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

2013 ◽  
Vol 82 (3) ◽  
pp. 1213-1221 ◽  
Author(s):  
L. M. Russo ◽  
A. R. Melton-Celsa ◽  
M. A. Smith ◽  
M. J. Smith ◽  
A. D. O'Brien
Keyword(s):  
Monoclonal Antibody ◽  
Shiga Toxin ◽  
Biological Activities ◽  
Lethal Dose ◽  
Content Type ◽  
Tubular Necrosis ◽  
Food Borne ◽  
Uremic Syndrome ◽  
A Subunit ◽  
Full Protection

ABSTRACTShiga toxin (Stx)-producingEscherichia coli(STEC) strains cause food-borne outbreaks of hemorrhagic colitis and, less commonly, a serious kidney-damaging sequela called the hemolytic uremic syndrome (HUS). Stx, the primary virulence factor expressed by STEC, is an AB5toxin with two antigenically distinct forms, Stx1a and Stx2a. Although both toxins have similar biological activities, Stx2a is more frequently produced by STEC strains that cause HUS than is Stx1a. Here we asked whether Stx1a and Stx2a act differently when delivered orally by gavage. We found that Stx2a had a 50% lethal dose (LD50) of 2.9 μg, but no morbidity occurred after oral intoxication with up to 157 μg of Stx1a. We also compared several biochemical and histological parameters in mice intoxicated orally versus intraperitoneally with Stx2a. We discovered that both intoxication routes caused similar increases in serum creatinine and blood urea nitrogen, indicative of kidney damage, as well as electrolyte imbalances and weight loss in the animals. Furthermore, kidney sections from Stx2a-intoxicated mice revealed multifocal, acute tubular necrosis (ATN). Of particular note, we detected Stx2a in kidney sections from orally intoxicated mice in the same region as the epithelial cell type in which ATN was detected. Lastly, we showed reduced renal damage, as determined by renal biomarkers and histopathology, and full protection of orally intoxicated mice with monoclonal antibody (MAb) 11E10 directed against the toxin A subunit; conversely, an irrelevant MAb had no therapeutic effect. Orally intoxicated mice could be rescued by MAb 11E10 6 h but not 24 h after Stx2a delivery.

scholarly journals Protective Efficacy and Pharmacokinetics of Human/Mouse Chimeric Anti-Stx1 and Anti-Stx2 Antibodies in Mice

2015 ◽  
Vol 22 (4) ◽  
pp. 448-455 ◽  
Author(s):  
Angela R. Melton-Celsa ◽  
H. M. Carvalho ◽  
Claire Thuning-Roberson ◽  
A. D. O'Brien
Keyword(s):  
Monoclonal Antibodies ◽  
Shiga Toxin ◽  
Protective Efficacy ◽  
Lethal Dose ◽  
The United States ◽  
Hemorrhagic Colitis ◽  
Pharmacokinetic Studies ◽  
Bacterial Challenge ◽  
Content Type ◽  
Uremic Syndrome

ABSTRACTIn the United States, Shiga toxin (Stx)-producingEscherichia coli(STEC) is the most frequent infectious cause of hemorrhagic colitis. Hemolytic uremic syndrome (HUS) is a serious sequela that may develop after STEC infection that can lead to renal failure and death in up to 10% of cases. STEC can produce one or more types of Stx, Stx1 and/or Stx2, and Stx1 and Stx2 are responsible for HUS-mediated kidney damage. We previously generated two monoclonal antibodies (MAbs) that neutralize the toxicity of Stx1 or Stx2. In this study, we evaluated the protective efficacy of human/mouse chimeric versions of those monoclonal antibodies, named cαStx1 and cαStx2. Mice given an otherwise lethal dose of Stx1 were protected from death when injected with cαStx1 either 1 h before or 1 h after toxin injection. Additionally, streptomycin-treated mice fed the mouse-lethal STEC strain B2F1 that produces the Stx2 variant Stx2d were protected when given a dose of 0.1 mg of cαStx2/kg of body weight administered up to 72 h post-oral bacterial challenge. Since many STEC strains produce both Stx1 and Stx2 and since either toxin may lead to the HUS, we also assessed the protective efficacy of the combined MAbs. We found that both antibodies were required to protect mice from the presence of both Stx1 and Stx2. Pharmacokinetic studies indicated that cαStx1 and cαStx2 had serum half-lives (t1/2) of about 50 and 145 h, respectively. We propose that cαStx1 and cαStx2, both of which have been tested for safety in humans, could be used therapeutically for prevention or treatment early in the development of HUS.

scholarly journals AB5Preassembly Is Not Required for Shiga Toxin Activity

2016 ◽  
Vol 198 (11) ◽  
pp. 1621-1630 ◽  
Author(s):  
Christine A. Pellino ◽  
Sayali S. Karve ◽  
Suman Pradhan ◽  
Alison A. Weiss
Keyword(s):  
Escherichia Coli ◽  
Enzymatic Activity ◽  
Hemolytic Uremic Syndrome ◽  
Shiga Toxin ◽  
Target Cells ◽  
Content Type ◽  
B Subunit ◽  
Uremic Syndrome ◽  
A Subunit ◽  
B Subunits

ABSTRACTShiga toxin (Stx)-producingEscherichia coli(STEC) is a major cause of foodborne illness, including the life-threatening complication hemolytic-uremic syndrome. The German outbreak in 2011 resulted in nearly 4,000 cases of infection, with 54 deaths. Two forms of Stx, Stx1 and Stx2, differ in potency, and subtype Stx2a is most commonly associated with fatal human disease. Stx is considered to be an AB5toxin. The single A (enzymatically active) subunit inhibits protein synthesis by cleaving a catalytic adenine from the eukaryotic rRNA. The B (binding) subunit forms a homopentamer and mediates cellular association and toxin internalization by binding to the glycolipid globotriaosylceramide (Gb3). Both subunits are essential for toxicity. Here we report that unlike other AB5toxin family members, Stx is produced by STEC as unassembled A and B subunits. A preformed AB5complex is not required for cellular toxicity orin vivotoxicity to mice, and toxin assembly likely occurs at the cell membrane. We demonstrate that disruption of A- and B-subunit association by use of A-subunit peptides that lack enzymatic activity can protect mice from lethal doses of toxin. Currently, no treatments have been proven to be effective for hemolytic-uremic syndrome. Our studies demonstrate that agents that interfere with A- and B-subunit assembly may have therapeutic potential. Shiga toxin (Stx) produced by pathogenicEscherichia coliis considered to be an AB5heterohexamer; however, no known mechanisms ensure AB5assembly. Stx released byE. coliis not in the AB5conformation and assembles at the receptor interface. Thus, unassembled Stx can impart toxicity. This finding shows that preventing AB5assembly is a potential treatment for Stx-associated illnesses.IMPORTANCEComplications due to Shiga toxin are frequently fatal, and at present, supportive care is the only treatment option. Furthermore, antibiotic treatment is contraindicated due to the ability of antibiotics to amplify bacterial expression of Shiga toxin. We report, contrary to prevailing assumptions, that Shiga toxin produced by STEC circulates as unassembled A and B subunits at concentrations that are lethal to mice. Similar to the case for anthrax toxin, assembly occurs on receptors expressed on the surfaces of mammalian target cells. Disruption of Shiga toxin assembly by use of A-subunit peptides that lack enzymatic activity protects mice from lethal challenge with Shiga toxin, suggesting a new approach for development of therapeutics.

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

2014 ◽  
Vol 82 (4) ◽  
pp. 1491-1499 ◽  
Author(s):  
María Pilar Mejias ◽  
Gabriel Cabrera ◽  
Romina Jimena Fernández-Brando ◽  
Ariela Baschkier ◽  
Giselle Ghersi ◽  
...  
Keyword(s):  
Shiga Toxin ◽  
Lethal Dose ◽  
Kidney Injury ◽  
Content Type ◽  
B Subunit ◽  
Maternal Immunization ◽  
Lumazine Synthase ◽  
Shiga Toxin 2 ◽  
Common Etiology ◽  
Uremic Syndrome

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

scholarly journals Virulence of the Shiga Toxin Type 2-Expressing Escherichia coli O104:H4 German Outbreak Isolate in Two Animal Models

2013 ◽  
Vol 81 (5) ◽  
pp. 1562-1574 ◽  
Author(s):  
Tonia Zangari ◽  
Angela R. Melton-Celsa ◽  
Aruna Panda ◽  
Nadia Boisen ◽  
Mark A. Smith ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Animal Models ◽  
Shiga Toxin ◽  
Morbidity And Mortality ◽  
Content Type ◽  
Tubular Necrosis ◽  
Eaec Strain ◽  
Food Borne ◽  
Outbreak Isolate

ABSTRACTIn May 2011, a large food-borne outbreak was traced to an unusual O104:H4 enteroaggregativeEscherichia coli(EAEC) strain that produced Shiga toxin (Stx) type 2 (Stx2). We developed a mouse model to study the pathogenesis and treatment for this strain and examined the virulence of the isolate for Dutch belted rabbits. O104:H4 strain C227-11 was gavaged into C57BL/6 mice at 109to 1011CFU/animal. The infected animals were then given water with ampicillin (Amp; 5 g/liter)ad libitum. The C227-11-infected, Amp-treated C57BL/6 mice exhibited both morbidity and mortality. Kidneys from mice infected with C227-11 showed acute tubular necrosis, a finding seen in mice infected with typical Stx-producingE. coli. We provided anti-Stx2 antibody after infection and found that all of the antibody-treated mice gained more weight than untreated mice and, in another study, that all of the antibody-treated animals lived, whereas 3/8 phosphate-buffered saline-treated mice died. We further compared the pathogenesis of C227-11 with that of an Stx-negative (Stx−) O104:H4 isolate, C734-09, and an Stx2−phage-cured derivative of C227-11. Whereas C227-11-infected animals lost weight or gained less weight over the course of infection and died, mice infected with either of the Stx−isolates did not lose weight and only one mouse died. When the Stx-positive (Stx+) and Stx2−O104:H4 strains were compared in rabbits, greater morbidity and mortality were observed in rabbits infected with the Stx2+isolates than the Stx2−isolates. In conclusion, we describe two animal models for EAEC pathogenesis, and these studies show that Stx2 is responsible for most of the virulence observed in C227-11-infected mice and rabbits.

scholarly journals Protection against Lethal Leptospirosis after Vaccination with LipL32 Coupled or Coadministered with the B Subunit of Escherichia coli Heat-Labile Enterotoxin

2012 ◽  
Vol 19 (5) ◽  
pp. 740-745 ◽  
Author(s):  
André A. Grassmann ◽  
Samuel R. Félix ◽  
Carolina Ximendes dos Santos ◽  
Marta G. Amaral ◽  
Amilton C. P. Seixas Neto ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Immune Response ◽  
Subunit Vaccine ◽  
Lethal Dose ◽  
Control Group ◽  
Hamster Model ◽  
Content Type ◽  
B Subunit ◽  
Heat Labile ◽  
A Subunit

ABSTRACTLeptospirosis, a worldwide zoonosis, lacks an effective, safe, and cross-protective vaccine. LipL32, the most abundant, immunogenic, and conserved surface lipoprotein present in all pathogenic species ofLeptospira, is a promising antigen candidate for a recombinant vaccine. However, several studies have reported a lack of protection when this protein is used as a subunit vaccine. In an attempt to enhance the immune response, we used LipL32 coupled to or coadministered with the B subunit of theEscherichia coliheat-labile enterotoxin (LTB) in a hamster model of leptospirosis. After homologous challenge with 5× the 50% lethal dose (LD50) ofLeptospira interrogans, animals vaccinated with LipL32 coadministered with LTB and LTB::LipL32 had significantly higher survival rates (P< 0.05) than animals from the control group. This is the first report of a protective immune response afforded by a subunit vaccine using LipL32 and represents an important contribution toward the development of improved leptospirosis vaccines.

scholarly journals Affinity-Based Screening of Tetravalent Peptides Identifies Subtype-Selective Neutralizers of Shiga Toxin 2d, a Highly Virulent Subtype, by Targeting a Unique Amino Acid Involved in Its Receptor Recognition

2016 ◽  
Vol 84 (9) ◽  
pp. 2653-2661 ◽  
Author(s):  
Takaaki Mitsui ◽  
Miho Watanabe-Takahashi ◽  
Eiko Shimizu ◽  
Baihao Zhang ◽  
Satoru Funamoto ◽  
...  
Keyword(s):  
Amino Acid ◽  
Receptor Binding ◽  
Shiga Toxin ◽  
Cellulose Membrane ◽  
Content Type ◽  
B Subunit ◽  
Human Disorders ◽  
Uremic Syndrome ◽  
Subtype Selective ◽  
Highly Virulent

Shiga toxin (Stx), a major virulence factor of enterohemorrhagicEscherichia coli(EHEC), can be classified into two subgroups, Stx1 and Stx2, each consisting of various closely related subtypes. Stx2 subtypes Stx2a and Stx2d are highly virulent and linked with serious human disorders, such as acute encephalopathy and hemolytic-uremic syndrome. Through affinity-based screening of a tetravalent peptide library, we previously developed peptide neutralizers of Stx2a in which the structure was optimized to bind to the B-subunit pentamer. In this study, we identified Stx2d-selective neutralizers by targeting Asn16 of the B subunit, an amino acid unique to Stx2d that plays an essential role in receptor binding. We synthesized a series of tetravalent peptides on a cellulose membrane in which the core structure was exactly the same as that of peptides in the tetravalent library. A total of nine candidate motifs were selected to synthesize tetravalent forms of the peptides by screening two series of the tetravalent peptides. Five of the tetravalent peptides effectively inhibited the cytotoxicity of Stx2a and Stx2d, and notably, two of the peptides selectively inhibited Stx2d. These two tetravalent peptides bound to the Stx2d B subunit with high affinity dependent on Asn16. The mechanism of binding to the Stx2d B subunit differed from that of binding to Stx2a in that the peptides covered a relatively wide region of the receptor-binding surface. Thus, this highly optimized screening technique enables the development of subtype-selective neutralizers, which may lead to more sophisticated treatments of infections by Stx-producing EHEC.

scholarly journals Inhibition of Water Absorption and Selective Damage to Human Colonic Mucosa Are Induced by Subtilase Cytotoxin Produced by Escherichia coli O113:H21

2013 ◽  
Vol 81 (8) ◽  
pp. 2931-2937 ◽  
Author(s):  
Elizabeth Gerhardt ◽  
Mariana Masso ◽  
Adrienne W. Paton ◽  
James C. Paton ◽  
Elsa Zotta ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Water Absorption ◽  
Shiga Toxin ◽  
Human Colon ◽  
Surface Epithelium ◽  
Inflammatory Infiltration ◽  
Content Type ◽  
Uremic Syndrome ◽  
Normal Water ◽  
Subtilase Cytotoxin

ABSTRACTShiga toxin-producingEscherichia coliO157:H7 (STEC) is by far the most prevalent serotype associated with hemolytic uremic syndrome (HUS) although many non-O157 STEC strains have been also isolated from patients with HUS. The main virulence factor of STEC is the Shiga toxin type 2 (Stx2) present in O157 and non-O157 strains. Recently, another toxin, named subtilase cytotoxin (SubAB), has been isolated from several non-O157 strains and may contribute to the pathogenesis of HUS. Here, we have demonstrated that an O113:H21 STEC strain expressing SubAB and Stx2 inhibits normal water absorption across human colon and causes damage to the surface epithelium, necrosis, mononuclear inflammatory infiltration, edema, and marked mucin depletion. This damage was less marked, but nevertheless significant, when purified SubAB orE. coliO113:H21 expressing only SubAB was assayed. This is the first study showing that SubAB may directly participate in the mechanisms of diarrhea in children infected with non-O157 STEC strains.

scholarly journals Behavior of Different Shiga Toxin-Producing Escherichia coli Serotypes in Various Experimentally Contaminated Raw-Milk Cheeses

2012 ◽  
Vol 79 (1) ◽  
pp. 150-158 ◽  
Author(s):  
Stéphane D. Miszczycha ◽  
Frédérique Perrin ◽  
Sarah Ganet ◽  
Emmanuel Jamet ◽  
Fanny Tenenhaus-Aziza ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Human Health Risk ◽  
Raw Milk ◽  
Public Health Implication ◽  
Content Type ◽  
Growth And Survival ◽  
The Public ◽  
Food Borne ◽  
Food Borne Illness

ABSTRACTShiga toxin-producingEscherichia coli(STEC) is an important cause of food-borne illness. The public health implication of the presence of STEC in dairy products remains unclear. Knowledge of STEC behavior in cheeses would help to evaluate the human health risk. The aim of our study was to observe the growth and survival of experimentally inoculated STEC strains in raw-milk cheeses manufactured and ripened according to five technological schemes: blue-type cheese, uncooked pressed cheese with long ripening and with short ripening steps, cooked cheese, and lactic cheese. Cheeses were contaminated with different STEC serotypes (O157:H7, O26:H11, O103:H2, and O145:H28) at the milk preparation stage. STEC growth and survival were monitored on selective media during the entire manufacturing process. STEC grew (2 to 3 log10CFU · g−1) in blue-type cheese and the two uncooked pressed cheeses during the first 24 h of cheese making. Then, STEC levels progressively decreased in cheeses that were ripened for more than 6 months. In cooked cheese and in lactic cheese with a long acidic coagulation step (pH < 4.5), STEC did not grow. Their levels decreased after the cooking step in the cooked cheese and after the coagulation step in the lactic cheese, but STEC was still detectable at the end of ripening and storage. A serotype effect was found: in all cheeses studied, serotype O157:H7 grew less strongly and was less persistent than the others serotypes. This study improves knowledge of the behavior of different STEC serotypes in various raw-milk cheeses.

scholarly journals Shiga Toxin-Producing Serogroup O91 Escherichia coli Strains Isolated from Food and Environmental Samples

2017 ◽  
Vol 83 (18) ◽  
Author(s):  
Peter C. H. Feng ◽  
Sabine Delannoy ◽  
David W. Lacher ◽  
Joseph M. Bosilevac ◽  
Patrick Fach ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Hemolytic Uremic Syndrome ◽  
Shiga Toxin ◽  
Severe Illness ◽  
Content Type ◽  
Virulence Potential ◽  
Short Palindromic Repeat ◽  
Uremic Syndrome ◽  
Severe Infections ◽  
Genetic Profiles

ABSTRACT Shiga toxin-producing Escherichia coli (STEC) strains of the O91:H21 serotype have caused severe infections, including hemolytic-uremic syndrome. Strains of the O91 serogroup have been isolated from food, animals, and the environment worldwide but are not well characterized. We used a microarray and other molecular assays to examine 49 serogroup O91 strains (environmental, food, and clinical strains) for their virulence potential and phylogenetic relationships. Most of the isolates were identified to be strains of the O91:H21 and O91:H14 serotypes, with a few O91:H10 strains and one O91:H9 strain being identified. None of the strains had the eae gene, which codes for the intimin adherence protein, and many did not have some of the genetic markers that are common in other STEC strains. The genetic profiles of the strains within each serotype were similar but differed greatly between strains of different serotypes. The genetic profiles of the O91:H21 strains that we tested were identical or nearly identical to those of the clinical O91:H21 strains that have caused severe diseases. Multilocus sequence typing and clustered regularly interspaced short palindromic repeat analyses showed that the O91:H21 strains clustered within the STEC 1 clonal group but the other O91 serotype strains were phylogenetically diverse. IMPORTANCE This study showed that food and environmental O91:H21 strains have similar genotypic profiles and Shiga toxin subtypes and are phylogenetically related to the O91:H21 strains that have caused hemolytic-uremic syndrome, suggesting that these strains may also have the potential to cause severe illness.

scholarly journals Effects of Antibiotics on Shiga Toxin 2 Production and Bacteriophage Induction by Epidemic Escherichia coli O104:H4 Strain

2012 ◽  
Vol 56 (6) ◽  
pp. 3277-3282 ◽  
Author(s):  
Martina Bielaszewska ◽  
Evgeny A. Idelevich ◽  
Wenlan Zhang ◽  
Andreas Bauwens ◽  
Frieder Schaumburg ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Therapy ◽  
Shiga Toxin ◽  
The Other ◽  
Emerging Pathogen ◽  
Outbreak Strain ◽  
Content Type ◽  
Shiga Toxin 2 ◽  
Uremic Syndrome

ABSTRACTThe role of antibiotics in treatment of enterohemorrhagicEscherichia coli(EHEC) infections is controversial because of concerns about triggering hemolytic-uremic syndrome (HUS) by increasing Shiga toxin (Stx) production. During the recent large EHEC O104:H4 outbreak, antibiotic therapy was indicated for some patients. We tested a diverse panel of antibiotics to which the outbreak strain is susceptible to interrogate the effects of subinhibitory antibiotic concentrations on induction ofstx2-harboring bacteriophages,stx2transcription, and Stx2 production in this emerging pathogen. Ciprofloxacin significantly increasedstx2-harboring phage induction and Stx2 production in outbreak isolates (Pvalues of <0.001 to <0.05), while fosfomycin, gentamicin, and kanamycin insignificantly influenced them (P> 0.1) and chloramphenicol, meropenem, azithromycin, rifaximin, and tigecycline significantly decreased them (P≤ 0.05). Ciprofloxacin and chloramphenicol significantly upregulated and downregulatedstx2transcription, respectively (P< 0.01); the other antibiotics had insignificant effects (P> 0.1). Meropenem, azithromycin, and rifaximin, which were used for necessary therapeutic or prophylactic interventions during the EHEC O104:H4 outbreak, as well as tigecycline, neither inducedstx2-harboring phages nor increasedstx2transcription or Stx2 production in the outbreak strain. These antibiotics might represent therapeutic options for patients with EHEC O104:H4 infection if antibiotic treatment is inevitable. We await further analysis of the epidemic to determine if usage of these agents was associated with an altered risk of developing HUS.

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