated outbreak of STEC O157:H7 resulted in higher hospitalization and HUS rates than previous STEC outbreaks. The spinach isolate, strain K3995, contains both stx_2a and stx_2c. We hypothesized that the enhanced virulence of K3995 reflects the combination of stx₂ alleles (carried on lysogenic phages) and/or the amount of Stx2 made by that strain. We compared the virulence of K3995 to those of other O157:H7 isolates and an isogenic Stx2 mutant in rabbits and mice. We also measured the relative levels of Stx2 produced from those strains with or without induction of the stx-carrying phage. Some rabbits infected with K3995 exhibited intestinal pathology and succumbed to infection, while none of those infected with O157:H7 strain 2812 (Stx1a⁺ Stx2a⁺) died or showed pathological signs. Rabbits infected with the isogenic Stx2a mutant K3995 stx_2a::cat were not colonized as well as those infected with K3995 and exhibited no signs of disease. In the streptomycin-treated mouse model, more animals infected with K3995 died than did those infected with O157:H7 strain 86-24 (Stx2a⁺). Additionally, K3995 produced higher levels of total Stx2 and toxin phage DNA in cultures after phage induction than did 86-24. Our results demonstrate the greater virulence of K3995 compared to other O157:H7 strains in rabbits and mice. We conclude that this enhanced virulence is linked to higher levels of Stx2 expression as a consequence of increased phage induction.     

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.     

Stx1-A亚单位的原核表达、复性及抗原性鉴定

目的构建志贺毒素1-A亚单位(Stx1-A)原核表达质粒,使其在大肠埃希菌中表达,对表达的重组蛋白进行纯化,并探讨其抗原性及应用价值。方法以大肠埃希菌O157DNA为模板扩增Stx1-A基因,重组克隆入原核表达载体pET32a( ),转化大肠埃希菌BL21(DE3),异丙基硫代-半乳糖苷(IPTG)诱导表达,包涵体经镍柱柱上复性和纯化,并对纯化产物进行Western印迹鉴定。结果重组质粒经双酶切鉴定和测序分析后证实构建成功。重组蛋白经十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)显示,其表达量占细菌总蛋白量的15%左右,主要以包涵体形式存在,通过镍柱柱上复性获得纯化的可溶性Stx1-A蛋白,纯度达95%以上,Western印迹检测显示特异性条带。结论成功构建了pET32a( )/Stx1-A重组质粒,并获得了具有抗原特异性的可溶性Stx1-A蛋白,为进一步制备抗Stx1-A抗体和研究其生物学功能奠定了基础。