Neutralization of Shiga Toxins Stx1, Stx2c, and Stx2e by Recombinant Bacteria Expressing Mimics of Globotriose and Globotetraose

ABSTRACT Strains of Escherichia coli producing Shiga toxins Stx1, Stx2, Stx2c, and Stx2d cause gastrointestinal disease and the hemolytic-uremic syndrome in humans. We have recently constructed a recombinant bacterium which displays globotriose (the receptor for these toxins) on its surface and adsorbs and neutralizes these Shiga toxins with very high efficiency. This agent has great potential for the treatment of humans with such infections. E. colistrains which cause edema disease in pigs produce a variant toxin, Stx2e, which has a different receptor specificity from that for the other members of the Stx family. We have now modified the globotriose-expressing bacterium such that it expresses globotetraose (the preferred receptor for Stx2e) by introducing additional genes encoding a N-acetylgalactosamine transferase and a UDP-N-acetylgalactosamine-4-epimerase. This bacterium had a reduced capacity to neutralize Stx1 and Stx2c in vitro, but remarkably, its capacity to bind Stx2e was similar to that of the globotriose-expressing construct; both constructs neutralized 98.4% of the cytotoxicity in lysates of E. coli JM109 expressing cloned stx 2e. These data suggest that either globotriose- or globotetraose-expressing constructs may be suitable for treatment and/or prevention of edema disease in pigs.

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Oral Administration of Formaldehyde-Killed Recombinant Bacteria Expressing a Mimic of the Shiga Toxin Receptor Protects Mice from Fatal Challenge with Shiga-ToxigenicEscherichia coli

Infection and Immunity ◽

10.1128/iai.69.3.1389-1393.2001 ◽

2001 ◽

Vol 69 (3) ◽

pp. 1389-1393 ◽

Cited By ~ 34

Author(s):

James C. Paton ◽

Trisha J. Rogers ◽

Renato Morona ◽

Adrienne W. Paton

Keyword(s):

Oral Administration ◽

Shiga Toxin ◽

High Efficiency ◽

Gastrointestinal Disease ◽

Protective Capacity ◽

Recombinant Bacteria ◽

Recombinant Bacterium ◽

Life Threatening ◽

Toxin Receptor ◽

Uremic Syndrome

ABSTRACT Gastrointestinal disease caused by Shiga toxin-producingEscherichia coli (STEC) is frequently complicated by life-threatening toxin-induced systemic sequelae, including the hemolytic uremic syndrome. We previously constructed a recombinant bacterium displaying a Shiga toxin receptor mimic on its surface which neutralized Shiga toxins with very high efficiency. Moreover, oral administration of the live bacterium completely protected mice from challenge with virulent STEC. In this study, we investigated the protective capacity of formaldehyde-killed receptor mimic bacteria, as these are likely to be safer for administration to humans. The killed bacteria completely protected STEC-challenged mice when administered three times daily; incomplete protection was achieved using two doses per day. Commencement of therapy could be delayed for up to 48 h after challenge without diminishing protection, depending on the virulence of the challenge strain. Thus, administration of this agent early in the course of human STEC disease may prevent progression to life-threatening complications.

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Contribution and Interaction of Shiga Toxin Genes to Escherichia coli O157:H7 Virulence

Toxins ◽

10.3390/toxins11100607 ◽

2019 ◽

Vol 11 (10) ◽

pp. 607 ◽

Cited By ~ 7

Author(s):

Gillian A.M. Tarr ◽

Taryn Stokowski ◽

Smriti Shringi ◽

Phillip I. Tarr ◽

Stephen B. Freedman ◽

...

Keyword(s):

Escherichia Coli ◽

Shiga Toxin ◽

Population Level ◽

Escherichia Coli O157 ◽

Additive Interaction ◽

Shiga Toxins ◽

E Coli ◽

Uremic Syndrome ◽

Shiga Toxin Genes

Escherichia coli O157:H7 is the predominant cause of diarrhea-associated hemolytic uremic syndrome (HUS) worldwide. Its cardinal virulence traits are Shiga toxins, which are encoded by stx genes, the most common of which are stx1a, stx2a, and stx2c. The toxins these genes encode differ in their in vitro and experimental phenotypes, but the human population-level impact of these differences is poorly understood. Using Shiga toxin-encoding bacteriophage insertion typing and real-time polymerase chain reaction, we genotyped isolates from 936 E. coli O157:H7 cases and verified HUS status via chart review. We compared the HUS risk between isolates with stx2a and those with stx2a and another gene and estimated additive interaction of the stx genes. Adjusted for age and symptoms, the HUS incidence of E. coli O157:H7 containing stx2a alone was 4.4% greater (95% confidence interval (CI) −0.3%, 9.1%) than when it occurred with stx1a. When stx1a and stx2a occur together, the risk of HUS was 27.1% lower (95% CI −87.8%, −2.3%) than would be expected if interaction were not present. At the population level, temporal or geographic shifts toward these genotypes should be monitored, and stx genotype may be an important consideration in clinically predicting HUS among E. coli O157:H7 cases.

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Molecular Characterization of the Locus Encoding Biosynthesis of the Lipopolysaccharide O Antigen of Escherichia coliSerotype O113

Infection and Immunity ◽

10.1128/iai.67.11.5930-5937.1999 ◽

1999 ◽

Vol 67 (11) ◽

pp. 5930-5937 ◽

Cited By ~ 45

Author(s):

Adrienne W. Paton ◽

James C. Paton

Keyword(s):

Immune Responses ◽

Gastrointestinal Disease ◽

Cosmid Library ◽

E Coli ◽

O Antigen ◽

Uremic Syndrome ◽

Human Immune ◽

K 12

ABSTRACT Shiga toxigenic Escherichia coli (STEC) strains are a diverse group of organisms capable of causing severe gastrointestinal disease in humans. Within the STEC family, eae-positive STEC strains, particularly those belonging to serogroups O157 and O111, appear to have greater virulence for humans. However, in spite of beingeae negative, STEC strains belonging to serogroup O113 have frequently been associated with cases of severe STEC disease, including hemolytic-uremic syndrome (HUS). Western blot analysis with convalescent-phase serum from a patient with HUS caused by an O113:H21 STEC strain indicated that human immune responses were directed principally against lipopolysaccharide O antigen. Accordingly, the serum was used to isolate a clone expressing O113 O antigen from a cosmid library of O113:H21 DNA constructed in E. coli K-12. Sequence analysis indicated that the O113 O-antigen biosynthesis (rfb) locus contains a cluster of nine genes which may be cotranscribed. Comparison with sequence databases identified candidate genes for four glycosyl transferases, an O-acetyl transferase, an O-unit flippase, and an O-antigen polymerase, as well as copies of galE and gnd. Two additional, separately transcribed genes downstream of the O113 rfbregion were predicted to encode enzymes involved in synthesis of activated sugar precursors, one of which (designated wbnF) was essential for O113 O-antigen synthesis, and so is clearly a part of the O113 rfb locus. Interestingly, expression of O113 O antigen by E. coli K-12 significantly increased in vitro adherence to both HEp-2 and Henle 407 cells.

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Creating Hybrid Genes by Homologous Recombination

Disease Markers ◽

10.1155/2000/596468 ◽

2000 ◽

Vol 16 (1-2) ◽

pp. 3-13 ◽

Cited By ~ 5

Author(s):

Peter L. Wang

Keyword(s):

Directed Evolution ◽

High Efficiency ◽

Genomic Sequence ◽

Sequence Data ◽

Strand Breaks ◽

E Coli ◽

Hybrid Genes ◽

Distinct Features

Recombination of homologous genes is a powerful mechanism for generating sequence diversity, and can be applied to protein analysis and directed evolution.In vitrorecombination methods such as DNA shuffling are very flexible and can give hybrid genes with multiple crossovers; they have been used extensively to evolve proteins with improved and novel properties.In vivorecombination in bothE. coliand yeast is greatly enhanced by double-strand breaks; forE. coli, mutant strains are often necessary to obtain high efficiency. Intra- and inter-molecular recombinationIn vivohave distinct features; both give hybrids with one or two crossovers, and have been used to study structure-function relationships of many proteins. Recentlyin vivorecombination has been used to generate diversity for directed evolution, creating a large phage display antibody library. Recombination methods will become increasingly useful in light of the explosion in genomic sequence data and potential for engineered proteins.

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Protection against Shiga-Toxigenic Escherichia coli by Non-Genetically Modified Organism Receptor Mimic Bacterial Ghosts

Infection and Immunity ◽

10.1128/iai.00669-15 ◽

2015 ◽

Vol 83 (9) ◽

pp. 3526-3533 ◽

Cited By ~ 5

Author(s):

Adrienne W. Paton ◽

Austen Y. Chen ◽

Hui Wang ◽

Lauren J. McAllister ◽

Florian Höggerl ◽

...

Keyword(s):

Escherichia Coli ◽

High Efficiency ◽

Genetically Modified ◽

Genetically Modified Organism ◽

Gastrointestinal Infections ◽

Content Type ◽

Recombinant Bacterium ◽

Bacterial Ghosts

Shiga-toxigenicEscherichia coli(STEC) causes severe gastrointestinal infections in humans that may lead to life-threatening systemic sequelae, such as the hemolytic uremic syndrome (HUS). Rapid diagnosis of STEC infection early in the course of disease opens a window of opportunity for therapeutic intervention, for example, by administration of agents that neutralize Shiga toxin (Stx) in the gut lumen. We previously developed a recombinant bacterium that expresses a mimic of the Stx receptor globotriaosyl ceramide (Gb3) on its surface through modification of the lipopolysaccharide (A. W. Paton, R. Morona, and J. C. Paton, Nat Med6:265–270, 2000,http://dx.doi.org/10.1038/73111). This construct was highly efficaciousin vivo, protecting mice from otherwise fatal STEC disease, but the fact that it is a genetically modified organism (GMO) has been a barrier to clinical development. In the present study, we have overcome this issue by development of Gb3 receptor mimic bacterial ghosts (BGs) that are not classified as GMOs. Gb3-BGs neutralized Stx1 and Stx2in vitrowith high efficiency, whereas alternative Gb3-expressing non-GMO subbacterial particles (minicells and outer membrane blebs) were ineffective. Gb3-BGs were highly efficacious in a murine model of STEC disease. All mice (10/10) treated with Gb3-BGs survived challenge with a highly virulent O113:H21 STEC strain and showed no pathological signs of renal injury. In contrast, 6/10 mice treated with control BGs succumbed to STEC challenge, and survivors exhibited significant weight loss, neutrophilia, and histopathological evidence of renal damage. Thus, Gb3-BGs offer a non-GMO approach to treatment of STEC infection in humans, particularly in an outbreak setting.

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Shiga Toxin Gene Loss and Transfer In Vitro and In Vivo during Enterohemorrhagic Escherichia coli O26 Infection in Humans

Applied and Environmental Microbiology ◽

10.1128/aem.02937-06 ◽

2007 ◽

Vol 73 (10) ◽

pp. 3144-3150 ◽

Cited By ~ 140

Author(s):

Martina Bielaszewska ◽

Rita Prager ◽

Robin Köck ◽

Alexander Mellmann ◽

Wenlan Zhang ◽

...

Keyword(s):

Escherichia Coli ◽

Shiga Toxin ◽

Gene Loss ◽

Pulsed Field Gel Electrophoresis ◽

Enterohemorrhagic Escherichia Coli ◽

Toxin Gene ◽

Shiga Toxins ◽

E Coli

ABSTRACT Escherichia coli serogroup O26 consists of enterohemorrhagic E. coli (EHEC) and atypical enteropathogenic E. coli (aEPEC). The former produces Shiga toxins (Stx), major determinants of EHEC pathogenicity, encoded by bacteriophages; the latter is Stx negative. We have isolated EHEC O26 from patient stools early in illness and aEPEC O26 from stools later in illness, and vice versa. Intrapatient EHEC and aEPEC isolates had quite similar pulsed-field gel electrophoresis (PFGE) patterns, suggesting that they might have arisen by conversion between the EHEC and aEPEC pathotypes during infection. To test this hypothesis, we asked whether EHEC O26 can lose stx genes and whether aEPEC O26 can be lysogenized with Stx-encoding phages from EHEC O26 in vitro. The stx 2 loss associated with the loss of Stx2-encoding phages occurred in 10% to 14% of colonies tested. Conversely, Stx2- and, to a lesser extent, Stx1-encoding bacteriophages from EHEC O26 lysogenized aEPEC O26 isolates, converting them to EHEC strains. In the lysogens and EHEC O26 donors, Stx2-converting bacteriophages integrated in yecE or wrbA. The loss and gain of Stx-converting bacteriophages diversifies PFGE patterns; this parallels findings of similar but not identical PFGE patterns in the intrapatient EHEC and aEPEC O26 isolates. EHEC O26 and aEPEC O26 thus exist as a dynamic system whose members undergo ephemeral interconversions via loss and gain of Stx-encoding phages to yield different pathotypes. The suggested occurrence of this process in the human intestine has diagnostic, clinical, epidemiological, and evolutionary implications.

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

Infection and Immunity ◽

10.1128/iai.00031-09 ◽

2009 ◽

Vol 77 (8) ◽

pp. 3234-3243 ◽

Cited By ~ 44

Author(s):

Sylvia Herold ◽

James C. Paton ◽

Adrienne W. Paton

Keyword(s):

Escherichia Coli ◽

Biofilm Formation ◽

Gastrointestinal Disease ◽

Pcr Analysis ◽

Systemic Complications ◽

E Coli ◽

Acid Protein ◽

Life Threatening ◽

Uremic Syndrome ◽

Enterocyte Effacement

ABSTRACT 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.

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308 IMMUNOLOCALIZATION AND GENE CLONING OF ZEATIN OXYLOSYLTRANSFERASE

HortScience ◽

10.21273/hortsci.29.5.474d ◽

1994 ◽

Vol 29 (5) ◽

pp. 474d-474

Author(s):

Ruth C. Martin ◽

Machteld C. Mok ◽

David W.S. Mok

Keyword(s):

Cdna Libraries ◽

E Coli ◽

Baculovirus Expression ◽

Expression Of Genes ◽

Immature Seeds ◽

Tissue Print ◽

Genes Encoding ◽

In Vitro Systems ◽

Transformation Systems

Cytokinins are widely used in tissue culture and transformation systems; however, little is known of their mode of action or the mechanisms regulating their levels in plant tissues. We are studying enzymes responsible for the metabolism of zeatin in immature seeds of Phaseolus. Selective expression of genes encoding such enzymes may regulate the level of active cytokinins during seed development as well as in in vitro systems. A zeatin O-xylosyltransferase, which mediates the formation of O-xylosylzeatin from trans-zeatin and UDP-xylose, has been isolated and monoclonal antibodies specific to the enzyme have been produced. Tissue print analyses demonstrated that the enzyme is primarily localized in the endosperm. ln situ localization and EM studies indicated that the enzyme is present in the cytoplasm and the nucleus. cDNA libraries were constructed from immature seed mRNA and immunopositive clones were selected. The products of these clones are being analyzed in E. coli and baculovirus expression systems.

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Interactions of polymyxin B in combination with aztreonam, minocycline, meropenem and rifampicin against Escherichia coli producing NDM and OXA-48-group carbapenemases

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01065-21 ◽

2021 ◽

Author(s):

Anna Olsson ◽

Marcus Hong ◽

Hissa Al-Farsi ◽

Christian G. Giske ◽

Pernilla Lagerbäck ◽

...

Keyword(s):

Escherichia Coli ◽

Lipid A ◽

Time Lapse ◽

Polymyxin B ◽

Positive Interactions ◽

Exact Test ◽

E Coli ◽

Genes Encoding ◽

Severe Infections

Objectives. Carbapenemase-producing Enterobacterales pose an increasing medical threat. Combination therapy is often used for severe infections; however, there is little evidence supporting the optimal selection of drugs. This study aimed to determine the in vitro effects of polymyxin B combinations against carbapenemase-producing Escherichia coli . Methods. The interactions of polymyxin B in combination with aztreonam, meropenem, minocycline or rifampicin against 20 clinical isolates of NDM and OXA-48-group-producing E. coli were evaluated using time-lapse microscopy. 24-h samples were spotted on plates with and without 4 x MIC polymyxin B for viable counts. Whole-genome sequencing was applied to identify resistance genes and mutations. Finally, potential associations between combination effects and bacterial genotypes were assessed using Fisher’s exact test. Results. Synergistic and bactericidal effects were observed with polymyxin B and minocycline against 11/20 strains and with polymyxin B and rifampicin against 9/20 strains. The combinations of polymyxin B and aztreonam or meropenem showed synergy against 2/20 strains. Negligible resistance development against polymyxin B was detected. Synergy with polymyxin B and minocycline was associated with genes involved in efflux (presence of tet(B) , wildtype soxR and the marB mutation H44Q) and lipopolysaccharide synthesis ( eptA C27Y, lpxB mutations and lpxK L323S). Synergy with polymyxin B and rifampicin was associated with sequence variations in arnT , which plays a role in lipid A modification. Conclusion. Polymyxin B in combination with minocycline or rifampicin frequently showed positive interactions against NDM- and OXA-48-group-producing E. coli . Synergy was associated with genes encoding efflux and components of the bacterial outer membrane.

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Bioaccumulation of Copper Ions by Escherichia coli Expressing Vanabin Genes from the Vanadium-Rich Ascidian Ascidia sydneiensis samea

Applied and Environmental Microbiology ◽

10.1128/aem.69.11.6442-6446.2003 ◽

2003 ◽

Vol 69 (11) ◽

pp. 6442-6446 ◽

Cited By ~ 19

Author(s):

Tatsuya Ueki ◽

Yasuhisa Sakamoto ◽

Nobuo Yamaguchi ◽

Hitoshi Michibata

Keyword(s):

Escherichia Coli ◽

Metal Binding ◽

Binding Proteins ◽

Copper Ions ◽

Maltose Binding Protein ◽

Molar Ratio ◽

Periplasmic Space ◽

E Coli ◽

Genes Encoding

ABSTRACT The genes encoding two vanadium-binding proteins, vanabin1 and vanabin2, from a vanadium-rich ascidian, Ascidia sydneiensis samea, were recently identified and cloned (T. Ueki, T. Adachi, S. Kawano, M. Aoshima, N. Yamaguchi, K. Kanamori, and H. Michibata, Biochim. Biophys. Acta 1626:43-50, 2003). The vanabins were found to bind vanadium(IV), and an excess of copper(II) ions inhibited the binding of vanadium(IV) to the vanabins in vitro. In this study, we constructed Escherichia coli strains that expressed vanabin1 or vanabin2 fused to maltose-binding protein (MBP) in the periplasmic space. We found that both strains accumulated about twenty times more copper(II) ions than the control BL21 strain, while no significant accumulation of vanadium was observed. The strains expressing either MBP-vanabin1 or MBP-vanabin2 absorbed approximately 70% of the copper ions in the medium to which 10 μM copper (II) ions were initially added. The MBP-vanabin1 and MBP-vanabin2 protein expressed in the periplasm bound to copper ions at a copper:protein molar ratio of 8:1 and 5:1, respectively, but MBP did not bind to copper ions. These data showed that the metal-binding proteins vanabin1 and vanabin2 bound copper ions directly and enhanced the bioaccumulation of copper ions by E. coli.

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