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.     

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.     

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.     

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.     

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.     

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.     

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.     

Rapid and Sensitive Detection of Shiga Toxin-Producing Escherichia coli from Nonenriched Stool Specimens by Real-Time PCR in Comparison to Enzyme Immunoassay and Culture

Shiga toxin (Stx)-producing Escherichia coli (STEC) bacteria are a frequent cause of food-borne gastroenteritis, hemorrhagic colitis, and hemolytic uremic syndrome. Because antimicrobial agents are generally contraindicated in patients infected with STEC, a sensitive and specific diagnostic test with rapid turnaround is essential. Current culture methods may fail to detect non-O157 STEC. We evaluated a Stx gene real-time PCR assay using hybridization probes and the LightCycler instrument with 204 prospectively collected stool specimens, which were also tested for Stx by enzyme immunoassay (EIA) (ProSpecT STEC; Remel, Lenexa, KS) and by culturing on chromogenic agar (Chromagar O157; BD BBL, Sparks, MD). In addition, 85 archived stool specimens previously tested for Stx (by EIA) and/or E. coli O157:H7 (by culture) were tested by PCR. Sample preparation for PCR included mixing the stool in sterile water and extraction of nucleic acid using the MagNA Pure LC instrument (Roche Diagnostics). The PCR assay had 100% sensitivity and specificity compared to EIA and culture for specimens collected prospectively (4 of 204 specimens were positive) and compared to culture and/or EIA for archival specimens (42 of 85 specimens were positive). Both the EIA and PCR produced positive results from a specimen containing an O103 serotype STEC in the prospective specimens, and the PCR test detected three positive specimens that contained nonviable STEC in the archived specimens. The PCR assay demonstrated 100% sensitivity and specificity compared to EIA and/or culture and more rapid turnaround than either EIA or culture.Shiga toxin (Stx)-producing Escherichia coli (STEC) is a frequent cause of food-borne outbreaks of diarrhea (15). Disease caused by STEC is characterized by abdominal pain and bloody diarrhea, and 5 to 15% of those individuals infected with serotype O157:H7 develop hemolytic uremic syndrome (HUS), a potentially life-threatening condition consisting of hemolytic anemia, thrombocytopenia, and kidney failure caused primarily by Stx (8). STEC may carry genes for one or both types of Stx, Stx1 and Stx2 (17).Although STEC strains are a diverse group of pathogens, up to the present, the most common serotype in the United States has been O157:H7. A common association is that of E. coli O157:H7 contaminating ground beef (3, 7), but recent large outbreaks have involved a variety of other foods, including leafy greens (6, 29). The diversity of potentially contaminated food means that patients may acquire STEC infection from many foodstuffs, far beyond the stereotypical risk of undercooked ground beef. The common denominator of tainted food products seems to be direct or indirect contamination from bovine feces. To best detect infected patients and potential outbreaks, clinical laboratories must have tools to quickly and accurately detect STEC in stool specimens. Culture on sorbitol MacConkey agar is an inexpensive, effective, and widely used method based on lack of sorbitol fermentation by E. coli O157:H7. Several drawbacks limit the utility of culture, including slow turnaround, false-negative results in antibiotic-treated patients, and false-negative results due to emerging serotypes of non-O157 STEC that ferment sorbitol (1, 14, 16, 29). Alternatively, a method that is increasingly utilized is detection of Stx antigen from stool, either directly or after broth enrichment. Our experience concurs with enzyme immunoassay (EIA) product insert data that optimal sensitivity and specificity are achieved only when a broth enrichment step is employed; this results in slow turnaround.Here, we describe a real-time PCR assay that can detect STEC using nucleic acid extracts of stool specimens. We evaluated the performance of this assay using both archived stool specimens and prospectively collected specimens and compared the results to those of culture and Stx antigen detection.(This study was presented in part at the 48th Annual Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, 25 to 28 October 2008.)     

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.     

OI-57, a Genomic Island of Escherichia coli O157, Is Present in Other Seropathotypes of Shiga Toxin-Producing E. coli Associated with Severe Human Disease

Strains of Shiga toxin-producing Escherichia coli (STEC) are a heterogeneous E. coli group that may cause severe disease in humans. STEC have been categorized into seropathotypes (SPTs) based on their phenotypic and molecular characteristics and the clinical features of the associated diseases. SPTs range from A to E, according to a decreasing rank of pathogenicity. To define the virulence gene asset (“virulome”) characterizing the highly pathogenic SPTs, we used microarray hybridization to compare the whole genomes of STEC belonging to SPTs B, C, and D with that of STEC O157 (SPT A). The presence of the open reading frames (ORFs) associated with SPTs A and B was subsequently investigated by PCR in a larger panel of STEC and in other E. coli strains. A genomic island termed OI-57 was present in SPTs A and B but not in the other SPTs. OI-57 harbors the putative virulence gene adfO, encoding a factor enhancing the adhesivity of STEC O157, and ckf, encoding a putative killing factor for the bacterial cell. PCR analyses showed that OI-57 was present in its entirety in the majority of the STEC genomes examined, indicating that it represents a stable acquisition of the positive clonal lineages. OI-57 was also present in a high proportion of the human enteropathogenic E. coli genomes assayed, suggesting that it could be involved in the attaching-and-effacing colonization of the intestinal mucosa. In conclusion, OI-57 appears to be part of the virulome of pathogenic STEC and further studies are needed to elucidate its role in the pathogenesis of STEC infections.Shiga toxin (Stx)-producing Escherichia coli (STEC) strains cause severe human diseases such as hemorrhagic colitis and the life-threatening hemolytic uremic syndrome (HUS) (20, 32). STEC strains are zoonotic pathogens, and ruminants, particularly cattle, are recognized as their main natural reservoir (3). STEC strains may belong to a large number of serotypes, but only some of them have been firmly associated with severe human infections (20, 36). E. coli O157:H7 causes most of the cases of severe disease worldwide, but infections sustained by STEC strains belonging to serogroups other than O157, such as O26, O111, O103, O145 and O121, have been increasingly reported (2, 3, 4, 26, 33, 36, 39).The pathogenicity of STEC mainly relies on the presence of virulence genes located on mobile genetic elements (MGEs). These include the lambdoid bacteriophages transducing the Stx-coding genes (22) and the large virulence plasmid carrying the genes governing the production of enterohemolysin (28) and other putative virulence factors. In STEC O157, this plasmid also contains toxB, a gene that encodes a protein likely involved in the colonization of the gut mucosa (30, 34). Other important virulence properties are carried by pathogenicity islands (PAIs) such as the “locus of enterocyte effacement” (LEE) (16), which governs the capability of STEC to colonize the intestinal mucosa of the host with a peculiar mechanism known as attaching-and-effacing (A/E) (16, 20). Another PAI, termed O island 122 (OI-122) in the sequence of STEC O157 strain EDL933 (24), has been frequently found in STEC strains usually associated with severe human disease (9, 11, 19). This PAI carries the large virulence gene efa1-lifA, which appears to be involved in the colonization of the intestinal mucosa and in the inhibition of the host immune response (10, 21, 31). Beside these MGEs, many horizontally acquired DNA regions carrying genes encoding putative virulence factors or proteins of unknown function have been described in the genome of the STEC strains fully or partially sequenced so far. More than 170 of these regions have been annotated as genomic islands (GIs) in the sequences of the STEC O157 strains EDL933 and RIMD0509952/VT2 Sakai (7, 25).Due to the high genomic plasticity of E. coli, MGEs can be easily exchanged among strains or lost, generating STEC clones with different genetic assets and, as a consequence, different degrees of virulence. Karmali et al. proposed to group STEC strains into five seropathotypes (SPTs), from A through E, according to their reported frequencies in human illness, their known association with outbreaks and severe disease, and the presence of MGEs such as the LEE and the OI-122 PAI (9). SPT A includes STEC strains of serotypes O157:H7 and O157:NM, associated with both outbreaks and sporadic cases of HUS. SPT B includes STEC strains that are capable of causing HUS and outbreaks but less commonly than STEC O157, such as those belonging to serotypes O26:H11, O103:H2, O111:NM, O121:H19, and O145:NM, whereas SPT C includes strains belonging to serotypes O91:H21 and O113:H21, which lack the LEE and have sometimes been associated with HUS (9). STEC serotypes associated with diarrhea but not with HUS, and STEC strains circulating only in animal reservoirs, have been included in SPTs D and E, respectively (9). This classification scheme takes into consideration the clinical aspects of the disease as well as the molecular features reported to play a role in STEC pathogenicity. However, the molecular bases of STEC infections have not been completely understood and the whole genetic asset conferring full virulence to SPTs A and B has not yet been completely identified.In an attempt to clarify this issue, we compared the entire genome of STEC O157 with those of STEC strains belonging to SPTs B, C, and D. This approach allowed us to identify a putative PAI (termed OI-57) in the sequence of the STEC O157 EDL933 strain (25) that was consistently present in STEC strains belonging to SPTs A and B but not in the less pathogenic SPTs C and D. The association of OI-57 with SPTs A and B was confirmed by examining a panel of STEC strains that included isolates belonging to SPTs from type A to type E as well as nonpathogenic E. coli strains.     

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.     

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.     

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.     

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.     

Sab,a Novel Autotransporter of Locus of Enterocyte Effacement-Negative Shiga-Toxigenic Escherichia coli O113:H21, Contributes to Adherence and Biofilm Formation

Shiga-toxigenic Escherichia coli (STEC) strains cause serious gastrointestinal disease, which can lead to potentially life-threatening systemic complications such as hemolytic-uremic syndrome. Although the production of Shiga toxin has been considered to be the main virulence trait of STEC for many years, the capacity to colonize the host intestinal epithelium is a crucial step in pathogenesis. In this study, we have characterized a novel megaplasmid-encoded outer membrane protein in locus of enterocyte effacement (LEE)-negative O113:H21 STEC strain 98NK2, termed Sab (for STEC autotransporter [AT] contributing to biofilm formation). The 4,296-bp sab gene encodes a 1,431-amino-acid protein with the features of members of the AT protein family. When expressed in E. coli JM109, Sab contributed to the diffuse adherence to human epithelial (HEp-2) cells and promoted biofilm formation on polystyrene surfaces. A 98NK2 sab deletion mutant was also defective in biofilm formation relative to its otherwise isogenic wild-type parent, and this was complemented by transformation with a sab-carrying plasmid. Interestingly, an unrelated O113:H21 STEC isolate that had a naturally occurring deletion in sab was similarly defective in biofilm formation. PCR analysis indicated that sab is present in LEE-negative STEC strains belonging to serotypes/groups O113:H21, O23, and O82:H8. These findings raise the possibility that Sab may contribute to colonization in a subset of LEE-negative STEC strains.Shiga-toxigenic Escherichia coli (STEC) strains are prominent food-borne pathogens that cause watery or bloody diarrhea and hemorrhagic colitis, which can progress to the life-threatening hemolytic-uremic syndrome (HUS) (15, 21, 29). In order to establish and maintain an infection, STEC strains are equipped with a diverse array of virulence factors. Among these factors, Shiga toxin has been considered to be a sine qua non of virulence, as reviewed previously (21, 29). However, attachment of STEC to the human intestinal mucosa is a critical first step in pathogenesis. Many STEC strains, including those of the highly prevalent O157:H7 serotype, carry the locus of enterocyte effacement (LEE) pathogenicity island, which encodes the capacity to produce attaching and effacing (A/E) lesions on the intestinal epithelium, similarly to those produced by enteropathogenic E. coli strains (11, 35). These STEC strains are often referred to as enterohemorrhagic E. coli (EHEC), although this classification is ill defined. A/E lesions are characterized by ultrastructural changes including the remodeling of the host cell cytoskeleton and intimate attachment of the bacteria to the cell surface (11, 35). The process of the generation of A/E lesions involves the expression of the eae gene, which encodes intimin, an outer membrane surface adhesin, and the delivery of the intimin receptor Tir and several other effector proteins into host cells via the LEE-encoded type III secretion apparatus (reviewed in references 5 and 11).However, many STEC isolates from cases of severe disease, including HUS, lack the LEE locus yet are clearly capable of efficient colonization of the human gut (28, 29). Several candidate adhesins have been identified in these strains, including the megaplasmid-encoded autoagglutinating adhesin Saa (26), the long polar fimbriae encoded by the lpf operon (10) (two distinct homologues of which are also present in STEC O157:H7 strains [39, 40]), and the immunoglobulin-binding protein EibG, which contributes to a chain-like adherence phenotype on HEp-2 cells (18). Tarr et al. (38) also previously identified Iha, a homologue of Vibrio cholerae IrgA, which promotes the adherence of STEC O157:H7 to HeLa cells and is widely distributed in LEE-positive and LEE-negative strains. STEC O157:H7 strains also produce a type IV pilus, HCP (47), and an E. coli common pilus, ECP (30), both of which contribute to in vitro adherence to intestinal epithelial cells. Additional putative adhesins from LEE-positive STEC strains include Efa1, which mediates the attachment of O111:NM STEC strains to Chinese hamster ovary cells (23). In addition, Torres et al. (41) previously identified a calcium-binding and heat-extractable AT protein of EHEC, termed Cah, which mediates aggregation and participates in biofilm formation. Recently, Wells et al. (45) also characterized the EHEC-encoded AT protein EhaA, which contributes to adherence to primary bovine epithelial cells (but not HeLa cells) and promotes biofilm formation as well.The AT proteins referred to above belong to a rapidly growing family of gram-negative surface proteins that are exported across the periplasmic space and either attached to the external face of the outer membrane or released by proteolysis into the environment (13). These large proteins share a characteristic structure comprising three distinct domains, namely, an N-terminal signal peptide, a divergent functional passenger domain (α-domain), and a conserved C-terminal domain which forms a β-barrel pore in the outer membrane (13, 46). This unique protein structure provides all the information required for transport to the cell surface, with the N-terminal sequence directing the protein to the periplasm via the sec pathway and the C-terminal domain mediating the translocation of the passenger domain to the external surface (14). The various α-domains confer a broad range of functions and/or phenotypes including aggregation, biofilm formation, adherence, invasion, serum resistance, and protease or esterase activity (7, 12).Research in our laboratory has focused on the identification of novel virulence factors of LEE-negative STEC strains associated with human disease. In this study, we describe the identification and characterization of a member of the AT family produced by hypervirulent LEE-negative O113:H21 STEC strain 98NK2, which confers adherence to human epithelial cells and mediates biofilm formation.     

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.     

Protection of Sheep against Rift Valley Fever Virus and Sheep Poxvirus with a Recombinant Capripoxvirus Vaccine

Rift Valley fever (RVF) is an epizootic viral disease of sheep that can be transmitted from sheep to humans, particularly by contact with aborted fetuses. A capripoxvirus (CPV) recombinant virus (rKS1/RVFV) was developed, which expressed the Rift Valley fever virus (RVFV) Gn and Gc glycoproteins. These expressed glycoproteins had the correct size and reacted with monoclonal antibodies (MAb) to native glycoproteins. Mice vaccinated with rKS1/RVFV were protected against RVFV challenge. Sheep vaccinated with rKS1/RVFV twice developed neutralizing antibodies and were significantly protected against RVFV and sheep poxvirus challenge. These findings further document the value of CPV recombinants as ruminant vaccine vectors and support the inclusion of RVFV genes encoding glycoproteins in multivalent recombinant vaccines to be used where RVF occurs.Rift Valley fever (RFV) virus (RVFV) is a mosquito-borne member of the genus Phlebovirus, family Bunyaviridae. It is widely distributed in Africa, causing endemic and epidemic disease in both humans and livestock, including sheep, cattle, and goats. RVF was first described in Kenya and was shown to be caused by a filterable virus transmissible via blood (9). Acute RVF in lambs is characterized by fever and death within 24 to 48 h of being detected (43). Signs in adult sheep include fever, mucopurulent nasal discharge, hemorrhagic diarrhea, and abortion in pregnant ewes (43). RVFV can be transmitted from infected sheep to humans, particularly when humans are exposed to aborted sheep fetuses and blood.Attenuated live RVFV vaccines are available for use in livestock. A mutagen-attenuated RVFV vaccine induces protective immune responses in lambs and appears to be safe (25); however, other studies documented teratogenic effects on lambs from vaccinated pregnant ewes similar to those caused by the attenuated RVFV strain Smithburn (18). An inactivated RVFV vaccine induces neutralizing antibody responses in humans (33), and its use in sheep would not induce teratogenic effects or abortions. However, the inactivated vaccine requires 3 doses (33) and is expensive to produce. Efforts to make RVFV vaccines without these disadvantages include an attenuated RVFV developed by reverse genetics and lacking the NSs and NSm genes (4) and other new-generation RVFV vaccines (reviewed in reference 19) that protect mice against virus challenge (7, 16, 24, 27).The middle (M) RNA segment of the RVFV genome encodes the viral glycoproteins Gn and Gc (8, 20), and recombinant vaccinia virus expressing these glycoproteins induces neutralizing antibody and protective immunity to RVFV in mice (7). Vaccinia virus is safe for animals, but there is some risk to humans, as it was reported previously to spread from human vaccinees to contacts (28, 55) and to cause serious clinical disease in human immunodeficiency virus-infected patients (36). Although modified vaccinia virus Ankara is a safer alternative for humans (6, 57), there are animal poxviruses with naturally restricted host ranges for vaccine vectors in animals (1, 13, 30, 31, 40, 46, 47, 52, 53).For ruminants, the genus Capripoxvirus (CPV) of the family Poxviridae has been an effective recombinant vector to induce protective immunity against several other viruses (3, 17, 29, 32, 40, 41, 51). This genus has three closely related species causing sheep pox, goat pox, and lumpy skin disease (LSD) of cattle. A recombinant LSD vaccine expressing the Gn and Gc glycoproteins of RVFV induced protection against RVFV challenge in mice (52, 53) and sheep (52). The three species of CPV have 96 to 97% nucleotide identity (49) and are restricted to ruminants, with no evidence of human infections (10, 11). Furthermore, attenuated CPV vaccines are in use in Africa and the Middle East to control ruminant poxvirus disease (11, 21). The use of a CPV vector to deliver virus vaccines to ruminants also induces immunity to the CPV vector, thus increasing the valence of the vaccine (3, 17, 39, 40). We report here the construction of a recombinant CPV that expresses the RVFV Gn and Gc glycoproteins and induces protective immunity against RVFV and sheep poxvirus (SPV) challenge in sheep.