1,677 results on '"Shiga Toxin 2"'
Search Results
2. Deletion of Sphingosine Kinase 2 Attenuates Acute Kidney Injury in Mice with Hemolytic-Uremic Syndrome.
- Author
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Müller T, Krieg N, Lange-Polovinkin AI, Wissuwa B, Gräler MH, Dennhardt S, and Coldewey SM
- Subjects
- Animals, Mice, Disease Models, Animal, Sphingolipids metabolism, Kidney pathology, Kidney metabolism, Mice, Inbred C57BL, Shiga Toxin 2, Gene Deletion, Male, Phosphotransferases (Alcohol Group Acceptor) genetics, Phosphotransferases (Alcohol Group Acceptor) metabolism, Acute Kidney Injury metabolism, Acute Kidney Injury pathology, Acute Kidney Injury etiology, Acute Kidney Injury genetics, Hemolytic-Uremic Syndrome pathology, Hemolytic-Uremic Syndrome genetics, Mice, Knockout
- Abstract
Typical hemolytic uremic syndrome (HUS) can occur as a severe systemic complication of infections with Shiga toxin (Stx)-producing Escherichia coli . Its pathology can be induced by Stx types, resulting in toxin-mediated damage to renal barriers, inflammation, and the development of acute kidney injury (AKI). Two sphingosine kinase (SphK) isozymes, SphK1 and SphK2, have been shown to be involved in barrier maintenance and renal inflammatory diseases. Therefore, we sought to determine their role in the pathogenesis of HUS. Experimental HUS was induced by the repeated administration of Stx2 in wild-type (WT) and SphK1 (SphK1
-/- ) or SphK2 (SphK2-/- ) null mutant mice. Disease severity was evaluated by assessing clinical symptoms, renal injury and dysfunction, inflammatory status and sphingolipid levels on day 5 of HUS development. Renal inflammation and injury were found to be attenuated in the SphK2-/- mice, but exacerbated in the SphK1-/- mice compared to the WT mice. The divergent outcome appeared to be associated with oppositely altered sphingolipid levels. This study represents the first description of the distinct roles of SphK1-/- and SphK2-/- in the pathogenesis of HUS. The identification of sphingolipid metabolism as a potential target for HUS therapy represents a significant advance in the field of HUS research.- Published
- 2024
- Full Text
- View/download PDF
3. Data on Escherichia coli O157:H7 Reported by Vincent Li and Colleagues (A challenging STEC strain isolation from patients' stools: an O166:H15 STEC strain with the stx2 gene).
- Subjects
ESCHERICHIA coli O157:H7 ,IMMUNOMAGNETIC separation ,ESCHERICHIA coli physiology ,BACTERIAL toxins ,TOXINS ,GLYCOSIDASES ,RIBOSOMES - Abstract
This article discusses a research study on Escherichia coli O157:H7, a strain of bacteria that can cause foodborne illnesses. The study focuses on two patients with acute gastroenteritis who tested positive for Shiga toxin-producing E. coli (STEC). The researchers used immunomagnetic separation (IMS) to isolate the STEC strains from the patients' stools, as routine culture methods failed. The strains were identified as O166:H15 STEC and were found to carry the Shiga toxin 2 encoding gene. The study highlights the challenges in isolating this strain and emphasizes the importance of characterizing virulence genes in these isolates. [Extracted from the article]
- Published
- 2024
4. Development of a homogeneous time-resolved FRET (HTRF) assay for the quantification of Shiga toxin 2 produced by E. coli
- Author
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Keiji Nakamura, Chikashi Tokuda, Hideyuki Arimitsu, Yoshiki Etoh, Mitsuhiro Hamasaki, Yuichiro Deguchi, Itsuki Taniguchi, Yasuhiro Gotoh, Yoshitoshi Ogura, and Tetsuya Hayashi
- Subjects
Shiga toxin 2 ,Shiga toxin-producing Escherichia coli ,Quantification method ,Homogeneous time-resolved fluorescence resonance energy transfer ,Medicine ,Biology (General) ,QH301-705.5 - Abstract
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.
- Published
- 2021
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5. A phase I study to evaluate the safety, tolerance and pharmacokinetics of anti-Shiga toxin hyperimmune equine F (ab') 2 fragments in healthy volunteers.
- Author
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Hiriart Y, Scibona P, Ferraris A, Belloso WH, Beruto V, Garcia Bournissen F, Zylberman V, Muñoz L, Goldbaum F, Spatz L, Colonna M, Sanguineti S, and Simonovich VA
- Subjects
- Child, Adult, Humans, Animals, Horses, Shiga Toxin 1, Healthy Volunteers, Single-Blind Method, Shiga Toxin 2, Hemolytic-Uremic Syndrome
- Abstract
Aims: Shiga toxin-producing Escherichia coli-haemolytic uraemic syndrome (STEC-HUS) is considered a toxaemic disorder in which early intervention with neutralizing antibodies may have therapeutic benefits. INM004, composed of F (ab')
2 fragments from equine immunoglobulins, neutralizes Stx1/Stx2, potentially preventing the onset of HUS., Methods: A single-centre, randomized, phase 1, single-blind, placebo-controlled clinical trial to evaluate INM004 safety, tolerance and pharmacokinetics (PK) in healthy adult volunteers, was conducted; in stage I, eight subjects were divided in two cohorts (n = 4) to receive a single INM004 dose of 2 or 4 mg kg-1 , or placebo (INM004:placebo ratio of 3:1). In stage II, six subjects received three INM004 doses of 4 mg kg-1 repeated every 24 h, or placebo (INM004:placebo ratio of 5:1)., Results: Eight subjects (57.1%) experienced mild treatment-emergent adverse events (TEAEs); most frequent were rhinitis, headache and flushing, resolved within 24 h without changes in treatment or additional intervention. No serious AEs were reported. Peak concentrations of INM004 occurred within 2 h after infusion, with median Cmax values of 45.1 and 77.7 μg mL-1 for 2 and 4 mg kg-1 , respectively. The serum concentration of INM004 declined in a biphasic manner (t1/2 range 30.7-52.9 h). Systemic exposures increased with each subsequent dose in a dose-proportional manner, exhibiting accumulation. Geometric median Cmax and AUC values were 149 and 10 300 μg h mL-1 , respectively, in the repeated dose regimen. Additionally, samples from subjects that received INM004 at 2 mg kg-1 showed neutralizing capacity against Stx1 and Stx2 in in vitro assays., Conclusions: The results obtained in this first-in-human study support progression into the phase 2 trial in children with HUS., (© 2024 British Pharmacological Society.)- Published
- 2024
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6. Development of a Rapid and Sensitive CANARY Biosensor Assay for the Detection of Shiga Toxin 2 from Escherichia coli .
- Author
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Tam CC, Wang Y, Du WX, Flannery AR, and He X
- Subjects
- Escherichia coli, Shiga Toxin, Biological Assay, Shiga Toxin 2, Abrin
- Abstract
Shiga-toxin-producing Escherichia coli (STEC) causes a wide spectrum of diseases including hemorrhagic colitis and hemolytic uremic syndrome (HUS). The current Food Safety Inspection Service (FSIS) testing methods for STEC use the Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) protocol, which includes enrichment, cell plating, and genomic sequencing and takes time to complete, thus delaying diagnosis and treatment. We wanted to develop a rapid, sensitive, and potentially portable assay that can identify STEC by detecting Shiga toxin (Stx) using the CANARY (Cellular Analysis and Notification of Antigen Risks and Yields) B-cell based biosensor technology. Five potential biosensor cell lines were evaluated for their ability to detect Stx2. The results using the best biosensor cell line (T5) indicated that this biosensor was stable after reconstitution with assay buffer covered in foil at 4 °C for up to 10 days with an estimated limit of detection (LOD) of ≈0.1-0.2 ng/mL for days up to day 5 and ≈0.4 ng/mL on day 10. The assay detected a broad range of Stx2 subtypes, including Stx2a, Stx2b, Stx2c, Stx2d, and Stx2g but did not cross-react with closely related Stx1, abrin, or ricin. Additionally, this assay was able to detect Stx2 in culture supernatants of STEC grown in media with mitomycin C at 8 and 24 h post-inoculation. These results indicate that the STEC CANARY biosensor developed in this study is sensitive, reproducible, specific, rapid (≈3 min), and may be applicable for surveillance of the environment and food to protect public health.
- Published
- 2024
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7. Environmental Cues Modulate Microglial Cell Behavior Upon Shiga Toxin 2 From Enterohemorrhagic Escherichia coli Exposure
- Author
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Clara Berdasco, Maite Duhalde Vega, María Victoria Rosato-Siri, and Jorge Goldstein
- Subjects
Shiga Toxin 2 ,microglial cell primary cultures ,LPS challenge ,heat shock exposure ,receptor Gb3-pathway ,Microbiology ,QR1-502 - Abstract
Shiga toxin (Stx) produced by enterohemorrhagic E. coli produces hemolytic uremic syndrome and encephalopathies in patients, which can lead to either reversible or permanent neurological abnormalities, or even fatal cases depending on the degree of intoxication. It has been observed that the inflammatory component plays a decisive role in the severity of the disease. Therefore, the objective of this work was to evaluate the behavior of microglial cell primary cultures upon Stx2 exposure and heat shock or lipopolysaccharide challenges, as cues which modulate cellular environments, mimicking fever and inflammation states, respectively. In these contexts, activated microglial cells incorporated Stx2, increased their metabolism, phagocytic capacity, and pro-inflammatory profile. Stx2 uptake was associated to receptor globotriaosylceramide (Gb3)-pathway. Gb3 had three clearly distinguishable distribution patterns which varied according to different contexts. In addition, toxin uptake exhibited both a Gb3-dependent and a Gb3-independent binding depending on those contexts. Altogether, these results suggest a fundamental role for microglial cells in pro-inflammatory processes in encephalopathies due to Stx2 intoxication and highlight the impact of environmental cues.
- Published
- 2020
- Full Text
- View/download PDF
8. Toxigenic properties and stx phage characterization of Escherichia coli O157 isolated from animal sources in a developing country setting
- Author
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Mahdia Rahman, Ashikun Nabi, Md Asadulghani, Shah M. Faruque, and Mohammad Aminul Islam
- Subjects
E. coli O157 ,Shiga toxin 2 ,Toxin non-producing ,stx phage ,Microbiology ,QR1-502 - Abstract
Abstract Background In many Asian countries including Bangladesh E. coli O157 are prevalent in animal reservoirs and in the food chain, but the incidence of human infection due to E. coli O157 is rare. One of the reasons could be inability of the organism from animal origin to produce sufficient amount of Shiga toxin (Stx), which is the main virulence factor associated with the severe sequelae of infection. This study aimed to fill out this knowledge gap by investigating the toxigenic properties and characteristics of stx phage of E. coli O157 isolated from animal sources in Bangladesh. Results We analysed 47 stx 2 positive E. coli O157 of food/animal origin for stx 2 gene variants, Shiga toxin production, presence of other virulence genes, stx phage insertion sites, presence of genes associated with functionality of stx phages (Q 933 and Q 21) and stx 2 upstream region. Of the 47 isolates, 46 were positive for both stx 2a and stx 2d while the remaining isolate was positive for stx 2d only. Reverse Passive Latex Agglutination assay (RPLA) showed that 42/47 isolates produced little or no toxin, while 5 isolates produced a high titre of toxin (64 to 128). 39/47 isolates were positive for the Toxin Non-Producing (TNP) specific regions in the stx 2 promoter. Additionally, all isolates were negative for antiterminator Q 933while a majority of isolates were positive for Q 21 gene suggesting the presence of defective stx phage. Of the yehV and wrbA phage insertion sites, yehV was found occupied in 11 isolates while wrbA site was intact in all the isolates. None of the isolates was positive for the virulence gene, cdt but all were positive for hlyA, katP, etpD and eae genes. Isolates that produced high titre Stx (n = 5) produced complete phage particles capable of infecting multiple bacterial hosts. One of these phages was shown to produce stable lysogens in host strains rendering the Stx2 producing ability. Conclusion Despite low frequency in the tested isolates, E. coli O157 isolates in Bangladesh carry inducible stx phages and have the capacity to produce Stx2, indicating a potential risk of E. coli O157 infection in humans.
- Published
- 2018
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9. New Findings Reported from Albany Describe Advances in Biosensors (Development of a Rapid and Sensitive CANARY Biosensor Assay for the Detection of Shiga Toxin 2 from Escherichia coli).
- Abstract
A recent report discusses research on the development of a rapid and sensitive biosensor assay for the detection of Shiga toxin 2 (Stx2) from Escherichia coli (E. coli). Shiga-toxin-producing E. coli can cause various diseases, including hemorrhagic colitis and hemolytic uremic syndrome. The researchers aimed to create an assay that could quickly identify STEC by detecting Stx using the CANARY B-cell based biosensor technology. The results showed that the biosensor was stable and had a limit of detection of 0.1-0.2 ng/mL. The assay was able to detect various subtypes of Stx2 and was specific and reproducible. The researchers concluded that this biosensor could be used for surveillance of the environment and food to protect public health. [Extracted from the article]
- Published
- 2024
10. Effect of phage variation on Shiga toxin 2 (Stx2) production and the virulence of Stx-producing Escherichia coli.
- Abstract
This article discusses the effect of phage variation on the production of Shiga toxin 2 (Stx2) and the virulence of Shiga toxin-producing Escherichia coli (STEC). Stx-producing E. coli can cause serious gastrointestinal illnesses, and Stx2 production is a major risk factor for severe infections. The study analyzed the genomic diversity of Stx phages in 71 strains of STEC and found that the production of Stx2 varied between strains. The research also identified specific types of Stx phages that promoted Stx2 production and virulence in host strains. This preprint has not yet undergone peer review. [Extracted from the article]
- Published
- 2024
11. High-fat and high-carbohydrate diets worsen the mouse brain susceptibility to damage produced by enterohemorrhagic Escherichia coli Shiga toxin 2.
- Subjects
ESCHERICHIA coli O157:H7 ,ESCHERICHIA coli toxins ,HIGH-carbohydrate diet ,HIGH-fat diet ,BRAIN damage ,FAT - Abstract
A recent study investigated the impact of high-fat and high-carbohydrate diets on the susceptibility of mouse brains to damage caused by enterohemorrhagic Escherichia coli (E. coli) Shiga toxin 2. The researchers found that both high-fat and high-carbohydrate diets worsened the deleterious effects of the toxin, with the combination of both diets yielding the most severe results. These effects included alterations in the neurovascular unit, neurodegeneration, and impairment of motor and sensitivity functions. The study suggests that poor nutrition may negatively influence patients affected by the toxin at a neurological level. However, it is important to note that these findings have not yet undergone peer review. [Extracted from the article]
- Published
- 2024
12. Environmental Cues Modulate Microglial Cell Behavior Upon Shiga Toxin 2 From Enterohemorrhagic Escherichia coli Exposure.
- Author
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Berdasco, Clara, Duhalde Vega, Maite, Rosato-Siri, María Victoria, and Goldstein, Jorge
- Subjects
ESCHERICHIA coli O157:H7 ,TOXINS ,CELL culture ,MICROGLIA - Abstract
Shiga toxin (Stx) produced by enterohemorrhagic E. coli produces hemolytic uremic syndrome and encephalopathies in patients, which can lead to either reversible or permanent neurological abnormalities, or even fatal cases depending on the degree of intoxication. It has been observed that the inflammatory component plays a decisive role in the severity of the disease. Therefore, the objective of this work was to evaluate the behavior of microglial cell primary cultures upon Stx2 exposure and heat shock or lipopolysaccharide challenges, as cues which modulate cellular environments, mimicking fever and inflammation states, respectively. In these contexts, activated microglial cells incorporated Stx2, increased their metabolism, phagocytic capacity, and pro-inflammatory profile. Stx2 uptake was associated to receptor globotriaosylceramide (Gb3)-pathway. Gb3 had three clearly distinguishable distribution patterns which varied according to different contexts. In addition, toxin uptake exhibited both a Gb3-dependent and a Gb3-independent binding depending on those contexts. Altogether, these results suggest a fundamental role for microglial cells in pro-inflammatory processes in encephalopathies due to Stx2 intoxication and highlight the impact of environmental cues. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
13. PIKfyve Regulation of Endosome‐Linked Pathways
- Author
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de Lartigue, Jane, Polson, Hannah, Feldman, Morri, Shokat, Kevan, Tooze, Sharon A, Urbé, Sylvie, and Clague, Michael J
- Subjects
Biotechnology ,Underpinning research ,1.1 Normal biological development and functioning ,Generic health relevance ,Androstadienes ,Animals ,Autophagy ,CD8 Antigens ,Endosomes ,ErbB Receptors ,Furin ,HeLa Cells ,Humans ,Membrane Glycoproteins ,Microtubule-Associated Proteins ,Molecular Structure ,Phosphatidylinositol 3-Kinases ,Phosphoinositide-3 Kinase Inhibitors ,Protein Kinase Inhibitors ,RNA ,Small Interfering ,Receptor Protein-Tyrosine Kinases ,Receptor ,IGF Type 2 ,Receptors ,Cytoplasmic and Nuclear ,Recombinant Fusion Proteins ,Shiga Toxin 2 ,Vacuoles ,Wortmannin ,trans-Golgi Network ,autophagy ,EGF receptor ,endocytosis ,phosphoinositide ,PIKfyve ,Hela Cells ,Biochemistry and Cell Biology ,Medical Microbiology ,Developmental Biology - Abstract
The phosphoinositide 5-kinase (PIKfyve) is a critical enzyme for the synthesis of PtdIns(3,5)P2, that has been implicated in various trafficking events associated with the endocytic pathway. We have now directly compared the effects of siRNA-mediated knockdown of PIKfyve in HeLa cells with a specific pharmacological inhibitor of enzyme activity. Both approaches induce changes in the distribution of CI-M6PR and trans-Golgi network (TGN)-46 proteins, which cycles between endosomes and TGN, leading to their accumulation in dispersed punctae, whilst the TGN marker golgin-245 retains a perinuclear disposition. Trafficking of CD8-CI-M6PR (retromer-dependent) and CD8-Furin (retromer-independent) chimeras from the cell surface to the TGN is delayed following drug administration, as is the transport of the Shiga toxin B-subunit. siRNA knockdown of PIKfyve produced no defect in epidermal growth factor receptor (EGFR) degradation, unless combined with knockdown of its activator molecule Vac14, suggesting that a low threshold of PtdIns(3,5)P2 is necessary and sufficient for this pathway. Accordingly pharmacological inhibition of PIKfyve results in a profound block to the lysosomal degradation of activated epidermal growth factor (EGF) and Met receptors. Immunofluorescence revealed EGF receptors to be trapped in the interior of a swollen endosomal compartment. In cells starved of amino acids, PIKfyve inhibition leads to the accumulation of the lipidated form of GFP-LC3, a marker of autophagosomal structures, which can be visualized as fluorescent punctae. We suggest that PIKfyve inhibition may render the late endosome/lysosome compartment refractory to fusion with both autophagosomes and with EGFR-containing multivesicular bodies.
- Published
- 2009
14. Differences in the Shiga Toxin (Stx) 2a Phage Regulatory Region Influence Stx2 Localization and Virulence of Stx-producing Escherichia Coli in Mice
- Author
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Microbiology & Immunology (MIC), SOM, Rama R. Atitkar, Angela R. Melton-Celsa, Microbiology & Immunology (MIC), SOM, Rama R. Atitkar, and Angela R. Melton-Celsa
- Abstract
Differential expression and localization of Shiga toxin type 2 (Stx2) from recA mutant derivatives of O157:H7 Escherichia coli strains Rama R. Atitkar1,2 , Angela R. Melton-Celsa1 Introduction Serotype O157:H7 STEC are associated with more severe disease, and they encode differing combinations of Stx types and subtypes. Encoded on the stx-phage, stx expression is linked to phage induction and the lytic cycle. Repressor-operator interactions within the phage regulatory region control the initiation and progression of the lytic cycle, and the RecA protein stimulates phage repressor cleavage. Genomic comparisons of stx-phages have uncovered regions of significant genetic diversity, including within the regulatory region. However, it is still unclear whether diversity within this region could influence STEC pathogenesis and virulence. 1 Uniformed Services University, Bethesda, Maryland, USA 2 Henry M. Jackson Foundation, Bethesda, Maryland, USA Correspondence: angela.melton-celsa@usuhs.edu STEC isolates exhibit differences in RecA-independent cytotoxicity Acknowledgements/Disclaimer This work was supported by National Institutes of Health grant R37 AI020148. Strains used in this study and related work were obtained from E. Dudley and M. Muniesa. The opinions or assertions contained herein are the private ones of the author/speaker and are not to be construed as official or reflecting the views of the Department of Defense, the Uniformed Services University of the Health Sciences, any other agency of the U.S. Government, or the Henry M. Jackson Foundation for the Advancement of Military Medicine. Methods The stx2a-phage regulatory region mediates differential RecA-independent cytotoxicity Background Studies about RecA and stx-phages have indirectly identified RecA-independent stx-phage induction and/or Stx production: - EDTA/chelation influences Stx production from recA-negative stx-phage lysogens (Imamovic et al, 2012). - Phage are detected from cultures of stx-phage ly, RITM0037416, Serotype O157:H7 STEC are associated with more severe disease, and they encode differing combinations of Stx types and subtypes. Encoded on the stx-phage, stx expression is linked to phage induction and the lytic cycle. Repressor-operator interactions within the phage regulatory region control the initiation and progression of the lytic cycle, and the RecA protein stimulates phage repressor cleavage. Genomic comparisons of stx-phages have uncovered regions of significant genetic diversity, including within the regulatory region. However, it is still unclear whether diversity within this region could influence STEC pathogenesis and virulence.
- Published
- 2023
15. Tamoxifen Derivatives Alter Retromer-Dependent Endosomal Tubulation and Sorting to Block Retrograde Trafficking of Shiga Toxins
- Author
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Andrey S. Selyunin, Karinel Nieves-Merced, Danyang Li, Stanton F. McHardy, and Somshuvra Mukhopadhyay
- Subjects
Shiga toxin ,Shiga toxin 2 ,tamoxifen ,retromer ,trafficking ,Golgi ,Medicine - Abstract
Shiga toxin 1 and 2 (STx1 and STx2) undergo retrograde trafficking to reach the cytosol of cells where they target ribosomes. As retrograde trafficking is essential for disease, inhibiting STx1/STx2 trafficking is therapeutically promising. Recently, we discovered that the chemotherapeutic drug tamoxifen potently inhibits the trafficking of STx1/STx2 at the critical early endosome-to-Golgi step. We further reported that the activity of tamoxifen against STx1/STx2 is independent of its selective estrogen receptor modulator (SERM) property and instead depends on its weakly basic chemical nature, which allows tamoxifen to increase endolysosomal pH and alter the recruitment of retromer to endosomes. The goal of the current work was to obtain a better understanding of the mechanism of action of tamoxifen against the more disease-relevant toxin STx2, and to differentiate between the roles of changes in endolysosomal pH and retromer function. Structure activity relationship (SAR) analyses revealed that a weakly basic amine group was essential for anti-STx2 activity. However, ability to deacidify endolysosomes was not obligatorily necessary because a tamoxifen derivative that did not increase endolysosomal pH exerted reduced, but measurable, activity. Additional assays demonstrated that protective derivatives inhibited the formation of retromer-dependent, Golgi-directed, endosomal tubules, which mediate endosome-to-Golgi transport, and the sorting of STx2 into these tubules. These results identify retromer-mediated endosomal tubulation and sorting to be fundamental processes impacted by tamoxifen; provide an explanation for the inhibitory effect of tamoxifen on STx2; and have important implications for the therapeutic use of tamoxifen, including its development for treating Shiga toxicosis.
- Published
- 2021
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16. A novel Shiga toxin 2a neutralizing antibody therapeutic with low immunogenicity and high efficacy.
- Author
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Kirkland ME, Patfield S, Hughes AC, Hernlem B, and He X
- Subjects
- Humans, Adult, Animals, Mice, Shiga Toxin therapeutic use, Shiga Toxin 2, Antibodies, Monoclonal, Humanized therapeutic use, Escherichia coli Infections drug therapy, Shiga-Toxigenic Escherichia coli, Hemolytic-Uremic Syndrome drug therapy
- Abstract
Shiga toxin-producing Escherichia coli infections are difficult to treat due to the risk of antibiotic-induced stress upregulating the production of toxins, medical treatment is consequently limited to supportive care to prevent the development of hemolytic uremic syndrome (HUS). Here, we introduce a potentially therapeutic humanized mouse monoclonal antibody (Hu-mAb 2-5) targeting Stx2a, the most common Shiga toxin subtype identified from outbreaks. We demonstrate that Hu-mAb 2-5 has low immunogenicity in healthy adults ex vivo and high neutralizing efficacy in vivo , protecting mice from mortality and HUS-related tissue damage., Competing Interests: The authors declare no conflict of interest.
- Published
- 2024
- Full Text
- View/download PDF
17. Red blood cell-derived arginase release in hemolytic uremic syndrome.
- Author
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Friberg N, Arvidsson I, Tontanahal A, Kristoffersson AC, Gram M, Kaplan BS, and Karpman D
- Subjects
- Humans, Child, Animals, Mice, Shiga Toxin 2, Endothelial Cells, Hemolysis, Arginase, Erythrocytes, Urea, Arginine, Ornithine, Lactate Dehydrogenases, Hemolytic-Uremic Syndrome complications, Hemolytic-Uremic Syndrome therapy, Thrombotic Microangiopathies complications, Escherichia coli O157, Renal Insufficiency, Escherichia coli Infections complications, Escherichia coli Infections therapy
- Abstract
Background: Hemolysis is a cardinal feature of hemolytic uremic syndrome (HUS) and during hemolysis excess arginase 1 is released from red blood cells. Increased arginase activity leads to reduced L-arginine, as it is converted to urea and L-ornithine, and thereby reduced nitric oxide bioavailability, with secondary vascular injury. The objective of this study was to investigate arginase release in HUS patients and laboratory models and correlate arginase levels to hemolysis and kidney injury., Methods: Two separate cohorts of patients (n = 47 in total) with HUS associated with Shiga toxin-producing enterohemorrhagic E. coli (EHEC) and pediatric controls (n = 35) were investigated. Two mouse models were used, in which mice were either challenged intragastrically with E. coli O157:H7 or injected intraperitoneally with Shiga toxin 2. An in vitro model of thrombotic microangiopathy was developed in which Shiga toxin 2- and E. coli O157 lipopolysaccharide-stimulated human blood cells combined with ADAMTS13-deficient plasma were perfused over glomerular endothelial cells. Two group statistical comparisons were performed using the Mann-Whitney test, multiple groups were compared using the Kruskal-Wallis test followed by Dunn's procedure, the Wilcoxon signed rank test was used for paired data, or linear regression for continuous variables., Results: HUS patients had excessively high plasma arginase 1 levels and activity (conversion of L-arginine to urea and L-ornithine) during the acute phase, compared to remission and controls. Arginase 1 levels correlated with lactate dehydrogenase activity, indicating hemolysis, as well as the need for dialysis treatment. Patients also exhibited high levels of plasma alpha-1-microglobulin, a heme scavenger. Both mouse models exhibited significantly elevated plasma arginase 1 levels and activity. Plasma arginase 1 levels correlated with lactate dehydrogenase activity, alpha-1-microglobulin and urea levels, the latter indicative of kidney dysfunction. In the in vitro model of thrombotic microangiopathy, bioactive arginase 1 was released and levels correlated to the degree of hemolysis., Conclusions: Elevated red blood cell-derived arginase was demonstrated in HUS patients and in relevant in vivo and in vitro models. The excessively high arginase levels correlated to the degree of hemolysis and kidney dysfunction. Thus, arginase inhibition should be investigated in HUS., (© 2024. The Author(s).)
- Published
- 2024
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18. Detection of Cleaved Stx2a in the Blood of STEC-Infected Patients.
- Author
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Varrone E, Carnicelli D, He X, Grasse M, Stampfer K, Huber S, Kellnerová S, Tazzari PL, Ricci F, Paterini P, Ardissino G, Morabito S, Orth-Höller D, Würzner R, and Brigotti M
- Subjects
- Humans, Shiga Toxin 2, Shiga Toxin, Neutrophils, Bacteria, Shiga-Toxigenic Escherichia coli, Escherichia coli Infections microbiology
- Abstract
Typical hemolytic uremic syndrome (HUS) is mainly caused by Shiga toxin-producing Escherichia coli (STEC) releasing Shiga toxin 2 (Stx2). Two different structures of this AB5 toxin have been described: uncleaved, with intact B and A chains, and cleaved, with intact B and a nicked A chain consisting of two fragments, A1 and A2, connected by a disulfide bond. Despite having the same toxic effect on sensitive cells, the two forms differ in their binding properties for circulating cells, serum components and complement factors, thus contributing to the pathogenesis of HUS differently. The outcome of STEC infections and the development of HUS could be influenced by the relative amounts of uncleaved or cleaved Stx2 circulating in patients' blood. Cleaved Stx2 was identified and quantified for the first time in four out of eight STEC-infected patients' sera by a method based on the inhibition of cell-free translation. Cleaved Stx2 was present in the sera of patients with toxins bound to neutrophils and in two out of three patients developing HUS, suggesting its involvement in HUS pathogenesis, although in association with other bacterial or host factors.
- Published
- 2023
- Full Text
- View/download PDF
19. Reduction is sufficient for the disassembly of ricin and Shiga toxin 1 but not Escherichia coli heat-labile enterotoxin.
- Author
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Guyette JL, Serrano A, Huhn Iii GR, Taylor M, Malkòm P, Curtis D, and Teter K
- Subjects
- Shiga Toxin 1, Protein Disulfide-Isomerases metabolism, Disulfides, Ricin toxicity, Ricin chemistry, Ricin metabolism
- Abstract
Many AB toxins contain an enzymatic A moiety that is anchored to a cell-binding B moiety by a disulfide bridge. After receptor-mediated endocytosis, some AB toxins undergo retrograde transport to the endoplasmic reticulum (ER) where reduction of the disulfide bond occurs. The reduced A subunit then dissociates from the holotoxin and enters the cytosol to alter its cellular target. Intoxication requires A chain separation from the holotoxin, but, for many toxins, it is unclear if reduction alone is sufficient for toxin disassembly. Here, we examined the link between reduction and disassembly for several ER-translocating toxins. We found disassembly of the reduced Escherichia coli heat-labile enterotoxin (Ltx) required an interaction with one specific ER-localized oxidoreductase: protein disulfide isomerase (PDI). In contrast, the reduction and disassembly of ricin toxin (Rtx) and Shiga toxin 1 (Stx1) were coupled events that did not require PDI and could be triggered by reductant alone. PDI-deficient cells accordingly exhibited high resistance to Ltx with continued sensitivity to Rtx and Stx1. The distinct structural organization of each AB toxin thus appears to determine whether holotoxin disassembly occurs spontaneously upon disulfide reduction or requires the additional input of PDI., Competing Interests: The authors declare no conflict of interest.
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- 2023
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20. Study Findings from Lund University Update Knowledge in Hemolytic Uremic Syndrome (Red blood cell-derived arginase release in hemolytic uremic syndrome).
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HEMOLYTIC-uremic syndrome ,ARGINASE ,BLOOD platelet disorders ,THROMBOTIC thrombocytopenic purpura ,BLOOD diseases ,LYMPHATIC diseases - Abstract
A study conducted by Lund University has found that hemolysis, the breakdown of red blood cells, is a key feature of hemolytic uremic syndrome (HUS). During hemolysis, excess arginase 1 is released from red blood cells, leading to reduced nitric oxide bioavailability and vascular injury. The study investigated arginase release in HUS patients and laboratory models and found that HUS patients had excessively high levels of plasma arginase 1 during the acute phase, which correlated with hemolysis and kidney dysfunction. The researchers suggest that further investigation into arginase inhibition as a potential treatment for HUS is warranted. [Extracted from the article]
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- 2024
21. Studies from Centers for Disease Control and Prevention in the Area of Escherichia coli Proteins Published (Identification and Characterization of ten Escherichia coli Strains Encoding Novel Shiga Toxin 2 Subtypes, Stx2n as Well as...).
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A new study on Escherichia coli proteins has been published by the Centers for Disease Control and Prevention (CDC). The study used publicly available genome sequences to detect a novel subtype of Shiga toxin, called Stx2n, in two clinical E. coli strains isolated in the USA. The researchers also identified other subtypes of Shiga toxin and analyzed their variability. The study emphasizes the importance of genomic surveillance to identify emerging variants of Shiga toxin, which has significant implications for public health. The researchers suggest designating one of the strains as the Stx2n type-strain. [Extracted from the article]
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- 2023
22. Isolation and characterization of Shiga toxigenic Escherichia coli of animal and bird origin by multiplex polymerase chain reaction
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S. Neher, A. K. Hazarika, L. M. Barkalita, P. Borah, D. P. Bora, and R. K. Sharma
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eae ,Escherichia coli ,Shiga toxigenic Escherichia coli ,Shiga toxin 1 ,Shiga toxin 2 ,Animal culture ,SF1-1100 ,Veterinary medicine ,SF600-1100 - Abstract
Aim: The purpose of this study was to determine the virulence genes and serotype of Shiga toxin producing Escherichia coli (STEC) strains isolated from animals and birds. Materials and Methods: A total of 226 different samples viz., fecal, intestinal content, rectal swab and heart blood were collected from different clinically affected/healthy animals and birds and were streaked on McConkeys’ lactose agar and eosin methylene blue agar for isolation of E. coli, confirmed by staining characteristics and biochemical tests. By polymerase chain reaction (PCR) all the E. coli isolates were screened for certain virulence genes, viz., Shiga toxin 1 (stx1), stx2 and eae and enterohemolytic (Ehly) phenotype was observed in washed sheep blood agar plate. All the isolated E. coli strains were forwarded to the National Salmonella and Escherichia Centre, Central Research Institute, Kasauli (Himachal Pradesh) for serotyping. Results: Out of 226 samples 138 yielded E. coli. All the isolates were screened for molecular detection of different virulent genes, viz. stx1, stx2 and eae, based on which 36 (26.08%) were identified as STEC. Among those STEC isolates, 15 (41.67%), 14 (38.89%), 1 (2.78%) exhibited eae, stx2, stx1 alone, respectively, whereas 4 (11.11%) and 2 (5.56%) carried both stx1 and stx2, stx2 and eae, respectively. Among the STEC isolates 22 were belonged to 15 different sero-groups, viz., O2, O20, O22, O25, O43, O60, O69, O90, O91, O95, O106, O118, O130, O162 and O170 and others were untypable. Ehly phenotype was observed in 10 (27.78%) the STEC isolates. Conclusion: The present study concluded that STEC could be isolated from both clinically affected as well as healthy animals and birds. Regular monitoring of more samples from animal and bird origin is important to identify natural reservoir of STEC to prevent zoonotic infection.
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- 2016
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23. The effect of two ribonucleases on the production of Shiga toxin and stx-bearing bacteriophages in Enterohaemorrhagic Escherichia coli
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Patricia B Lodato
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Virulence Factors ,Mitomycin ,Science ,Virulence ,Viral Plaque Assay ,medicine.disease_cause ,Shiga Toxin 1 ,Shiga Toxin 2 ,Microbiology ,Article ,Shiga Toxin ,Ribonucleases ,fluids and secretions ,STX2 ,hemic and lymphatic diseases ,medicine ,Bacteriophages ,Gene ,Prophage ,Escherichia coli Infections ,Multidisciplinary ,biology ,Toxin ,Shiga toxin ,Gene Expression Regulation, Bacterial ,Bacterial pathogenesis ,biochemical phenomena, metabolism, and nutrition ,bacterial infections and mycoses ,Transcription antitermination ,Lytic cycle ,Enterohemorrhagic Escherichia coli ,biology.protein ,bacteria ,Medicine ,Plasmids - Abstract
Enterohaemorrhagic Escherichia coli (EHEC) comprise a group of intestinal pathogens responsible for a range of illnesses, including kidney failure and neurological compromise. EHEC produce critical virulence factors, Shiga toxin (Stx) 1 or 2, and the synthesis of Stx2 is associated with worse disease manifestations. Infected patients only receive supportive treatment because some conventional antibiotics enable toxin production. Shiga toxin 2 genes (stx2) are carried in λ-like bacteriophages (stx2-phages) inserted into the EHEC genome as prophages. Factors that cause DNA damage induce the lytic cycle of stx2-phages, leading to Stx2 production. The phage Q protein is critical for transcription antitermination of stx2 and phage lytic genes. This study reports that deficiency of two endoribonucleases (RNases), E and G, significantly delayed cell lysis and impaired production of both Stx2 and stx2-phages, unlike deficiency of either enzyme alone. Moreover, scarcity of both enzymes reduced the concentrations of Q and stx2 transcripts and slowed cell growth.
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- 2021
24. Shiga toxin 2eB-transgenic lettuce vaccine: N-glycosylation is important for protecting against porcine edema disease
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HAMABATA, Takashi, SATO, Toshio, TAKITA, Eiji, MATSUI, Takeshi, KAWABATA, Takahiro, IMAOKA, Taishi, NAKANISHI, Nobuo, TSUKAHARA, Takamitsu, and SAWADA, Kazutoshi
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colostrum-deprived piglet infection model ,Glycosylation ,Full Paper ,Shiga-Toxigenic Escherichia coli ,Swine ,animal diseases ,Bacteriology ,porcine edema disease ,Shiga toxin 2e-producing Escherichia coli ,Lettuce ,stx2eB-transgenic lettuce vaccine ,Shiga Toxin 2 ,Shiga Toxin ,fluids and secretions ,Bacterial Vaccines ,Animals ,Edema ,Escherichia coli Infections - Abstract
Porcine edema disease (ED) is a life-threatening toxemia caused by enteric infection with Shiga toxin 2e (Stx2e)-producing Escherichia coli (STEC) in weaned piglets. We previously reported that the stx2eB-transgenic lettuce 2BH strain shows potential for use as an oral vaccine candidate against ED. However, the 2BH strain expressed a hemagglutinin (HA)-tag together with Stx2eB and contained non-canonical N-glycosylation. Therefore, we developed two Stx2eB-lettuce strains, the 3 (G+) strain in which the HA-tag was removed from 2BH, and the 3 (G-) lettuce strain, in which the 73rd Asn was replaced with Ser to prevent non-canonical N-glycosylation of Stx2eB from the 3 (G+) strain. We examined the protective effect of these newly developed two strains compared with the previous 2BH strain against ED using a colostrum-deprived piglet STEC infection model. We found that the N-glycosylated 2BH and 3 (G+) strains relieved the pathogenic symptoms of ED in STEC-challenged piglets, whereas the non-glycosylated 3 (G-) strain did not. N-Glycosylation of the Stx2eB product in lettuce may be involved in the immune response in piglets.
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- 2021
25. Apyrase decreases phage induction and Shiga toxin release from E. coli O157:H7 and has a protective effect during infection
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Ida Arvidsson, Ashmita Tontanahal, Karl Johansson, Ann-Charlotte Kristoffersson, Sára Kellnerová, Michael Berger, Ulrich Dobrindt, and Diana Karpman
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Lipopolysaccharides ,Microbiology (medical) ,Mice, Inbred BALB C ,Apyrase ,Gastroenterology ,Escherichia coli O157 ,Shiga Toxin 2 ,Microbiology ,Gastrointestinal Microbiome ,Shiga Toxin ,Mice ,Adenosine Triphosphate ,Infectious Diseases ,Animals ,Humans ,Bacteriophages ,Escherichia coli Infections - Abstract
Shiga toxin (Stx)-producing enterohemorrhagiciEscherichia coli/i(EHEC) cause gastrointestinal infection and, in severe cases, hemolytic uremic syndrome which may lead to death. There is, to-date, no therapy for this infection. Stx induces ATP release from host cells and ATP signaling mediates its cytotoxic effects. Apyrase cleaves and neutralizes ATP and its effect on Stx and EHEC infection was therefore investigated. Apyrase decreased bacterial RecA and dose-dependently decreased toxin release fromiE. coli/iO157:H7iin vitro/i, demonstrated by reduced phage DNA and protein levels. The effect was investigated in a mouse model ofiE. coli/iO157:H7 infection. BALB/c mice infected with Stx2-producingiE. coli/iO157:H7 were treated with apyrase intraperitoneally, on days 0 and 2 post-infection, and monitored for 11 days. Apyrase-treated mice developed disease two days later than untreated mice. Untreated infected mice lost significantly more weight than those treated with apyrase. Apyrase-treated mice exhibited less colonic goblet cell depletion and apoptotic cells, as well as lower fecal ATP and Stx2, compared to untreated mice. Apyrase also decreased platelet aggregation induced by co-incubation of human platelet-rich-plasma with Stx2 andiE. coli/iO157 lipopolysaccharide in the presence of collagen. Thus, apyrase had multiple protective effects, reducing RecA levels,istx2/iand toxin release from EHEC, reducing fecal Stx2 and protecting mouse intestinal cells, as well as decreasing platelet activation, and could thereby delay the development of disease.
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- 2022
26. Anti‐Shiga toxin 2 antibodies in enterohemorrhagic Escherichia coli O104:H4 infected patients may predict hemolytic uremic syndrome.
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Dammermann, Werner, Mihajlov, Valentin, Middendorf, Barbara, Mellmann, Alexander, Karch, Helge, Lüth, Stefan, and Ullrich, Sebastian
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VEROCYTOTOXINS , *ESCHERICHIA coli O157:H7 , *ENTEROCOLITIS , *CRITICAL care medicine , *PLASMAPHERESIS - Abstract
Abstract: Background and Aim: An outbreak of Shiga toxin 2 (Stx2) producing enterohemorrhagic and enteroaggregative Escherichia coli O104:H4 infection in May 2011 in Germany caused enterocolitis and an unprecedented high 22% rate of hemolytic uremic syndrome (HUS). We hypothesized that anti‐Stx2 IgM or IgG titers might predict HUS development. Methods: Thirty‐two patients infected with enterohemorrhagic Escherichia coli O104:H4 (HUS: n = 23; non‐HUS: n = 9) were retrospectively screened for anti‐Stx2 IgM/IgG and matched with clinical data regarding HUS development, fever, superinfection, dialysis, neurological symptoms, intensive care, antibiotic treatment, and plasmapheresis. Results: Only HUS patients showed a prominent Stx2‐specific humoral response in the early acute phase. Despite a strong trend towards prediction of HUS development, statistical analysis revealed no significant correlation between high IgM/IgG titers and further key clinical parameters such as fever, superinfection, neurological symptoms, antibiotic treatment, and plasmapheresis. Conclusions: Anti‐Stx2 antibodies seem to accompany or even precede HUS development. [ABSTRACT FROM AUTHOR]
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- 2018
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27. Soluble CD40 Ligand and Oxidative Response Are Reciprocally Stimulated during Shiga Toxin-Associated Hemolytic Uremic Syndrome.
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Recalde, Maria J. Abrey, Alvarez, Romina S., Alberto, Fabiana, Mejias, Maria P., Ramos, Maria V., Brando, Romina J. Fernandez, Bruballa, Andrea C., Exeni, Ramon A., Alconcher, Laura, Ibarra, Cristina A., Amaral, María M., and Palermo, Marina S.
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- *
HEMOLYTIC-uremic syndrome , *OXIDATIVE stress , *BLOOD platelets , *PATHOGENIC bacteria , *BACTERIAL toxins , *ENDOTHELIUM diseases , *GLOMERULAR filtration rate - Abstract
Shiga toxin (Stx), produced by Escherichia coli, is the main pathogenic factor of diarrheaassociated hemolytic uremic syndrome (HUS), which is characterized by the obstruction of renal microvasculature by platelet-fibrin thrombi. It is well known that the oxidative imbalance generated by Stx induces platelet activation, contributing to thrombus formation. Moreover, activated platelets release soluble CD40 ligand (sCD40L), which in turn contributes to oxidative imbalance, triggering the release of reactive oxidative species (ROS) on various cellular types. The aim of this work was to determine if the interaction between the oxidative response and platelet-derived sCD40L, as consequence of Stx-induced endothelium damage, participates in the pathogenic mechanism during HUS. Activated human glomerular endothelial cells (HGEC) by Stx2 induced platelets to adhere to them. Although platelet adhesion did not contribute to endothelial damage, high levels of sCD40L were released to the medium. The release of sCD40L by activated platelets was inhibited by antioxidant treatment. Furthermore, we found increased levels of sCD40L in plasma from HUS patients, which were also able to trigger the respiratory burst in monocytes in a sCD40L-dependent manner. Thus, we concluded that platelet-derived sCD40L and the oxidative response are reciprocally stimulated during Stx2-associated HUS. This process may contribute to the evolution of glomerular occlusion and the microangiopathic lesions. [ABSTRACT FROM AUTHOR]
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- 2017
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28. Eliglustat prevents Shiga toxin 2 cytotoxic effects in human renal tubular epithelial cells
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Claudia Silberstein, Daiana Soledad Sanchez, Lilian Karina Fischer Sigel, Alejandro Balestracci, María Marta Amaral, and Cristina Ibarra
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Pyrrolidines ,Globotriaosylceramide ,Shiga Toxin 2 ,Shiga Toxin ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,030225 pediatrics ,medicine ,Humans ,Cytotoxic T cell ,Viability assay ,Child ,Cells, Cultured ,Kidney ,Gaucher Disease ,biology ,business.industry ,Acute kidney injury ,Epithelial Cells ,Shiga toxin ,medicine.disease ,medicine.anatomical_structure ,chemistry ,Apoptosis ,Pediatrics, Perinatology and Child Health ,Cancer research ,biology.protein ,business ,030217 neurology & neurosurgery ,Eliglustat - Abstract
Background Shiga toxin-producing Escherichia coli is responsible for post-diarrheal (D+) hemolytic uremic syndrome (HUS), which is a cause of acute renal failure in children. The glycolipid globotriaosylceramide (Gb3) is the main receptor for Shiga toxin (Stx) in kidney target cells. Eliglustat (EG) is a specific and potent inhibitor of glucosylceramide synthase, first step of glycosphingolipid biosynthesis, actually used for the treatment of Gaucher's disease. The aim of the present work was to evaluate the efficiency of EG in preventing the damage caused by Stx2 in human renal epithelial cells. Methods Human renal tubular epithelial cell (HRTEC) primary cultures were pre-treated with different dilutions of EG followed by co-incubation with EG and Stx2 at different times, and cell viability, proliferation, apoptosis, tubulogenesis, and Gb3 expression were assessed. Results In HRTEC, pre-treatments with 50 nmol/L EG for 24 h, or 500 nmol/L EG for 6 h, reduced Gb3 expression and totally prevented the effects of Stx2 on cell viability, proliferation, and apoptosis. EG treatment also allowed the development of tubulogenesis in 3D-HRTEC exposed to Stx2. Conclusions EG could be a potential therapeutic drug for the prevention of acute kidney injury caused by Stx2. Impact For the first time, we have demonstrated that Eliglustat prevents Shiga toxin 2 cytotoxic effects on human renal epithelia, by reducing the expression of the toxin receptor globotriaosylceramide. The present work also shows that Eliglustat prevents Shiga toxin 2 effects on tubulogenesis of renal epithelial cells. Eliglustat, actually used for the treatment of patients with Gaucher's disease, could be a therapeutic strategy to prevent the renal damage caused by Shiga toxin.
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- 2021
29. Antibiotic-mediated expression analysis of Shiga toxin 1 and 2 in multi-drug-resistant Shiga toxigenic Escherichia coli
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Aniqa Rehman, Khalid Mehmood, Zeeshan Mustafa, Danish Gul, Saadia Andleeb, and Sidra Rahmat Ullah
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medicine.drug_class ,Antibiotics ,Tigecycline ,Fosfomycin ,Shiga Toxin 1 ,Shiga Toxin 2 ,Microbiology ,Meropenem ,03 medical and health sciences ,Antibiotic resistance ,STX2 ,medicine ,Humans ,Escherichia coli Infections ,030304 developmental biology ,0303 health sciences ,Shiga-Toxigenic Escherichia coli ,biology ,030306 microbiology ,Gene Expression Profiling ,Shiga toxin ,Gene Expression Regulation, Bacterial ,General Medicine ,biochemical phenomena, metabolism, and nutrition ,Anti-Bacterial Agents ,Ciprofloxacin ,biology.protein ,medicine.drug - Abstract
Shiga toxin-producing Escherichia coli (STEC) is an important foodborne pathogens, known to cause enteric infections especially diarrhea, mainly attributed to Shiga toxins (Stxs). The use of certain antibiotics for treating this infection is controversial, owing to an increased risk for producing Stxs (Stx 1 and Stx 2). Increased antibiotic resistance is also thought to be involved in the pathogenesis of STEC diseases. The purpose of this study was to analyze the effects of antibiotics on induction of Stx 1 and Stx 2 in clinical STEC isolates and to investigate the relationships between increased resistance and Stx production. Fifteen clinical isolates were treated with sub minimum inhibitory concentrations (Sub MIC) of clinically used antibiotics (ciprofloxacin, fosfomycin, tigecycline, and meropenem), and the changes in expression levels of stx1 and stx2 genes were estimated using qRT-PCR. The expressions of Shiga toxins were found to be increased up to 6.5- and eightfold under ciprofloxacin and tigecycline Sub MIC, respectively. Fosfomycin had weak induction effect of up to twofold, whereas meropenem had the weakest influence on such expression. Resistant isolates were found to be more prone to increased expression of toxins.
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- 2021
30. A nontoxigenic form of Shiga toxin 2 suppresses the production of amyloid β by altering the intracellular transport of amyloid precursor protein through its receptor-binding B-subunit
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Satoru Funamoto, Koichi Furukawa, Waka Sato, Takashi Hamabata, Kiyotaka Nishikawa, and Miho Watanabe-Takahashi
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0301 basic medicine ,Cell Survival ,Endosome ,Protein subunit ,Biophysics ,CHO Cells ,Endosomes ,Endocytosis ,Shiga Toxin 2 ,Biochemistry ,Amyloid beta-Protein Precursor ,03 medical and health sciences ,symbols.namesake ,Cricetulus ,0302 clinical medicine ,Catalytic Domain ,Extracellular ,Amyloid precursor protein ,Animals ,Humans ,Molecular Biology ,Amyloid beta-Peptides ,Globosides ,biology ,Chemistry ,Trihexosylceramides ,Endoplasmic reticulum ,Cell Membrane ,Shiga toxin ,Cell Biology ,Golgi apparatus ,Recombinant Proteins ,Cell biology ,Protein Transport ,030104 developmental biology ,030220 oncology & carcinogenesis ,Mutation ,Phosphatidylcholines ,symbols ,biology.protein ,Lysosomes - Abstract
Accumulation of amyloid-β peptide (Aβ) in neuronal cells and in the extracellular regions in the brain is a major cause of Alzheimer's disease (AD); therefore, inhibition of Aβ accumulation offers a promising approach for therapeutic strategies against AD. Aβ is produced by sequential proteolysis of amyloid precursor protein (APP) in late/recycling endosomes after endocytosis of APP located in the plasma membrane. Aβ is then released from cells in a free form or in an exosome-bound form. Shiga toxin (Stx) is a major virulence factor of enterohemorrhagic Escherichia coli. Recently, we found that one of the Stx subtypes, Stx2a, has a unique intracellular transport route after endocytosis through its receptor-binding B-subunit. A part of Stx2a can be transported to late/recycling endosomes and then degraded in a lysosomal acidic compartment, although in general Stx is transported to the Golgi and then to the endoplasmic reticulum in a retrograde manner. In this study, we found that treatment of APP-expressing cells with a mutant Stx2a (mStx2a), lacking cytotoxic activity because of mutations in the catalytic A-subunit, stimulated the transport of APP to the acidic compartment, which led to degradation of APP and a reduction in the amount of Aβ. mStx2a-treatment also inhibited the extracellular release of Aβ. Therefore, mStx2a may provide a new strategy to inhibit the production of Aβ by modulating the intracellular transport of APP.
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- 2021
31. New Enterohemorrhagic Escherichia coli Study Findings Recently Were Reported by Researchers at Third Affiliated Hospital of Chongqing Medical University (Shiga toxin 2 A-subunit induces mitochondrial damage, mitophagy and apoptosis via the...).
- Abstract
Apoptosis, Bacterial Toxins, Biological Factors, Caco-2 Cells, Cellular Physiology, Cellular Structures, Cytoplasm, Cytoplasmic Structures, Enterotoxins, Escherichia coli Proteins, Foodborne Diseases and Conditions, Genetics, Glycoside Hydrolases, Health and Medicine, Intracellular Space, Mitochondria, N-Glycosyl Hydrolases, Organelles, Ribosome Inactivating Proteins, Shiga Toxin 2, Enterohemorrhagic Escherichia coli, Shiga Toxins, Subcellular Fractions, Type 2 Ribosome Inactivating Proteins Keywords: Apoptosis; Bacterial Toxins; Biological Factors; Caco-2 Cells; Cellular Physiology; Cellular Structures; Cytoplasm; Cytoplasmic Structures; Enterohemorrhagic Escherichia coli; Enterotoxins; Escherichia coli Proteins; Foodborne Diseases and Conditions; Genetics; Glycoside Hydrolases; Health and Medicine; Intracellular Space; Mitochondria; N-Glycosyl Hydrolases; Organelles; Ribosome Inactivating Proteins; Shiga Toxin 2; Shiga Toxins; Subcellular Fractions; Type 2 Ribosome Inactivating Proteins EN Apoptosis Bacterial Toxins Biological Factors Caco-2 Cells Cellular Physiology Cellular Structures Cytoplasm Cytoplasmic Structures Enterohemorrhagic Escherichia coli Enterotoxins Escherichia coli Proteins Foodborne Diseases and Conditions Genetics Glycoside Hydrolases Health and Medicine Intracellular Space Mitochondria N-Glycosyl Hydrolases Organelles Ribosome Inactivating Proteins Shiga Toxin 2 Shiga Toxins Subcellular Fractions Type 2 Ribosome Inactivating Proteins 699 699 1 10/03/23 20231006 NES 231006 2023 OCT 6 (NewsRx) -- By a News Reporter-Staff News Editor at Genomics & Genetics Weekly -- Researchers detail new data in enterohemorrhagic Escherichia coli. [Extracted from the article]
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- 2023
32. Intestinal mucus-derived metabolites modulate virulence of a clade 8 enterohemorrhagic
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Nicolás, Garimano, María Luján, Scalise, Fernando, Gómez, María Marta, Amaral, and Cristina, Ibarra
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Virulence ,Virulence Factors ,Escherichia coli Proteins ,Mucins ,Galactose ,Escherichia coli O157 ,Shiga Toxin 2 ,Gastrointestinal Microbiome ,Intestines ,Mucus ,Enterohemorrhagic Escherichia coli ,Humans ,Escherichia coli Infections ,Expectorants ,Fucose - Abstract
The human colonic mucus is mainly composed of mucins, which are highly glycosylated proteins. The normal commensal colonic microbiota has mucolytic activity and is capable of releasing the monosaccharides contained in mucins, which can then be used as carbon sources by pathogens such as Enterohemorrhagic
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- 2022
33. Shiga Toxin 2 Triggers C3a-Dependent Glomerular and Tubular Injury through Mitochondrial Dysfunction in Hemolytic Uremic Syndrome
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Simona Buelli, Monica Locatelli, Claudia Elisa Carminati, Daniela Corna, Domenico Cerullo, Barbara Imberti, Luca Perico, Maurizio Brigotti, Mauro Abbate, Carlamaria Zoja, Ariela Benigni, Giuseppe Remuzzi, Marina Morigi, Buelli, Simona, Locatelli, Monica, Carminati, Claudia Elisa, Corna, Daniela, Cerullo, Domenico, Imberti, Barbara, Perico, Luca, Brigotti, Maurizio, Abbate, Mauro, Zoja, Carlamaria, Benigni, Ariela, Remuzzi, Giuseppe, and Morigi, Marina
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podocyte ,mitochondrial damage ,Animal ,Podocytes ,Kidney Glomerulus ,chemical and pharmacologic phenomena ,General Medicine ,urologic and male genital diseases ,Shiga Toxin 2 ,C3a/C3aR signaling ,Mitochondria ,Receptors, Complement ,podocytes ,proximal tubular epithelial cells ,complement ,Mice ,Hemolytic-Uremic Syndrome ,Animals ,Kidney Glomerulu ,proximal tubular epithelial cell - Abstract
Shiga toxin (Stx)-producing Escherichia coli is the predominant offending agent of post-diarrheal hemolytic uremic syndrome (HUS), a rare disorder of microvascular thrombosis and acute kidney injury possibly leading to long-term renal sequelae. We previously showed that C3a has a critical role in the development of glomerular damage in experimental HUS. Based on the evidence that activation of C3a/C3a receptor (C3aR) signaling induces mitochondrial dysregulation and cell injury, here we investigated whether C3a caused podocyte and tubular injury through induction of mitochondrial dysfunction in a mouse model of HUS. Mice coinjected with Stx2/LPS exhibited glomerular podocyte and tubular C3 deposits and C3aR overexpression associated with cell damage, which were limited by C3aR antagonist treatment. C3a promoted renal injury by affecting mitochondrial wellness as demonstrated by data showing that C3aR blockade reduced mitochondrial ultrastructural abnormalities and preserved mitochondrial mass and energy production. In cultured podocytes and tubular cells, C3a caused altered mitochondrial fragmentation and distribution, and reduced anti-oxidant SOD2 activity. Stx2 potentiated the responsiveness of renal cells to the detrimental effects of C3a through increased C3aR protein expression. These results indicate that C3aR may represent a novel target in Stx-associated HUS for the preservation of renal cell integrity through the maintenance of mitochondrial function.
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- 2022
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34. Detection of Shiga Toxin-Producing
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María Luján, Scalise, Nicolás, Garimano, Marcelo, Sanz, Nora Lia, Padola, Patricia, Leonino, Adriana, Pereyra, Roberto, Casale, María Marta, Amaral, Flavia, Sacerdoti, and Cristina, Ibarra
- Subjects
Shiga-Toxigenic Escherichia coli ,Virulence Factors ,Escherichia coli Proteins ,Pregnancy Outcome ,Cervix Uteri ,Escherichia coli O157 ,Shiga Toxin 2 ,Rats ,Pregnancy ,Risk Factors ,Chlorocebus aethiops ,Animals ,Humans ,Female ,Pregnant Women ,Vero Cells ,HeLa Cells - Abstract
The presence of
- Published
- 2022
35. Absence of interleukin-10 reduces progression of shiga toxin-induced hemolytic uremic syndrome
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María Florencia Todero, María Victoria Ramos, Catalina D. Alba-Soto, Gonzalo Ezequiel Pineda, Alan Mauro Bernal, Andrea Cecilia Bruballa, Marina S. Palermo, Elsa Zotta, Bárbara Rearte, Martín A. Isturiz, and Romina Jimena Fernandez-Brando
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0301 basic medicine ,Hemolytic anemia ,Neutrophils ,medicine.medical_treatment ,030106 microbiology ,Kidney ,urologic and male genital diseases ,medicine.disease_cause ,Shiga Toxin 2 ,03 medical and health sciences ,chemistry.chemical_compound ,Transforming Growth Factor beta ,medicine ,Animals ,Mice, Knockout ,Mice, Inbred BALB C ,Creatinine ,biology ,Interleukin-6 ,Tumor Necrosis Factor-alpha ,Toxin ,business.industry ,Shiga toxin ,General Medicine ,medicine.disease ,Interleukin-10 ,Survival Rate ,Interleukin 10 ,030104 developmental biology ,Cytokine ,medicine.anatomical_structure ,chemistry ,Hemolytic-Uremic Syndrome ,Immunology ,biology.protein ,Tumor necrosis factor alpha ,Corticosterone ,business - Abstract
Hemolytic Uremic Syndrome (HUS), a disease triggered by Shiga toxin (Stx), is characterized by hemolytic anemia, thrombocytopenia and renal failure. The inflammatory response mediated by polymorphonuclear neutrophils (PMNs) and monocytes is essential to HUS onset. Still, the role of anti-inflammatory cytokines is less clear. The deficiency of IL-10, an anti-inflammatory cytokine, leads to severe pathology in bacterial infections but also to beneficial effects in models of sterile injury. The aim of this work was to analyze the role of IL-10 during HUS. Control and IL-10 lacking mice (IL-10−/−) were intravenously injected with Stx type 2 (Stx2) and survival rate was evaluated. PMN and circulating and renal pro- and anti-inflammatory factors were analyzed by FACS and enzyme-linked immunosorbent assay (ELISA) respectively. IL-10−/− mice showed a higher survival associated with lower renal damage reflected by reduced plasma urea and creatinine levels than control mice. Circulating PMN increased at 72 h in both mouse strains accompanied by an up-regulation of CD11b in control mice. In parallel, renal PMN were significantly increased only in control mice after toxin. Plasma TNF-α, IL-6 and corticosterone levels were higher increased in IL-10−/− than control mice. Simultaneously renal TNF-α raised constantly but was accompanied by increased TGF-β levels in IL-10−/− mice. These results demonstrate that the profile of circulating and renal cytokines after Stx2 differed between strains suggesting that balance of these factors could participate in renal protection. We conclude that IL-10 absence has a protective role in an experimental model of HUS by reducing PMN recruitment into kidney and renal damage, and increasing mice survival.
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- 2021
36. Evaluation of immunochromatographic test of Shiga toxin 2e in enrichment cultures of swine edema disease clinical samples
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Masafumi Mukamoto, Tomoko Kohda, Hideyuki Arimitsu, and Masahiro Kusumoto
- Subjects
Swine ,Immunochromatographic test ,Edema Disease of Swine ,medicine.disease_cause ,Shiga Toxin 2 ,Shiga Toxin ,Microbiology ,sandwich ELISA ,Chlorocebus aethiops ,immunochromatography ,Escherichia coli ,medicine ,Animals ,Mesenteric lymph nodes ,Vero Cells ,Escherichia coli Infections ,Feces ,Swine Diseases ,shiga toxin 2e ,Edema disease ,Full Paper ,General Veterinary ,biology ,Toxin ,Chemistry ,Bacteriology ,Shiga toxin ,edema disease ,medicine.anatomical_structure ,Vero cell ,biology.protein ,Cell culture supernatant - Abstract
To simplify the diagnosis of swine edema disease, overnight culture supernatants of swine clinical samples were assayed using immunochromatographic test strips we developed previously. Small-intestinal contents, mesenteric lymph nodes, and fecal samples were cultured in casamino acid-yeast extract broth overnight, after which supernatants were loaded onto immunochromatographic test strips to determine whether they could detect Shiga toxin 2e (Stx2e). Among 23 clinical samples in which PCR-identified stx2e-positive E. coli were isolated, samples from seven of ten small-intestinal contents, one of three mesenteric lymph nodes and six of ten fecal samples showed Stx2e-positive reactions in the protein-based immunochromatographic test. Additionally, one small-intestinal content sample, in which stx2e-positive E. coli were not isolated, showed an Stx2e-positive reaction. Furthermore, the immunochromatographic test results of the samples were associated with the toxin concentration determined by sandwich ELISA and cytotoxicity assay results on Vero cells. The toxin concentration range of the samples with positive and negative reactions were 2.1–196.2 ng/ml and 0–12.8 ng/ml, respectively. The sensitivity and specificity of this immunochromatographic test strip calculated from all clinical samples analyzed in this study were 60.9% and 94.4%, respectively. Our immunochromatographic test strip has strong potential for simple and accurate diagnosis for edema disease by detecting toxin expression, complementing the PCR method.
- Published
- 2021
37. Occurrence of ‘gang of five’ Shiga toxin‐producing Escherichia coli serogroups on Belgian dairy cattle farms by overshoe sampling
- Author
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F. Engelen, Damien Thiry, Jacques Mainil, L. De Zutter, Brecht Devleesschauwer, and Eric Cox
- Subjects
0106 biological sciences ,Veterinary medicine ,Farms ,animal diseases ,Virulence ,Biology ,Serogroup ,medicine.disease_cause ,Shiga Toxin 2 ,01 natural sciences ,Applied Microbiology and Biotechnology ,Shiga Toxin ,law.invention ,Foodborne Diseases ,Feces ,03 medical and health sciences ,fluids and secretions ,Belgium ,STX2 ,law ,010608 biotechnology ,medicine ,Animals ,Adhesins, Bacterial ,Shiga toxin-producing Escherichia coli ,Escherichia coli ,Escherichia coli Infections ,Dairy cattle ,Polymerase chain reaction ,0303 health sciences ,Shiga-Toxigenic Escherichia coli ,Immunomagnetic Separation ,030306 microbiology ,Escherichia coli Proteins ,Outbreak ,biochemical phenomena, metabolism, and nutrition ,bacterial infections and mycoses ,Food Microbiology ,bacteria ,Cattle ,Acid treatment - Abstract
Shiga toxin-producing Escherichia coli (STEC) are foodborne pathogens responsible for global outbreaks. This study was conducted to investigate the occurrence of ‘gang of five’ STEC serogroups (O26, O103, O111, O145, O157) on Belgian dairy cattle farms by overshoe (OVS) sampling, and to evaluate the presence of virulence genes in the obtained isolates. A total of 88 OVS, collected from the pen beddings of 19 Belgian dairy cattle farms, were selectively enriched in mTSBn, followed by immunomagnetic separation and plating onto CT-SMAC for O157 STEC isolation, as well as in Brila broth, followed by a selective acid treatment and plating onto CHROMagar TM STEC and chromID TM EHEC for non-O157 STEC isolation. Overall, 11 out of 19 farms (58%) tested positive for presence of ‘gang of five’ STEC. O26 STEC was most frequently isolated from OVS (11/88; 12.5%), followed by O157 (10/88; 11.5%), O145 (3/88; 3.5%) and O103 (3/88; 3.5%). Additionally, 35% of the OVS collected from pens housing young cattle 1-24 months of age tested positive for ‘gang of five’ STEC, indicating that this age category is more likely to harbour STEC compared to new-born and adult cattle. Importantly, half of the obtained ‘gang of five’ STEC isolates (48%) possessed the eae and stx2 gene, suggesting a high pathogenic potential to humans.
- Published
- 2020
38. Structural basis for the interaction of Shiga toxin 2a with a C-terminal peptide of ribosomal P stalk proteins
- Author
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Nilgun E. Tumer, Xiao-Ping Li, Michael J. Rudolph, and Simon A. Davis
- Subjects
Ribosomal Proteins ,0301 basic medicine ,Trichosanthin ,Protein subunit ,Ricin ,Molecular Dynamics Simulation ,Crystallography, X-Ray ,medicine.disease_cause ,Shiga Toxin 2 ,Microbiology ,Biochemistry ,Ribosome ,03 medical and health sciences ,chemistry.chemical_compound ,medicine ,Humans ,Amino Acid Sequence ,Molecular Biology ,Escherichia coli ,Binding Sites ,030102 biochemistry & molecular biology ,biology ,Chemistry ,Ribosome-inactivating protein ,Shiga toxin ,Translation (biology) ,Cell Biology ,Elongation factor ,030104 developmental biology ,biology.protein ,Peptides ,Ribosomes ,Protein Binding - Abstract
The principal virulence factor of human pathogenic enterohemorrhagic Escherichia coli is Shiga toxin (Stx). Shiga toxin 2a (Stx2a) is the subtype most commonly associated with severe disease outcomes such as hemorrhagic colitis and hemolytic uremic syndrome. The catalytic A1 subunit (Stx2A1) binds to the conserved elongation factor binding C-terminal domain (CTD) of ribosomal P stalk proteins to inhibit translation. Stx2a holotoxin also binds to the CTD of P stalk proteins because the ribosome-binding site is exposed. We show here that Stx2a binds to an 11-mer peptide (P11) mimicking the CTD of P stalk proteins with low micromolar affinity. We cocrystallized Stx2a with P11 and defined their interactions by X-ray crystallography. We found that the last six residues of P11 inserted into a shallow pocket on Stx2A1 and interacted with Arg-172, Arg-176, and Arg-179, which were previously shown to be critical for binding of Stx2A1 to the ribosome. Stx2a formed a distinct P11-binding mode within a different surface pocket relative to ricin toxin A subunit and trichosanthin, suggesting different ribosome recognition mechanisms for each ribosome inactivating protein (RIP). The binding mode of Stx2a to P11 is also conserved among the different Stx subtypes. Furthermore, the P stalk protein CTD is flexible and adopts distinct orientations and interaction modes depending on the structural differences between the RIPs. Structural characterization of the Stx2a-ribosome complex is important for understanding the role of the stalk in toxin recruitment to the sarcin/ricin loop and may provide a new target for inhibitor discovery.
- Published
- 2020
39. Gene expression profile and injury sites in mice treated with Shiga toxin 2 and lipopolysaccharide as a Shiga toxin-associated hemolytic uremic syndrome model
- Author
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Yohei Kume, Hayato Go, Ryo Maeda, Kazuhide Suyama, Tsutomu Mori, Yukihiko Kawasaki, Koichi Hashimoto, and Mitsuaki Hosoya
- Subjects
Lipopolysaccharides ,Male ,Mice ,Aquaporin 2 ,Physiology ,Hemolytic-Uremic Syndrome ,Genetics ,Animals ,Solute Carrier Family 12, Member 3 ,urologic and male genital diseases ,Transcriptome ,Shiga Toxin 2 ,Shiga Toxin - Abstract
Shiga toxin 2 (Stx2) and lipopolysaccharide (LPS) contribute to the development of hemolytic uremic syndrome (HUS). Mouse models of HUS induced by LPS/Stx2 have been used for elucidating HUS pathophysiology and for therapeutic development. However, the underlying molecular mechanisms and detailed injury sites in this model remain unknown. We analyzed mouse kidneys after LPS/Stx2 administration using microarrays. Decreased urinary osmolality and urinary potassium were observed after LPS/Stx2 administration, suggestive of distal nephron disorders. A total of 1,212 and 1,016 differentially expressed genes were identified in microarrays at 6 h and 72 h after LPS/Stx2 administration, respectively, compared with those in controls. Ingenuity pathway analysis revealed activation of TNFR1/2, iNOS, and IL-6 signaling at both time points, and inhibition of pathways associated with lipid metabolism at 72 h only. The strongly downregulated genes in the 72-h group were expressed in the distal nephrons. In particular, genes associated with distal convoluted tubule (DCT) 2/connecting tubule (CNT) and principal cells of the cortical collecting duct (CCD) were downregulated to a greater extent than those associated with DCT1 and intercalated cells. Stx receptor globotriaosylceramide 3 (Gb3) revealed no colocalization with DCT1-specific PVALB and intercalated cell-specific SLC26A4 but did present colocalization with SLC12A3 (present in both DCT1 and DCT2), and AQP2 in principal cells. Gb3 localization tended to coincide with the segment in which the downregulated genes were present. Thus, the LPS/Stx2-induced kidney injury model represents damage to DCT2/CNT and principal cells in the CCD, based on molecular, biological, and physiological findings.
- Published
- 2022
40. A unique peptide-based pharmacophore identifies an inhibitory compound against the A-subunit of Shiga toxin
- Author
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Miho Watanabe-Takahashi, Miki Senda, Ryunosuke Yoshino, Masahiro Hibino, Shinichiro Hama, Tohru Terada, Kentaro Shimizu, Toshiya Senda, and Kiyotaka Nishikawa
- Subjects
Multidisciplinary ,Enterohemorrhagic Escherichia coli ,Receptors, Drug ,Humans ,Peptides ,Shiga Toxin 2 ,Escherichia coli Infections ,Shiga Toxin - Abstract
Shiga toxin (Stx), a major virulence factor of enterohemorrhagic Escherichia coli (EHEC), can cause fatal systemic complications. Recently, we identified a potent inhibitory peptide that binds to the catalytic A-subunit of Stx. Here, using biochemical structural analysis and X-ray crystallography, we determined a minimal essential peptide motif that occupies the catalytic cavity and is required for binding to the A-subunit of Stx2a, a highly virulent Stx subtype. Molecular dynamics simulations also identified the same motif and allowed determination of a unique pharmacophore for A-subunit binding. Notably, a series of synthetic peptides containing the motif efficiently inhibit Stx2a. In addition, pharmacophore screening and subsequent docking simulations ultimately identified nine Stx2a-interacting molecules out of a chemical compound database consisting of over 7,400,000 molecules. Critically, one of these molecules markedly inhibits Stx2a both in vitro and in vivo, clearly demonstrating the significance of the pharmacophore for identifying therapeutic agents against EHEC infection.
- Published
- 2022
41. Genome Sequence Analysis and Characterization of Shiga Toxin 2 Production by
- Author
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Mark, Eppinger, Sonia, Almería, Anna, Allué-Guardia, Lori K, Bagi, Anwar A, Kalalah, Joshua B, Gurtler, and Pina M, Fratamico
- Subjects
Humans ,Escherichia coli O157 ,Laboratory Infection ,Sequence Analysis ,Shiga Toxin 2 ,Escherichia coli Infections ,Anti-Bacterial Agents - Abstract
A laboratory-acquired
- Published
- 2022
42. Cytokines expression from altered motor thalamus and behavior deficits following sublethal administration of Shiga toxin 2a involve the induction of the globotriaosylceramide receptor
- Author
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David Arenas-Mosquera, Alipio Pinto, Natacha Cerny, Clara Berdasco, Adriana Cangelosi, Patricia Andrea Geoghegan, Emilio Luis Malchiodi, Mauricio De Marzi, and Jorge Goldstein
- Subjects
Lipopolysaccharides ,Mice ,Thalamus ,Trihexosylceramides ,Escherichia coli ,Animals ,Cytokines ,Toxicology ,Shiga Toxin 2 ,Escherichia coli Infections ,Shiga Toxin - Abstract
Encephalopathy associated with hemolytic uremic syndrome is produced by enterohemorrhagic E. coli (EHEC) infection, which releases the virulence factors Shiga toxin (Stx) and lipopolysaccharide (LPS). Neurological compromise is a poor prognosis and mortality factor of the disease, and the thalamus is one of the brain areas most frequently affected. We have previously demonstrated the effectiveness of anti-inflammatory drugs to ameliorate the deleterious effects of these toxins. However, the thalamic production of cytokines involved in pro-inflammatory processes has not yet been acknowledged. The aim of this work attempts to determine whether systemic sublethal Stx2a or co-administration of Stx2a with LPS are able to rise a proinflammatory profile accompanying alterations of the neurovascular unit in anterior and lateral ventral nuclei of the thalamus (VA-VL) and motor behavior in mice. After 4 days of treatment, Stx2a affected the lectin-bound microvasculature distribution while increasing the expression of GFAP in reactive astrocytes and producing aberrant NeuN distribution in degenerative neurons. In addition, increased swimming latency was observed in a motor behavioral test. All these alterations were heightened when Stx2a was co-administered with LPS. The expression of pro-inflammatory cytokines TNFα, INF-γ and IL-2 was detected in VA-VL. All these effects were concomitant with increased expression of the Stx receptor globotriaosylceramide (Gb3), which hints at receptor involvement in the neuroinflammatory process as a key finding of this study. In conclusion, Stx2a to Gb3 may be determinant in triggering a neuroinflammatory event, which may resemble clinical outcomes and should thus be considered in the development of preventive strategies.
- Published
- 2022
43. Therapeutic Antibodies Against Shiga Toxins: Trends and Perspectives
- Author
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Izabella de Macedo Henrique, Flavia Sacerdoti, Raissa Lozzardo Ferreira, Camila Henrique, Maria Marta Amaral, Roxane Maria Fontes Piazza, and Daniela Luz
- Subjects
trends ,Microbiology (medical) ,therapy ,Shiga-Toxigenic Escherichia coli ,Shiga toxin-producing E. coli ,Immunology ,Stx toxins ,Shiga Toxins ,Shiga Toxin 2 ,Microbiology ,QR1-502 ,Shiga Toxin ,Infectious Diseases ,Hemolytic-Uremic Syndrome ,Humans ,Immunologic Factors ,antibodies ,Escherichia coli Infections - Abstract
Shiga toxins (Stx) are AB5-type toxins, composed of five B subunits which bind to Gb3 host cell receptors and an active A subunit, whose action on the ribosome leads to protein synthesis suppression. The two Stx types (Stx1 and Stx2) and their subtypes can be produced by Shiga toxin-producing Escherichia coli strains and some Shigella spp. These bacteria colonize the colon and induce diarrhea that may progress to hemorrhagic colitis and in the most severe cases, to hemolytic uremic syndrome, which could lead to death. Since the use of antibiotics in these infections is a topic of great controversy, the treatment remains supportive and there are no specific therapies to ameliorate the course. Therefore, there is an open window for Stx neutralization employing antibodies, which are versatile molecules. Indeed, polyclonal, monoclonal, and recombinant antibodies have been raised and tested in vitro and in vivo assays, showing differences in their neutralizing ability against deleterious effects of Stx. These molecules are in different phases of development for which we decide to present herein an updated report of these antibody molecules, their source, advantages, and disadvantages of the promising ones, as well as the challenges faced until reaching their applicability.
- Published
- 2022
- Full Text
- View/download PDF
44. Pectic oligosaccharide structure-function relationships: Prebiotics, inhibitors of Escherichia coli O157:H7 adhesion and reduction of Shiga toxin cytotoxicity in HT29 cells.
- Author
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Di, Rong, Vakkalanka, Malathi S., Onumpai, Chatchaya, Chau, Hoa K., White, Andre, Rastall, Robert A., Yam, Kit, and Jr.Hotchkiss, Arland T.
- Subjects
- *
OLIGOSACCHARIDE structure , *PREBIOTICS , *ESCHERICHIA coli O157:H7 , *BACTERIAL adhesion , *BACTERIAL toxins , *CELL-mediated cytotoxicity - Abstract
Shiga toxin (Stx)-producing, food-contaminating Escherichia coli (STEC) is a major health concern. Plant-derived pectin and pectic-oligosaccharides (POS) have been considered as prebiotics and for the protection of humans from Stx. Of five structurally different citrus pectic samples, POS1, POS2 and modified citrus pectin 1 (MCP1) were bifidogenic with similar fermentabilities in human faecal cultures and arabinose-rich POS2 had the greatest prebiotic potential. Pectic oligosaccharides also enhanced lactobacilli growth during mixed batch faecal fermentation. We demonstrated that all pectic substrates were anti-adhesive for E. coli O157:H7 binding to human HT29 cells. Lower molecular weight and deesterification enhanced the anti-adhesive activity. We showed that all pectic samples reduced Stx2 cytotoxicity in HT29 cells, as measured by the reduction of human rRNA depurination detected by our novel TaqMan-based RT-qPCR assay, with POS1 performing the best. POS1 competes with Stx2 binding to the Gb3 receptor based on ELISA results, underlining the POS anti-STEC properties. [ABSTRACT FROM AUTHOR]
- Published
- 2017
- Full Text
- View/download PDF
45. Differences in Ribosome Binding and Sarcin/Ricin Loop Depurination by Shiga and Ricin Holotoxins.
- Author
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Xiao-Ping Li and Tumer, Nilgun E.
- Subjects
- *
RICIN , *BACTERIAL toxins , *RIBOSOMES , *BINDING sites , *BIOCHEMISTRY - Abstract
Both ricin and Shiga holotoxins display no ribosomal activity in their native forms and need to be activated to inhibit translation in a cell-free translation inhibition assay. This is because the ribosome binding site of the ricin A chain (RTA) is blocked by the B subunit in ricin holotoxin. However, it is not clear why Shiga toxin 1 (Stx1) or Shiga toxin 2 (Stx2) holotoxin is not active in a cell-free system. Here, we compare the ribosome binding and depurination activity of Stx1 and Stx2 holotoxins with the A1 subunits of Stx1 and Stx2 using either the ribosome or a 10-mer RNA mimic of the sarcin/ricin loop as substrates. Our results demonstrate that the active sites of Stx1 and Stx2 holotoxins are blocked by the A2 chain and the B subunit, while the ribosome binding sites are exposed to the solvent. Unlike ricin, which is enzymatically active, but cannot interact with the ribosome, Stx1 and Stx2 holotoxins are enzymatically inactive but can interact with the ribosome. [ABSTRACT FROM AUTHOR]
- Published
- 2017
- Full Text
- View/download PDF
46. Host response to the subtilase cytotoxin produced by locus of enterocyte effacement‐negative Shiga‐toxigenic Escherichia coli
- Author
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Kohei Ogura, Kinnosuke Yahiro, Hiroyasu Tsutsuki, and Joel Moss
- Subjects
Virulence Factors ,Protein subunit ,Immunology ,Apoptosis ,Biology ,Endoplasmic Reticulum ,Shiga Toxin 1 ,medicine.disease_cause ,Shiga Toxin 2 ,Microbiology ,Subtilase ,Foodborne Diseases ,Mice ,03 medical and health sciences ,Virology ,medicine ,Animals ,Humans ,Subtilisins ,Endoplasmic Reticulum Chaperone BiP ,Escherichia coli ,030304 developmental biology ,0303 health sciences ,Shiga-Toxigenic Escherichia coli ,030306 microbiology ,Escherichia coli Proteins ,Endoplasmic reticulum ,Shiga toxin ,Endoplasmic Reticulum Stress ,Chaperone (protein) ,Unfolded protein response ,biology.protein ,Locus of enterocyte effacement - Abstract
Shiga-toxigenic Escherichia coli (STEC) is a major bacterium responsible for disease resulting from foodborne infection, including bloody diarrhea and hemolytic uremic syndrome. STEC produces important virulence factors such as Shiga toxin (Stx) 1 and/or 2. In the STEC family, some locus of enterocyte effacement-negative STEC produce two different types of cytotoxins, namely, Stx2 and subtilase cytotoxin (SubAB). The Stx2 and SubAB cytotoxins are structurally similar and composed of one A subunit and pentamer of B subunits. The catalytically active A subunit of SubAB is a subtilase-like serine protease and specifically cleaves an endoplasmic reticulum (ER) chaperone 78-kDa glucose-regulated protein (GRP78/BiP), a monomeric ATPase that is crucial in protein folding and quality control. The B subunit binds to cell surface receptors. SubAB recognizes sialic carbohydrate-modified cell surface proteins as a receptor. After translocation into cells, SubAB is delivered to the ER, where it cleaves GRP78/BiP. SubAB-catalyzed BiP cleavage induces ER stress, which causes various cell events including inhibition of protein synthesis, suppression of nuclear factor-kappa B activation, apoptotic cell death, and stress granules formation. In this review, we describe SubAB, the SubAB receptor, and the mechanism of cell response to the toxin.
- Published
- 2020
47. Involvement of NF-κB1 and the Non-canonical NF-κB Signaling Pathway in the Pathogenesis of Acute Kidney Injury in Shiga-toxin-2-induced Hemolytic-uremic Syndrome in Mice
- Author
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Nadine Krieg, Sina M. Coldewey, Isabelle V Sobbe, and Sophie Dennhardt
- Subjects
Thrombotic microangiopathy ,030204 cardiovascular system & hematology ,Critical Care and Intensive Care Medicine ,Shiga Toxin 2 ,CCL5 ,Proinflammatory cytokine ,Pathogenesis ,Mice ,03 medical and health sciences ,0302 clinical medicine ,medicine ,Animals ,biology ,business.industry ,RELB ,NF-kappa B ,030208 emergency & critical care medicine ,Shiga toxin ,Acute Kidney Injury ,medicine.disease ,CXCL1 ,Disease Models, Animal ,Hemolytic-Uremic Syndrome ,Emergency Medicine ,Cancer research ,biology.protein ,Cytokines ,Signal transduction ,business ,Signal Transduction - Abstract
The hemolytic-uremic syndrome (HUS) is a thrombotic microangiopathy which can occur as a severe systemic complication after an infection with Shiga-toxin-(Stx)-producing Escherichia coli (STEC). Elevated levels of proinflammatory cytokines associated with the classical nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB) signaling pathway were detected in the urine of HUS patients. Thus, we hypothesize that the immune response of the infected organism triggered by Stx can affect the kidneys and contributes to acute kidney injury. Hitherto, the role of the classical and non-canonical NF-κB signaling pathway in HUS has not been evaluated systematically in vivo. We aimed to investigate in a murine model of Shiga toxin-induced HUS-like disease, whether one or both pathways are involved in the renal pathology in HUS. In kidneys of mice subjected to Stx or sham-treated mice, protein or gene expression analyses were performed to assess the expression of receptors activating the classical and non-canonical pathway, such as Fn14 and CD40, levels of NF-κB1/RelA and NF-κB2/RelB including its upstream signaling proteins, and expression of cytokines as target molecules of both pathways. In line with a higher expression of Fn14 and CD40, we detected an enhanced translocation of NF-κB1 and RelA as well as NF-κB2 and RelB into the nucleus accompanied by an increased gene expression of the NF-κB1-target cytokines Ccl20, Cxcl2, Ccl2, Cxcl1, IL-6, TNF-α, Cxcl10, and Ccl5, indicating an activation of the classical and non-canonical NF-κB pathway. Thereby, we provide, for the first time, in vivo evidence for an involvement of both NF-κB signaling pathways in renal pathophysiology of STEC-HUS.
- Published
- 2020
48. Rescue from Stx2-Producing E.?coli-Associated Encephalopathy by Intravenous Injection of Muse Cells in NOD-SCID Mice
- Author
-
Ryo Ozuru, Sohkichi Matsumoto, Shinsuke Kato, Kosuke Tashiro, Kaori Yasuda, Takahiro Tsuji, Muhammad Y. Amuran, Kimiharu Iwadate, Jun Fujii, Naoki Nishida, Yoichi Kurozawa, Noriko Konishi, Shohei Wakao, Junko Isobe, Morio Iino, Naoya Ohara, Takashi Matsuba, Eijiro Yamashita, Mari Dezawa, Arisato Yadoiwa, Misato Hida, and Sari Matsumoto
- Subjects
Male ,Cell Transplantation ,Mice, SCID ,Shiga Toxin 2 ,Immunoglobulin G ,Disease Outbreaks ,Mice ,0302 clinical medicine ,Japan ,Mice, Inbred NOD ,Shiga toxin-producing Escherichia coli ,Drug Discovery ,Escherichia coli Infections ,Aged, 80 and over ,0303 health sciences ,Brain Diseases ,Mice, Inbred ICR ,biology ,Shiga-Toxigenic Escherichia coli ,Brain ,Eculizumab ,Treatment Outcome ,030220 oncology & carcinogenesis ,Injections, Intravenous ,acute encephalopathy ,Molecular Medicine ,Female ,Original Article ,Stem cell ,medicine.drug ,Adult ,medicine.drug_class ,Encephalopathy ,Muse cells ,Monoclonal antibody ,Mesenchymal Stem Cell Transplantation ,Sudden death ,03 medical and health sciences ,Genetics ,medicine ,Animals ,Humans ,Immunoadsorption ,Molecular Biology ,030304 developmental biology ,Pharmacology ,Severe combined immunodeficiency ,business.industry ,medicine.disease ,Mice, Inbred C57BL ,Disease Models, Animal ,Immunology ,biology.protein ,business - Abstract
Shiga toxin-producing Escherichia coli (STEC) causes hemorrhagic colitis, hemolytic uremic syndrome, and acute encephalopathies that may lead to sudden death or severe neurologic sequelae. Current treatments, including immunoglobulin G (IgG) immunoadsorption, plasma exchange, steroid pulse therapy, and the monoclonal antibody eculizumab, have limited effects against the severe neurologic sequelae. Multilineage-differentiating stress-enduring (Muse) cells are endogenous reparative non-tumorigenic stem cells that naturally reside in the body and are currently under clinical trials for regenerative medicine. When administered intravenously, Musecells accumulate to the damaged tissue, where they exert anti-inflammatory, anti-apoptotic, anti-fibrotic, and immunomodulatory effects, and replace damaged cells by differentiating into tissue-constituent cells. Here, severely immunocompromised non-obese diabetic/severe combined immunodeficiency (NOD-SCID) mice orally inoculated with 9 × 109 colony-forming units of STEC O111 and treated 48 h later with intravenous injection of 5 × 104 Muse cells exhibited 100% survival and no severe after-effects of infection. Suppression of granulocyte-colony-stimulating factor (G-CSF) by RNAi abolished the beneficial effects of Muse cells, leading to a 40% death and significant body weight loss, suggesting the involvement of G-CSF in the beneficial effects of Muse cells in STEC-infected mice. Thus, intravenous administration of Muse cells could be a candidate therapeutic approach for preventing fatal encephalopathy after STEC infection., Graphical Abstract, NOD-SCID mice orally inoculated with 9 × 109 colony-forming units of Shiga toxin-producing Escherichia coli (STEC) O111 and treated 48 h later with an intravenous injection of 5 × 104 human bone marrow-derived multilineage-differentiating stress-enduring (Muse) cells exhibited 100% survival. Thus, the intravenous administration of Muse cells might be a candidate therapeutic approach for preventing acute encephalopathy after STEC infection.
- Published
- 2020
49. Prevalence of Virulence Genes of Diarrheagenic Escherichia coli in Fecal Samples Obtained from Cattle, Poultry and Diarrheic Patients in Bangladesh
- Author
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Mst Misrat Masuma Parvez, Eriko Kage-Nakadai, Mst. Nusrat Zahan, Wahedul Karim Ansari, Yoshikazu Nishikawa, Ashraful Alam, Sharif Uddin, Tanvir Rahman, Sayduzzaman Jowel, Mio Shono, and Shafiullah Parvej
- Subjects
Diarrhea ,0301 basic medicine ,Microbiology (medical) ,Virulence Factors ,030106 microbiology ,Cattle Diseases ,Virulence ,Enterotoxin ,Shiga Toxin 1 ,medicine.disease_cause ,Shiga Toxin 2 ,Poultry ,Microbiology ,Feces ,03 medical and health sciences ,0302 clinical medicine ,STX2 ,Escherichia coli ,Prevalence ,medicine ,Animals ,Humans ,Shigella ,030212 general & internal medicine ,Escherichia coli Infections ,Poultry Diseases ,Bangladesh ,biology ,Shiga toxin ,General Medicine ,biology.organism_classification ,Infectious Diseases ,biology.protein ,Cattle ,medicine.symptom ,Bacteria - Abstract
Using multiplex real-time PCR, 960 fecal samples collected from poultry, cattle, and patients with diarrhea in Bangladesh were screened for diarrheagenic Escherichia coli (DEC). The invasion-related gene virB showed the highest prevalence in human patients (41%) and was shown to be positively correlated first with afaB with regards to diffuse adhesion and second with aggR with regards to aggregative adhesion. These three genes were specific to human patients. In contrast, the Shiga toxin genes stx1 (57%) and stx2 (40%) were prevalent in cattle samples. The eae gene, which is associated with attaching and effacing lesion formation, and the elt and est genes, which are associated with enterotoxins, were detected from all three sample sources. Heat map construction and hierarchical clustering assigned the samples into five different clusters, with the patient samples positive for virB and afaB being placed together in one cluster. Although the detection of virulence genes cannot be a direct indication of the distribution of diarrheagenic organisms, their detection suggests that Shigella spp. or enteroinvasive E. coli are the most prevalent diarrheagenic bacteria in Bangladesh and that diffusely adherent E. coli is concomitantly present with these bacteria. eae-possessing organisms in patients may come from cattle and poultry sources. The small number of stx-positive patients could be explained by the small number of animal samples that were positive for both eae and stx.
- Published
- 2020
50. Stx2 Induces Differential Gene Expression and Disturbs Circadian Rhythm Genes in the Proximal Tubule
- Author
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Fumiko Obata, Ryo Ozuru, Takahiro Tsuji, Takashi Matsuba, and Jun Fujii
- Subjects
Lipopolysaccharides ,Circadian Rhythm Signaling Peptides and Proteins ,Health, Toxicology and Mutagenesis ,Protein Array Analysis ,Epithelial Cells ,Toxicology ,Shiga Toxin 2 ,Circadian Rhythm ,Kidney Tubules, Proximal ,Mice ,Shiga toxin type 2 (Stx2) ,renal proximal tubule ,mouse ,human renal proximal tubular epithelial cell (RPTEC) ,microarray ,circadian rhythm ,Gene Expression Regulation ,Glycosuria ,Animals ,Humans - Abstract
Shiga toxin-producing Escherichia coli (STEC) causes proximal tubular defects in the kidney. However, factors altered by Shiga toxin (Stx) within the proximal tubules are yet to be shown. We determined Stx receptor Gb3 in murine and human kidneys and confirmed the receptor expression in the proximal tubules. Stx2-injected mouse kidney tissues and Stx2-treated human primary renal proximal tubular epithelial cell (RPTEC) were collected and microarray analysis was performed. We compared murine kidney and RPTEC arrays and selected common 58 genes that are differentially expressed vs. control (0 h, no toxin-treated). We found that the most highly expressed gene was GDF15, which may be involved in Stx2-induced weight loss. Genes associated with previously reported Stx2 activities such as src kinase Yes phosphorylation pathway activation, unfolded protein response (UPR) and ribotoxic stress response (RSR) showed differential expressions. Moreover, circadian clock genes were differentially expressed, suggesting Stx2-induced renal circadian rhythm disturbance. Circadian rhythm-regulated proximal tubular Na+-glucose transporter SGLT1 (SLC5A1) was down-regulated, indicating proximal tubular functional deterioration, and mice developed glucosuria confirming proximal tubular dysfunction. Stx2 alters gene expression in murine and human proximal tubules through known activities and newly investigated circadian rhythm disturbance, which may result in proximal tubular dysfunctions.
- Published
- 2022
- Full Text
- View/download PDF
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