Recombinant Shiga Toxin B-Subunit-Keyhole Limpet Hemocyanin Conjugate Vaccine Protects Mice from Shigatoxemia

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) causes hemorrhagic colitis in humans and, in a subgroup of infected subjects, a more serious condition called hemolytic-uremic syndrome (HUS). These conditions arise because EHEC produces two antigenically distinct forms of Shiga toxin (Stx), called Stx1 and Stx2. Despite this, the production of Stx2 by virtually all EHEC serotypes and the documented role this toxin plays in HUS make it an attractive vaccine candidate. Previously, we assessed the potential of a purified recombinant Stx2 B-subunit preparation to prevent Shigatoxemia in rabbits. This study revealed that effective immunization could be achieved only if endotoxin was included with the vaccine antigen. Since the presence of endotoxin would be unacceptable in a human vaccine, the object of the studies described herein was to investigate ways to safely augment, in mice, the immunogenicity of the recombinant Stx2 B subunit containing <1 endotoxin unit per ml. The study revealed that sera from mice immunized with such a preparation, conjugated to keyhole limpet hemocyanin and administered with the Ribi adjuvant system, displayed the highest Shiga toxin 2 B-subunit-specific immunoglobulin G1 (IgG1) and IgG2a enzyme-linked immunosorbent assay titers and cytotoxicity-neutralizing activities in Ramos B cells. As well, 100% of the mice vaccinated with this preparation were subsequently protected from a lethal dose of Stx2 holotoxin. These results support further evaluation of a Stx2 B-subunit-based human EHEC vaccine.

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

Infection and Immunity ◽

10.1128/iai.00027-14 ◽

2014 ◽

Vol 82 (4) ◽

pp. 1491-1499 ◽

Cited By ~ 16

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.

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Switching Shiga Toxin (Stx) Type from Stx2d to Stx2a but Not Stx2c Alters Virulence of Stx-Producing Escherichia coli (STEC) Strain B2F1 in Streptomycin (Str)-Treated Mice

Toxins ◽

10.3390/toxins13010064 ◽

2021 ◽

Vol 13 (1) ◽

pp. 64

Author(s):

Beth A. McNichol ◽

Rebecca A. Bova ◽

Kieron Torres ◽

Lan N. Preston ◽

Angela R. Melton-Celsa

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Oral Administration ◽

Amino Acid Composition ◽

Shiga Toxin ◽

Vero Cells ◽

Toxin B ◽

B Subunit ◽

Intestinal Mucus ◽

Binding Subunit

Shiga toxin (Stx)-producing Escherichia coli (STEC) strain B2F1 produces Stx type 2d, a toxin that becomes more toxic towards Vero cells in the presence of intestinal mucus. STEC that make Stx2d are more pathogenic to streptomycin (Str)-treated mice than most STEC that produce Stx2a or Stx2c. However, purified Stx2d is only 2- or 7-fold more toxic by the intraperitoneal route than Stx2a or Stx2c, respectively. We hypothesized, therefore, that the toxicity differences among Stx2a, Stx2c, and Stx2d occur at the level of delivery from the intestine. To evaluate that hypothesis, we altered the toxin type produced by stx2d+ mouse virulent O91:H21 clinical isolate B2F1 to Stx2a or Stx2c. Because B2F1 encodes two copies of stx2d, we did these studies in a derivative of B2F1 in which stx2d1 was deleted. Although the strains were equivalently virulent to the Str-treated mice at the 1010 dose, the B2F1 strain that produced Stx2a was attenuated relative to the ones that produced Stx2d or Stx2c when administered at 103 CFU/mouse. We next compared the oral toxicities of purified Stx2a, Stx2c, and Stx2d. We found that purified Stx2d is more toxic than Stx2a or Stx2c upon oral administration at 4 µg/mouse. Taken together, these studies suggest that Stx2 toxins are most potent when delivered directly from the bacterium. Furthermore, because Stx2d and Stx2c have the identical amino acid composition in the toxin B subunit, our results indicate that the virulence difference between Stx2a and Stx2d and Stx2c resides in the B or binding subunit of the toxins.

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A System for Production and Rapid Purification of Large Amounts of the Shiga Toxin/Shiga-Like Toxin B Subunit

Molecular Pathogenesis of Gastrointestinal Infections ◽

10.1007/978-1-4684-5982-1_39 ◽

1991 ◽

pp. 299-301

Author(s):

Arthur Donohue-Rolfe ◽

David W. K. Acheson ◽

Gerald T. Keusch ◽

Marcia B. Goldberg ◽

Stephanie A. Boyko ◽

...

Keyword(s):

Shiga Toxin ◽

Toxin B ◽

B Subunit ◽

Rapid Purification

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Shiga toxin–binding site for host cell receptor GPP130 reveals unexpected divergence in toxin-trafficking mechanisms

Molecular Biology of the Cell ◽

10.1091/mbc.e13-01-0057 ◽

2013 ◽

Vol 24 (15) ◽

pp. 2311-2318 ◽

Cited By ~ 28

Author(s):

Somshuvra Mukhopadhyay ◽

Brendan Redler ◽

Adam D. Linstedt

Keyword(s):

Shiga Toxin ◽

Transmembrane Domain ◽

Cell Receptor ◽

Toxin B ◽

Toxin Binding ◽

B Subunit ◽

Host Cell Receptor ◽

Developed World ◽

Trafficking Receptor ◽

Shed Light

Shiga toxicosis is caused by retrograde trafficking of one of three types of Shiga toxin (STx), STx, STx1, or STx2. Trafficking depends on the toxin B subunits, which for STx and STx1 are identical and bind GPP130, a manganese (Mn)-sensitive intracellular trafficking receptor. Elevated Mn down-regulates GPP130, rendering STx/STx1 harmless. Its effectiveness against STx2, however, which is a serious concern in the developed world, is not known. Here we show that Mn-induced GPP130 down-regulation fails to block STx2 trafficking. To shed light on this result, we tested the purified B subunit of STx2 for binding to GPP130 and found that it failed to interact. We then mapped residues at the interface of the GPP130-STx/STx1 complex. In GPP130, binding mapped to a seven-residue stretch in its lumenal stem domain next to the transmembrane domain. This stretch was required for STx/STx1 transport. In STx/STx1, binding mapped to a histidine–asparagine pair on a surface-exposed loop of the toxin B subunit. Significantly, these residues are not conserved in STx2, explaining the lack of effectiveness of Mn against STx2. Together our results imply that STx2 uses an evolutionarily distinct trafficking mechanism and that Mn as a potential therapy should be focused on STx/STx1 outbreaks, which account for the vast majority of cases worldwide.

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Shiga Toxin 2 Reduces Complement Inhibitor CD59 Expression on Human Renal Tubular Epithelial and Glomerular Endothelial Cells

Infection and Immunity ◽

10.1128/iai.01079-12 ◽

2013 ◽

Vol 81 (8) ◽

pp. 2678-2685 ◽

Cited By ~ 23

Author(s):

Silvia Ehrlenbach ◽

Alejandra Rosales ◽

Wilfried Posch ◽

Doris Wilflingseder ◽

Martin Hermann ◽

...

Keyword(s):

Endothelial Cells ◽

Cell Surface ◽

Shiga Toxin ◽

Human Kidney ◽

Enzyme Linked Immunosorbent Assay ◽

Content Type ◽

Protein Levels ◽

Glomerular Endothelial Cells ◽

Shiga Toxin 2 ◽

Renal Tubular

ABSTRACTInfections with enterohemorrhagicEscherichia coli(EHEC) are a primary cause of hemolytic-uremic syndrome (HUS). Recently, Shiga toxin 2 (Stx2), the major virulence factor of EHEC, was reported to interact with complement, implying that the latter is involved in the pathogenesis of EHEC-induced HUS. The aim of the present study was to investigate the effect of Stx2 on the expression of membrane-bound complement regulators CD46, CD55, and CD59 on proximal tubular epithelial (HK-2) and glomerular endothelial (GEnC) cells derived from human kidney cells that are involved in HUS. Incubation with Stx2 did not influence the amount of CD46 or CD55 on the surface of HK-2 and GEnC cells, as determined by fluorescence-activated cell sorter analysis. In contrast, CD59 was significantly reduced by half on GEnC cells, but the reduction on HK-2 cells was less pronounced. With increasing amounts of Stx2, reduction of CD59 also reached significance in HK-2 cells. Enzyme-linked immunosorbent assay analyses showed that CD59 was not present in the supernatant of Stx2-treated cells, implying that CD59 reduction was not caused by cleavage from the cell surface. In fact, reverse transcription-quantitative PCR analyses showed downregulation of CD59 mRNA as the likely reason for CD59 cell surface reduction. In addition, a significant increase in terminal complement complex deposition on HK-2 cells was observed after treatment with Stx2, as a possible consequence of CD59 downregulation. In summary, Stx2 downregulates CD59 mRNA and protein levels on tubular epithelial and glomerular endothelial cells, and this downregulation likely contributes to complement activation and kidney destruction in EHEC-associated HUS.

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Shiga Toxin Uptake and Sequestration in Extracellular Vesicles Is Mediated by Its B-Subunit

Toxins ◽

10.3390/toxins12070449 ◽

2020 ◽

Vol 12 (7) ◽

pp. 449 ◽

Cited By ~ 1

Author(s):

Annie Willysson ◽

Anne-lie Ståhl ◽

Daniel Gillet ◽

Julien Barbier ◽

Jean-Christophe Cintrat ◽

...

Keyword(s):

Extracellular Vesicles ◽

Hela Cells ◽

Shiga Toxin ◽

Blood Cells ◽

Continuous Release ◽

B Subunit ◽

Uremic Syndrome ◽

Translocation Assay ◽

Synthase Inhibitor ◽

Toxin Uptake

Shiga toxin (Stx)-stimulated blood cells shed extracellular vesicles (EVs) which can transfer the toxin to the kidneys and lead to hemolytic uremic syndrome. The toxin can be taken up by renal cells within EVs wherein the toxin is released, ultimately leading to cell death. The mechanism by which Stx is taken up, translocated, and sequestered in EVs was addressed in this study utilizing the B-subunit that binds to the globotriaosylceramide (Gb3) receptor. We found that Stx1B was released in EVs within minutes after stimulation of HeLa cells or red blood cells, detected by live cell imaging and flow cytometry. In the presence of Retro-2.1, an inhibitor of intracellular retrograde trafficking, a continuous release of Stx-positive EVs occurred. EVs from HeLa cells possess the Gb3 receptor on their membrane, and EVs from cells that were treated with a glycosylceramide synthase inhibitor, to reduce Gb3, bound significantly less Stx1B. Stx1B was detected both on the membrane and within the shed EVs. Stx1B was incubated with EVs derived from blood cells, in the absence of cells, and was shown to bind to, and be taken up by, these EVs, as demonstrated by electron microscopy. Using a membrane translocation assay we demonstrated that Stx1B was taken up by blood cell- and HeLa-derived EVs, an effect enhanced by chloropromazine or methyl-ß-cyclodextrin, suggesting toxin transfer within the membrane. This is a novel mechanism by which EVs derived from blood cells can sequester their toxic content, possibly to evade the host response.

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Prevalence, Characterization, and Genotypic Analysis of Escherichia coli O157:H7/NM fromSelected Beef Exporting Abattoirs of Argentina

Journal of Food Protection ◽

10.4315/0362-028x-73.4.649 ◽

2010 ◽

Vol 73 (4) ◽

pp. 649-656 ◽

Cited By ~ 35

Author(s):

M. O. MASANA ◽

G. A. LEOTTA ◽

L. L. DEL CASTILLO ◽

B. A. D'ASTEK ◽

P. M. PALLADINO ◽

...

Keyword(s):

Escherichia Coli ◽

Shiga Toxin ◽

Phage Type ◽

Escherichia Coli O157 ◽

Phage Types ◽

Clonal Relatedness ◽

Genotypic Analysis ◽

Shiga Toxin 2 ◽

Uremic Syndrome ◽

Fecal Content

In Argentina, Escherichia coli O157:H7/NM (STEC O157) is the prevalent serotype associated with hemolytic uremic syndrome (HUS), which is endemic in the country with more than 400 cases per year. In order to estimate the prevalence and characteristics of STEC O157 in beef cattle at slaughter, a survey of 1,622 fecal and carcass samples was conducted in nine beef exporting abattoirs from November 2006 to April 2008. A total of 54 samples were found positive for STEC O157, with an average prevalence of 4.1% in fecal content and 2.6% in carcasses. Calves and heifers presented higher percentages of prevalence in feces, 10.5 and 8.5%, respectively. All STEC O157 isolates harbored stx2 (Shiga toxin 2), eae (intimin), ehxA (enterohemolysin), and fliCH7 (H7 flagellin) genes, while stx1 (Shiga toxin 1) was present in 16.7% of the strains. The prevalent (56%) stx genotype identified was stx2 combined with variant stx2c (vh-a), the combination of which is also prevalent (>90%) in STEC O157 post–enteric HUS cases in Argentina. The clonal relatedness of STEC O157 strains was established by phage typing and pulsed-field gel electrophoresis (PFGE). The 54 STEC isolates were categorized into 12 different phage types and in 29 XbaI-PFGE patterns distributed in 27 different lots. STEC O157 strains isolated from 5 of 21 carcasses were identical by PFGE (100% similarity) to strains of the fecal content of the same or a contiguous bovine in the lot. Five phage type–PFGE–stx profiles of 10 strains isolated in this study matched with the profiles of the strains recovered from 18 of 122 HUS cases that occurred in the same period.

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Cytotoxic effect of Shiga toxin-2 holotoxin and its B subunit on human renal tubular epithelial cells

Microbes and Infection ◽

10.1016/j.micinf.2005.07.005 ◽

2006 ◽

Vol 8 (2) ◽

pp. 410-419 ◽

Cited By ~ 35

Author(s):

Virginia Pistone Creydt ◽

Claudia Silberstein ◽

Elsa Zotta ◽

Cristina Ibarra

Keyword(s):

Epithelial Cells ◽

Shiga Toxin ◽

Cytotoxic Effect ◽

Tubular Epithelial Cells ◽

Renal Tubular Epithelial Cells ◽

B Subunit ◽

Shiga Toxin 2 ◽

Renal Tubular

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Induction by Sphingomyelinase of Shiga Toxin Receptor and Shiga Toxin 2 Sensitivity in Human Microvascular Endothelial Cells

Infection and Immunity ◽

10.1128/iai.71.2.845-849.2003 ◽

2003 ◽

Vol 71 (2) ◽

pp. 845-849 ◽

Cited By ~ 12

Author(s):

T. G. Obrig ◽

R. M. Seaner ◽

M. Bentz ◽

C. A. Lingwood ◽

B. Boyd ◽

...

Keyword(s):

Endothelial Cells ◽

Shiga Toxin ◽

Microvascular Endothelial Cells ◽

Cytotoxic Action ◽

Ceramide Level ◽

Shiga Toxins ◽

Shiga Toxin 2 ◽

Toxin Receptor ◽

Uremic Syndrome ◽

Microvascular Endothelial

ABSTRACT Shiga toxin-producing enterohemorrhagic Escherichia coli is the major cause of acute renal failure in young children. The interaction of Shiga toxins 1 and 2 (Stx1 and Stx2) with endothelial cells is an important step in the renal coagulation and thrombosis observed in hemolytic uremic syndrome. Previous studies have shown that bacterial lipopolysaccharide and host cytokines slowly sensitize endothelial cells to Shiga toxins. In the present study, bacterial neutral sphingomyelinase (SMase) rapidly (1 h) sensitized human dermal microvascular endothelial cells (HDMEC) to the cytotoxic action of Stx2. Exposure of endothelial cells to neutral SMase (0.067 U/ml) caused a rapid increase of intracellular ceramide that persisted for hours. Closely following the change in ceramide level was an increase in the expression of globotriaosylceramide (Gb3), the receptor for Stx2. A rapid increase was also observed in the mRNA for ceramide:glucosyltransferase (CGT), the first of three glycosyltransferase enzymes of the Gb3 biosynthetic pathway. The product of CGT (glucosylceramide) was also increased. In contrast, mRNA for the third enzyme of the pathway, Gb3 synthase, was constitutively produced and was not influenced by SMase treatment of HDMEC. These results describe a rapid response mechanism by which extracellular neutral SMase derived from either bacteria or eukaryotic cells may signal endothelial cells to become sensitive to Shiga toxins.

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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

Infection and Immunity ◽

10.1128/iai.00149-16 ◽

2016 ◽

Vol 84 (9) ◽

pp. 2653-2661 ◽

Cited By ~ 2

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.

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