scholarly journals Cytotoxic and Apoptotic Effects of Recombinant Subtilase Cytotoxin Variants of Shiga Toxin-Producing Escherichia coli

2015 ◽  
Vol 83 (6) ◽  
pp. 2338-2349 ◽  
Author(s):  
J. Funk ◽  
N. Biber ◽  
M. Schneider ◽  
E. Hauser ◽  
S. Enzenmüller ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Vero Cells ◽  
Toxic Activity ◽  
Content Type ◽  
B Subunits ◽  
Induced Apoptosis ◽  
The Individual ◽  
Apoptotic Effects ◽  
Subtilase Cytotoxin

In this study, the cytotoxicity of the recently described subtilase variant SubAB2-2of Shiga toxin-producingEscherichia coliwas determined and compared to the plasmid-encoded SubAB1and the chromosome-encoded SubAB2-1variant. The genes for the respective enzymatic active (A) subunits and binding (B) subunits of the subtilase toxins were amplified and cloned. The recombinant toxin subunits were expressed and purified. Their cytotoxicity on Vero cells was measured for the single A and B subunits, as well as for mixtures of both, to analyze whether hybrids with toxic activity can be identified. The results demonstrated that all three SubAB variants are toxic for Vero cells. However, the values for the 50% cytotoxic dose (CD50) differ for the individual variants. Highest cytotoxicity was shown for SubAB1. Moreover, hybrids of subunits from different subtilase toxins can be obtained which cause substantial cytotoxicity to Vero cells after mixing the A and B subunits prior to application to the cells, which is characteristic for binary toxins. Furthermore, higher concentrations of the enzymatic subunit SubA1exhibited cytotoxic effects in the absence of the respective B1subunit. A more detailed investigation in the human HeLa cell line revealed that SubA1alone induced apoptosis, while the B1subunit alone did not induce cell death.

scholarly journals Transcription of the Subtilase Cytotoxin Gene subAB1 in Shiga Toxin-Producing Escherichia coli Is Dependent on hfq and hns

2019 ◽  
Vol 85 (20) ◽  
Author(s):  
Laura Heinisch ◽  
Katharina Zoric ◽  
Maike Krause ◽  
Herbert Schmidt
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Shiga Toxin ◽  
Promoter Activity ◽  
Deletion Mutants ◽  
Content Type ◽  
E Coli ◽  
Intensive Investigation ◽  
Subtilase Cytotoxin

ABSTRACT Certain foodborne Shiga toxin-producing Escherichia coli (STEC) strains carry genes encoding the subtilase cytotoxin (SubAB). Although the mode of action of SubAB is under intensive investigation, information about the regulation of subAB gene expression is currently not available. In this study, we investigated the regulation of the chromosomal subAB1 gene in laboratory E. coli strain DH5α and STEC O113:H21 strain TS18/08 using a luciferase reporter gene assay. Special emphasis was given to the role of the global regulatory protein genes hfq and hns in subAB1 promoter activity. Subsequently, quantitative real-time PCR was performed to analyze the expression of Shiga toxin 2a (Stx2a), SubAB1, and cytolethal distending toxin V (Cdt-V) genes in STEC strain TS18/08 and its isogenic hfq and hns deletion mutants. The deletion of hfq led to a significant increase of up to 2-fold in subAB1 expression, especially in the late growth phase, in both strains. However, deletion of hns showed different effects on the promoter activity during the early and late exponential growth phases in both strains. Furthermore, upregulation of stx2a and cdt-V was demonstrated in hfq and hns deletion mutants in TS18/08. These data showed that the expression of subAB1, stx2a, and cdt-V is integrated in the regulatory network of global regulators Hfq and H-NS in Escherichia coli. IMPORTANCE Shiga toxin-producing Escherichia coli (STEC) strains are responsible for outbreaks of foodborne diseases, such as hemorrhagic colitis and the hemolytic uremic syndrome. The pathogenicity of those strains can be attributed to, among other factors, the production of toxins. Recently, the subtilase cytotoxin was detected in locus of enterocyte effacement (LEE)-negative STEC, and it was confirmed that it contributes to the cytotoxicity of those STEC strains. Although the mode of action of SubAB1 is under intensive investigation, the regulation of gene expression is currently not known. The global regulatory proteins H-NS and Hfq have impact on many cellular processes and have been described to regulate virulence factors as well. Here, we investigate the role of hns and hfq in expression of subAB1 as well as stx2a and cdt-V in an E. coli laboratory strain as well as in wild-type STEC strain TS18/08.

scholarly journals Draft Genome Sequences of Five Shiga Toxin-Producing Escherichia coli Isolates Harboring the New and Recently Described Subtilase Cytotoxin Allelic Variant subAB2-3

2017 ◽  
Vol 5 (8) ◽  
Author(s):  
Taurai Tasara ◽  
Lisa Fierz ◽  
Jochen Klumpp ◽  
Herbert Schmidt ◽  
Roger Stephan
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Sequence Data ◽  
Draft Genome ◽  
Allelic Variant ◽  
Genome Sequences ◽  
Content Type ◽  
Subtilase Cytotoxin

ABSTRACT We present here the draft genome sequences of five Shiga toxin-producing Escherichia coli (STEC) strains which tested positive in a primary subAB screening. Assembly and annotation of the draft genomes revealed that all strains harbored the recently described allelic variant subAB 2-3 . Based on the sequence data, primers were designed to identify and differentiate this variant.

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 Prevalences of Shiga Toxin Subtypes and Selected Other Virulence Factors among Shiga-Toxigenic Escherichia coli Strains Isolated from Fresh Produce

2013 ◽  
Vol 79 (22) ◽  
pp. 6917-6923 ◽  
Author(s):  
Peter C. H. Feng ◽  
Shanker Reddy
Keyword(s):  
Escherichia Coli ◽  
Virulence Factors ◽  
Virulence Factor ◽  
Shiga Toxin ◽  
Fresh Produce ◽  
Putative Virulence Factor ◽  
Content Type ◽  
Human Illness ◽  
Subtilase Cytotoxin ◽  
Virulence Factor Genes

ABSTRACTShiga-toxigenicEscherichia coli(STEC) strains were isolated from a variety of fresh produce, but mostly from spinach, with an estimated prevalence rate of 0.5%. A panel of 132 produce STEC strains were characterized for the presence of virulence and putative virulence factor genes and for Shiga toxin subtypes. About 9% of the isolates were found to have theeaegene, which encodes the intimin binding protein, and most of these belonged to known pathogenic STEC serotypes, such as O157:H7 and O26:H11, or to serotypes that reportedly have caused human illness. Among theeae-negative strains, there were three O113:H21 strains and one O91:H21 strain, which historically have been implicated in illness and therefore may be of concern as well. TheehxAgene, which encodes enterohemolysin, was found in ∼60% of the isolates, and thesaaandsubABgenes, which encode STEC agglutinating adhesin and subtilase cytotoxin, respectively, were found in ∼30% of the isolates. However, the precise roles of these three putative virulence factors in STEC pathogenesis have not yet been fully established. Thestx1aandstx2asubtypes were present in 22% and 56%, respectively, of the strains overall and were the most common subtypes among produce STEC strains. Thestx2dsubtype was the second most common subtype (28% overall), followed bystx2c(7.5%), and only 2 to 3% of the produce STEC strains had thestx2eandstx2gsubtypes. Almost half of the produce STEC strains had only partial serotypes or were untyped, and most of those that were identified belonged to unremarkable serotypes. Considering the uncertainties of some of these Stx subtypes and putative virulence factors in causing human illness, it is difficult to determine the health risk of many of these produce STEC strains.

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 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 Novel Subtilase Cytotoxin Produced by Shiga-Toxigenic Escherichia coli Induces Apoptosis in Vero Cells via Mitochondrial Membrane Damage

2009 ◽  
Vol 77 (7) ◽  
pp. 2919-2924 ◽  
Author(s):  
Gen Matsuura ◽  
Naoko Morinaga ◽  
Kinnosuke Yahiro ◽  
Reiko Komine ◽  
Joel Moss ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Mitochondrial Membrane ◽  
Caspase 3 ◽  
Membrane Damage ◽  
Annexin V ◽  
Vero Cells ◽  
Caspase 8 ◽  
Caspase Inhibitors ◽  
Induced Apoptosis ◽  
Subtilase Cytotoxin

ABSTRACT 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.

scholarly journals 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 ◽  
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.

scholarly journals Phylogenetic structure of Shiga toxin-producing Escherichia coli O157:H7 from sub-lineage to SNPs

2021 ◽  
Author(s):  
Timothy J. Dallman ◽  
David R. Greig ◽  
Saheer E. Gharbia ◽  
Claire Jenkins
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Sequence Similarity ◽  
Evidence Base ◽  
Point Sources ◽  
Single Linkage ◽  
Phylogenetic Structure ◽  
Content Type ◽  
Geographically Dispersed ◽  
Cluster Threshold

Sequence similarity of pathogen genomes can infer the relatedness between isolates as the fewer genetic differences identified between pairs of isolates, the less time since divergence from a common ancestor. Clustering based on hierarchical single linkage clustering of pairwise SNP distances has been employed to detect and investigate outbreaks. Here, we evaluated the evidence-base for the interpretation of phylogenetic clusters of Shiga toxin-producing Escherichia coli (STEC) O157:H7. Whole genome sequences of 1193 isolates of STEC O157:H7 submitted to Public Health England between July 2015 and December 2016 were mapped to the Sakai reference strain. Hierarchical single linkage clustering was performed on the pairwise SNP difference between all isolates at descending distance thresholds. Cases with known epidemiological links fell within 5-SNP single linkage clusters. Five-SNP single linkage community clusters where an epidemiological link was not identified were more likely to be temporally and/or geographically related than sporadic cases. Ten-SNP single linkage clusters occurred infrequently and were challenging to investigate as cases were few, and temporally and/or geographically dispersed. A single linkage cluster threshold of 5-SNPs has utility for the detection of outbreaks linked to both persistent and point sources. Deeper phylogenetic analysis revealed that the distinction between domestic UK and imported isolates could be inferred at the sub-lineage level. Cases associated with domestically acquired infection that fall within clusters that are predominantly travel associated are likely to be caused by contaminated imported food.

scholarly journals Subtilase Cytotoxin-Coding Genes in Verotoxin-Producing Escherichia coli Strains from Sheep and Goats Differ from Those from Cattle

2011 ◽  
Vol 77 (23) ◽  
pp. 8259-8264 ◽  
Author(s):  
José A. Orden ◽  
Pilar Horcajo ◽  
Ricardo de la Fuente ◽  
José A. Ruiz-Santa-Quiteria ◽  
Gustavo Domínguez-Bernal ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Human Disease ◽  
Virulence Genes ◽  
Small Ruminants ◽  
Human Beings ◽  
Content Type ◽  
Sheep And Goats ◽  
E Coli ◽  
Healthy Cattle ◽  
Subtilase Cytotoxin

ABSTRACTSubtilase cytotoxin (SubAB) from verotoxin (VT)-producingEscherichia coli(VTEC) strains was first described in the 98NK2 strain and has been associated with human disease. However, SubAB has recently been found in two VT-negativeE. colistrains (ED 591 and ED 32). SubAB is encoded by two closely linked, cotranscribed genes (subAandsubB). In this study, we investigated the presence ofsubABgenes in 52 VTEC strains isolated from cattle and 209 strains from small ruminants, using PCR. Most (91.9%) VTEC strains from sheep and goats and 25% of the strains from healthy cattle possessedsubABgenes. The presence ofsubABin a high percentage of the VTEC strains from small ruminants might increase the pathogenicity of these strains for human beings. Some differences in the results of PCRs and in the association with some virulence genes suggested the existence of different variants ofsubAB. We therefore sequenced thesubAgene in 12 strains and showed that thesubAgene in most of thesubAB-positive VTEC strains from cattle was almost identical (about 99%) to that in the 98NK2 strain, while thesubAgene in most of thesubAB-positive VTEC strains from small ruminants was almost identical to that in the ED 591 strain. We propose the termssubAB1to describe the SubAB-coding genes resembling that in the 98NK2 strain andsubAB2to describe those resembling that in the ED 591 strain.

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