scholarly journals The A1 Subunit of Shiga Toxin 2 Has Higher Affinity for Ribosomes and Higher Catalytic Activity than the A1 Subunit of Shiga Toxin 1

2015 ◽  
Vol 84 (1) ◽  
pp. 149-161 ◽  
Author(s):  
Debaleena Basu ◽  
Xiao-Ping Li ◽  
Jennifer N. Kahn ◽  
Kerrie L. May ◽  
Peter C. Kahn ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Shiga Toxin ◽  
The United States ◽  
Stem Loop ◽  
Content Type ◽  
Shiga Toxin 2 ◽  
Life Threatening ◽  
Uremic Syndrome ◽  
Ab5 Toxins

Shiga toxin (Stx)-producingEscherichia coli(STEC) infections can lead to life-threatening complications, including hemorrhagic colitis (HC) and hemolytic-uremic syndrome (HUS), which is the most common cause of acute renal failure in children in the United States. Stx1 and Stx2 are AB5 toxins consisting of an enzymatically active A subunit associated with a pentamer of receptor binding B subunits. Epidemiological evidence suggests that Stx2-producingE. colistrains are more frequently associated with HUS than Stx1-producing strains. Several studies suggest that the B subunit plays a role in mediating toxicity. However, the role of the A subunits in the increased potency of Stx2 has not been fully investigated. Here, using purified A1 subunits, we show that Stx2A1 has a higher affinity for yeast and mammalian ribosomes than Stx1A1. Biacore analysis indicated that Stx2A1 has faster association and dissociation with ribosomes than Stx1A1. Analysis of ribosome depurination kinetics demonstrated that Stx2A1 depurinates yeast and mammalian ribosomes and an RNA stem-loop mimic of the sarcin/ricin loop (SRL) at a higher catalytic rate and is a more efficient enzyme than Stx1A1. Stx2A1 depurinated ribosomes at a higher levelin vivoand was more cytotoxic than Stx1A1 inSaccharomyces cerevisiae. Stx2A1 depurinated ribosomes and inhibited translation at a significantly higher level than Stx1A1 in human cells. These results provide the first direct evidence that the higher affinity for ribosomes in combination with higher catalytic activity toward the SRL allows Stx2A1 to depurinate ribosomes, inhibit translation, and exhibit cytotoxicity at a significantly higher level than Stx1A1.

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.

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 Host-Like Carbohydrates Promote Bloodstream Survival of Vibrio vulnificusIn Vivo

2015 ◽  
Vol 83 (8) ◽  
pp. 3126-3136 ◽  
Author(s):  
Jean-Bernard Lubin ◽  
Warren G. Lewis ◽  
Nicole M. Gilbert ◽  
Cory M. Weimer ◽  
Salvador Almagro-Moreno ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Vibrio Vulnificus ◽  
Polymyxin B ◽  
The United States ◽  
Sialic Acids ◽  
Natural Resistance ◽  
Bacterial Survival ◽  
Content Type ◽  
Life Threatening

Sialic acids are found on all vertebrate cell surfaces and are part of a larger class of molecules known as nonulosonic acids. Many bacterial pathogens synthesize related nine-carbon backbone sugars; however, the role(s) of these non-sialic acid molecules in host-pathogen interactions is poorly understood.Vibrio vulnificusis the leading cause of seafood-related death in the United States due to its ability to quickly access the host bloodstream, which it can accomplish through gastrointestinal or wound infection. However, little is known about how this organism persists systemically. Here we demonstrate that sialic acid-like molecules are present on the lipopolysaccharide ofV. vulnificus, are required for full motility and biofilm formation, and also contribute to the organism's natural resistance to polymyxin B. Further experiments in a murine model of intravenousV. vulnificusinfection demonstrated that expression of nonulosonic acids had a striking benefit for bacterial survival during bloodstream infection and dissemination to other tissuesin vivo. In fact, levels of bacterial persistence in the blood corresponded to the overall levels of these molecules expressed byV. vulnificusisolates. Taken together, these results suggest that molecules similar to sialic acids evolved to facilitate the aquatic lifestyle ofV. vulnificusbut that their emergence also resulted in a gain of function with life-threatening potential in the human host.

scholarly journals A Novel Tail-Associated O91-Specific Polysaccharide Depolymerase from a Podophage Reveals Lytic Efficacy of Shiga Toxin-Producing Escherichia coli

2020 ◽  
Vol 86 (9) ◽  
Author(s):  
Yibao Chen ◽  
Xiangmin Li ◽  
Shuang Wang ◽  
Lingyu Guan ◽  
Xinxin Li ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Hemolytic Uremic Syndrome ◽  
Proinflammatory Cytokines ◽  
Shiga Toxin ◽  
Hemorrhagic Colitis ◽  
Content Type ◽  
Increased Risk ◽  
Specific Polysaccharide ◽  
Life Threatening ◽  
Uremic Syndrome

ABSTRACT Shiga toxin-producing Escherichia coli (STEC) strains are important zoonotic foodborne pathogens, causing diarrhea, hemorrhagic colitis, and life-threatening hemolytic uremic syndrome (HUS) in humans. However, antibiotic treatment of STEC infection is associated with an increased risk of HUS. Therefore, there is an urgent need for early and effective therapeutic strategies. Here, we isolated lytic T7-like STEC phage PHB19 and identified a novel O91-specific polysaccharide depolymerase (Dep6) in the C terminus of the PHB19 tailspike protein. Dep6 exhibited strong hydrolase activity across wide ranges of pH (pH 4 to 8) and temperature (20 to 60°C) and degraded polysaccharides on the surface of STEC strain HB10. In addition, both Dep6 and PHB19 degraded biofilms formed by STEC strain HB10. In a mouse STEC infection model, delayed Dep6 treatment (3 h postinfection) resulted in only 33% survival, compared with 83% survival when mice were treated simultaneously with infection. In comparison, pretreatment with Dep6 led to 100% survival compared with that of the control group. Surprisingly, a single PHB19 treatment resulted in 100% survival in all three treatment protocols. Moreover, a significant reduction in the levels of proinflammatory cytokines was observed at 24 h postinfection in Dep6- or PHB19-treated mice. These results demonstrated that Dep6 or PHB19 might be used as a potential therapeutic agent to prevent STEC infection. IMPORTANCE Shiga toxin-producing Escherichia coli (STEC) is an important foodborne pathogen worldwide. The Shiga-like toxin causes diarrhea, hemorrhagic colitis, and life-threatening hemolytic uremic syndrome (HUS) in humans. Although antibiotic therapy is still used for STEC infections, this approach may increase the risk of HUS. Phages or phage-derived depolymerases have been used to treat bacterial infections in animals and humans, as in the case of the “San Diego patient” treated with a phage cocktail. Here, we showed that phage PHB19 and its O91-specific polysaccharide depolymerase Dep6 degraded STEC biofilms and stripped the lipopolysaccharide (LPS) from STEC strain HB10, which was subsequently killed by serum complement in vitro. In a mouse model, PHB19 and Dep6 protected against STEC infection and caused a significant reduction in the levels of proinflammatory cytokines. This study reports the use of an O91-specific polysaccharide depolymerase for the treatment of STEC infection in mice.

scholarly journals Human Serum Amyloid P Component Protects againstEscherichia coliO157:H7 Shiga Toxin 2 In Vivo: Therapeutic Implications for Hemolytic‐Uremic Syndrome

2006 ◽  
Vol 193 (8) ◽  
pp. 1120-1124 ◽  
Author(s):  
Glen D. Armstrong ◽  
George L. Mulvey ◽  
Paola Marcato ◽  
Thomas P. Griener ◽  
Melvyn C. Kahan ◽  
...  
Keyword(s):  
Human Serum ◽  
Shiga Toxin ◽  
Amyloid P Component ◽  
Therapeutic Implications ◽  
Serum Amyloid P ◽  
Serum Amyloid P Component ◽  
Shiga Toxin 2 ◽  
Uremic Syndrome ◽  
Amyloid P

scholarly journals Development of a homogeneous time-resolved FRET (HTRF) assay for the quantification of Shiga toxin 2 produced by E. coli

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e11871
Author(s):  
Keiji Nakamura ◽  
Chikashi Tokuda ◽  
Hideyuki Arimitsu ◽  
Yoshiki Etoh ◽  
Mitsuhiro Hamasaki ◽  
...  
Keyword(s):  
Shiga Toxin ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Assay System ◽  
Time Resolved ◽  
Virulence Potential ◽  
Shiga Toxin 2 ◽  
Quantification Analysis ◽  
Life Threatening ◽  
Uremic Syndrome

Shiga toxin-producing Escherichia coli (STEC) is a major intestinal pathogen and causes serious gastrointestinal illness, which includes diarrhea, hemorrhagic colitis, and life-threatening hemolytic uremic syndrome. The major virulence factors of STEC are Shiga toxins (Stx1 and Stx2), which belong to the AB-type toxin family. Among several subtypes of Stx1 and Stx2, the production of Stx2a is thought to be a risk factor for severe STEC infections, but Stx2a production levels vary markedly between STEC strains, even strains with the same serotype. Therefore, quantitative analyses of Stx2 production by STEC strains are important to understand the virulence potential of specific lineages or sublineages. In this study, we developed a novel Stx2 quantification method by utilizing homogeneous time-resolved fluorescence resonance energy transfer (HTRF) technology. To determine suitable “sandwich” assay conditions, we tested 6 combinations of fluorescence-labeled monoclonal antibodies (mAbs) specific to Stx2 and compared the HTRF signal intensities obtained at various incubation times. Through this analysis, we selected the most suitable mAb pair, one recognizing the A subunit and the other recognizing the B subunit, thus together detecting Stx holotoxins. The optimal incubation time was also determined (18 h). Then, we optimized the concentrations of the two mAbs based on the range for linearity. The established HTRF assay detected 0.5 ng/ml of the highly purified recombinant Stx2a and Stx2e proteins and the working range was 1–64 ng/ml for both Stx2a and Stx2e. Through the quantification analysis of Stx proteins in STEC cell lysates, we confirmed that other Stx2 subtypes (Stx2b, Stx2c, Stx2d and Stx2g) can also be quantified at a certain level of accuracy, while this assay system does not detect Stx2f, which is highly divergent in sequence from other Stx2 subtypes, and Stx1. As the HTRF protocol we established is simple, this assay system should prove useful for the quantitative analysis of Stx2 production levels of a large number of STEC strains.

scholarly journals Development and Evaluation of a Novel VHH-Based Immunocapture Assay for High-Sensitivity Detection of Shiga Toxin Type 2 (Stx2) in Stool Samples

2019 ◽  
Vol 58 (3) ◽  
Author(s):  
Luciano J. Melli ◽  
Vanesa Zylberman ◽  
Yanina Hiriart ◽  
Constanza E. Lauche ◽  
Ariela Baschkier ◽  
...  
Keyword(s):  
Shiga Toxin ◽  
High Sensitivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Content Type ◽  
Clinical Sensitivity ◽  
Life Threatening ◽  
Uremic Syndrome ◽  
Stool Samples ◽  
High Yields ◽  
Postdiarrheal Hemolytic Uremic Syndrome

ABSTRACT Shiga toxin (Stx)-producing Escherichia coli (STEC) is the main cause of postdiarrheal hemolytic-uremic syndrome (HUS), a life-threatening clinical complication characterized by hemolytic anemia, thrombocytopenia, and acute renal failure that mainly affects children. A relevant feature of STEC strains is the production of Stx, and all of them express Stx1 and/or Stx2 regardless of the strain serotype. Therefore, Stx detection assays are considered the most suitable methods for the early detection of STEC infections. Single-domain antibodies from camelids (VHHs) exhibit several advantages in comparison with conventional antibodies, making them promising tools for diagnosis. In this work, we have exploited VHH technology for the development of an immunocapture assay for Stx2 detection. Thirteen anti-Stx2 VHHs previously obtained from a variable-domain repertoire library were selected and evaluated in 130 capture-detection pair combinations for Stx detection. Based on this analysis, two VHHs were selected and a double VHH-based biotin-streptavidin capture enzyme-linked immunosorbent assay (ELISA) with spectrophotometric detection was developed and optimized for Stx2 detection. This assay showed an excellent analytical and clinical sensitivity in both STEC culture supernatants and stool samples even higher than the sensitivity of a commercial ELISA. Furthermore, based on the analysis of stool samples, the VHH-based ELISA showed high correlation with stx2 detection by PCR and a commercial rapid membrane-based immunoassay. The intrinsic properties of VHHs (high target affinity and specificity, stability, and ease of expression at high yields in recombinant bacteria) and their optimal performance for Stx detection make them attractive tools for the diagnosis of HUS related to STEC (STEC-HUS).

scholarly journals AnIn VitroCombined Antibiotic-Antibody Treatment Eliminates Toxicity from Shiga Toxin-Producing Escherichia coli

2015 ◽  
Vol 59 (9) ◽  
pp. 5435-5444 ◽  
Author(s):  
Craig Skinner ◽  
Guodong Zhang ◽  
Stephanie Patfield ◽  
Xiaohua He
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Shiga Toxin ◽  
Neutralizing Antibodies ◽  
Vero Cells ◽  
Antibody Treatment ◽  
Gastrointestinal Infections ◽  
Content Type ◽  
Life Threatening ◽  
Uremic Syndrome

ABSTRACTTreating Shiga toxin-producingEscherichia coli(STEC) gastrointestinal infections is difficult. The utility of antibiotics for STEC treatment is controversial, since antibiotic resistance among STEC isolates is widespread and certain antibiotics dramatically increase the expression of Shiga toxins (Stxs), which are some of the most important virulence factors in STEC. Stxs contribute to life-threatening hemolytic uremic syndrome (HUS), which develops in considerable proportions of patients with STEC infections. Understanding the antibiotic resistance profiles of STEC isolates and the Stx induction potential of promising antibiotics is essential for evaluating any antibiotic treatment of STEC. In this study, 42 O157:H7 or non-O157 STEC isolates (including the “big six” serotypes) were evaluated for their resistance against 22 antibiotics by using an antibiotic array. Tigecycline inhibited the growth of all of the tested STEC isolates and also inhibited the production of Stxs (Stx2 in particular). In combination with neutralizing antibodies to Stx1 and Stx2, the tigecycline-antibody treatment fully protected Vero cells from Stx toxicity, even when the STEC bacteria and the Vero cells were cultured together. The combination of an antibiotic such as tigecycline with neutralizing antibodies presents a promising strategy for future STEC treatments.

scholarly journals Promoter Sequence of Shiga Toxin 2 (Stx2) Is Recognized In Vivo, Leading to Production of Biologically Active Stx2

mBio ◽  
2013 ◽  
Vol 4 (5) ◽  
Author(s):  
Leticia V. Bentancor ◽  
Maria P. Mejías ◽  
Alípio Pinto ◽  
Marcos F. Bilen ◽  
Roberto Meiss ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Central Nervous System ◽  
Nervous System ◽  
Hemolytic Uremic Syndrome ◽  
Shiga Toxin ◽  
Biologically Active ◽  
Eukaryotic Cells ◽  
Content Type ◽  
Uremic Syndrome

ABSTRACTShiga toxins (Stx) are the main agent responsible for the development of hemolytic-uremic syndrome (HUS), the most severe and life-threatening systemic complication of infection with enterohemorrhagicEscherichia coli(EHEC) strains. We previously described Stx2 expression by eukaryotic cells after they were transfectedin vitrowith thestx2gene cloned into a prokaryotic plasmid (pStx2). The aim of this study was to evaluate whether mammalian cells were also able to express Stx2in vivoafter pStx2 injection. Mice were inoculated by hydrodynamics-based transfection (HBT) with pStx2. We studied the survival, percentage of polymorphonuclear leukocytes in plasma, plasma urea levels, and histology of the kidneys and the brains of mice. Mice displayed a lethal dose-related response to pStx2. Stx2 mRNA was recovered from the liver, and Stx2 cytotoxic activity was observed in plasma of mice injected with pStx2. Stx2 was detected by immunofluorescence in the brains of mice inoculated with pStx2, and markers of central nervous system (CNS) damage were observed, including increased expression of glial fibrillary acidic protein (GFAP) and fragmentation of NeuN in neurons. Moreover, anti-Stx2B-immunized mice were protected against pStx2 inoculation. Our results show that Stx2 is expressedin vivofrom the wildstx2gene, reproducing pathogenic damage induced by purified Stx2 or secondary to EHEC infection.IMPORTANCEEnterohemorrhagic Shiga toxin (Stx)-producingEscherichia coli(EHEC) infections are a serious public health problem, and Stx is the main pathogenic agent associated with typical hemolytic-uremic syndrome (HUS). In contrast to the detailed information describing the molecular basis for EHEC adherence to epithelial cells, very little is known about how Stx is released from bacteria in the gut, reaching its target tissues, mainly the kidney and central nervous system (CNS). In order to develop an efficient treatment for EHEC infections, it is necessary to understand the mechanisms involved in Stx expression. In this regard, the present study demonstrates that mammals can synthesize biologically active Stx using the natural promoter associated with the Stx-converting bacteriophage genome. These results could impact the comprehension of EHEC HUS, since local eukaryotic cells transduced and/or infected by bacteriophage encoding Stx2 could be an alternative source of Stx production.

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.

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