RESUMEN
Shiga toxin-producing Escherichia coli (STEC) causes a wide spectrum of diseases including hemorrhagic colitis and hemolytic uremic syndrome (HUS). Previously, we developed a rapid, sensitive, and potentially portable assay that identified STEC by detecting Shiga toxin (Stx) using a B-cell based biosensor platform. We applied this assay to detect Stx2 present in food samples that have been implicated in previous STEC foodborne outbreaks (milk, lettuce, and beef). The STEC enrichment medium, modified Tryptone Soy Broth (mTSB), inhibited the biosensor assay, but dilution with the assay buffer relieved this effect. Results with Stx2a toxoid-spiked food samples indicated an estimated limit of detection (LOD) of ≈4 ng/mL. When this assay was applied to food samples inoculated with STEC, it was able to detect 0.4 CFU/g or 0.4 CFU/mL of STEC at 16 h post incubation (hpi) in an enrichment medium containing mitomycin C. Importantly, this assay was even able to detect STEC strains that were high expressors of Stx2 at 8 hpi. These results indicate that the STEC CANARY biosensor assay is a rapid and sensitive assay applicable for detection of STEC contamination in food with minimal sample processing that can complement the current Food Safety Inspection Service (US) methodologies for STEC.
Asunto(s)
Técnicas Biosensibles , Microbiología de Alimentos , Lactuca , Escherichia coli Shiga-Toxigénica , Escherichia coli Shiga-Toxigénica/aislamiento & purificación , Técnicas Biosensibles/métodos , Lactuca/microbiología , Contaminación de Alimentos/análisis , Leche/microbiología , Animales , Toxina Shiga II/análisis , Toxina Shiga II/genética , Límite de Detección , Carne Roja/microbiología , BovinosRESUMEN
The rectal-anal junction (RAJ) is the major colonization site of Shiga toxin-producing Escherichia coli (STEC) O157 in beef cattle, leading to transmission of this foodborne pathogen from farms to food chains. To date, there is limited understanding regarding whether the mucosa-attached microbiome has a profound impact on host-STEC interactions. In this study, the active RAJ mucosa-attached microbiota and its potential role in host immunity-STEC commensal interactions were investigated using RAJ mucosal biopsies collected from calves orally challenged with two STEC O157 strains with or without functional stx2a (stx2a+ or stx2a-). The results revealed that shifts of microbial diversity, topology, and assembly patterns were subjected to stx2a production post-challenge and Paeniclostridium and Gallibacterium were the keystone taxa for both microbial interactions and assembly. Additional mucosal transcriptome profiling showed stx2a-dependent host immune responses (i.e. B- and T-cell signaling and antigen processing and presentation) post-challenge. Further integrated analysis revealed that mucosa-attached beneficial microbes (i.e. Provotella, Faecalibacterium, and Dorea) interacted with host immune genes pre-challenge to maintain host homeostasis; however, opportunistic pathogenic microbes (i.e. Paeniclostridium) could interact with host immune genes after the STEC O157 colonization and interactions were stx2a-dependent. Furthermore, predicted bacterial functions involved in pathogen (O157 and Paeniclostridium) colonization and metabolism were related to host immunity. These findings suggest that during pathogen colonization, host-microbe interactions could shift from beneficial to opportunistic pathogenic bacteria driven and be dependent on the production of particular virulence factors, highlighting the potential regulatory role of mucosa-attached microbiota in affecting pathogen-commensal host interactions in calves with STEC O157 infection.
Asunto(s)
Infecciones por Escherichia coli , Escherichia coli O157 , Mucosa Intestinal , Recto , Animales , Escherichia coli O157/inmunología , Escherichia coli O157/genética , Recto/microbiología , Bovinos , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/veterinaria , Mucosa Intestinal/microbiología , Mucosa Intestinal/inmunología , Enfermedades de los Bovinos/microbiología , Enfermedades de los Bovinos/inmunología , Microbioma Gastrointestinal , Interacciones Huésped-Patógeno , Interacciones Microbiota-Huesped/inmunología , Toxina Shiga II/genética , Toxina Shiga II/inmunologíaRESUMEN
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.
Asunto(s)
Lesión Renal Aguda , Síndrome Hemolítico-Urémico , Ratones Noqueados , Fosfotransferasas (Aceptor de Grupo Alcohol) , Animales , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Lesión Renal Aguda/metabolismo , Lesión Renal Aguda/patología , Lesión Renal Aguda/etiología , Lesión Renal Aguda/genética , Ratones , Síndrome Hemolítico-Urémico/patología , Síndrome Hemolítico-Urémico/genética , Modelos Animales de Enfermedad , Esfingolípidos/metabolismo , Riñón/patología , Riñón/metabolismo , Ratones Endogámicos C57BL , Toxina Shiga II , Eliminación de Gen , MasculinoRESUMEN
Shiga toxins are the main virulence factors of Shiga toxin producing E. coli (STEC) and S. dysenteriae. There is no effective therapy to counter the disease caused by these toxins. The A1 subunits of Shiga toxins bind the C-termini of ribosomal P-stalk proteins to depurinate the sarcin/ricin loop. The ribosome binding site of Shiga toxin 2 has not been targeted by small molecules. We screened a fragment library against the A1 subunit of Shiga toxin 2 (Stx2A1) and identified a fragment, BTB13086, which bound at the ribosome binding site and mimicked the binding mode of the P-stalk proteins. We synthesized analogs of BTB13086 and identified a series of molecules with similar affinity and inhibitory activity. These are the first compounds that bind at the ribosome binding site of Stx2A1 and inhibit activity. These compounds hold great promise for further inhibitor development against STEC infection.
Asunto(s)
Ribosomas , Toxina Shiga II , Ribosomas/metabolismo , Ribosomas/efectos de los fármacos , Toxina Shiga II/antagonistas & inhibidores , Toxina Shiga II/metabolismo , Toxina Shiga II/química , Sitios de Unión , Unión Proteica , Escherichia coli Shiga-Toxigénica/efectos de los fármacos , Escherichia coli Shiga-Toxigénica/metabolismo , HumanosRESUMEN
Ricin is one of the most toxic substances known and a type B biothreat agent. Shiga toxins (Stxs) produced by E. coli (STEC) and Shigella dysenteriae are foodborne pathogens. There is no effective therapy against ricin or STEC and there is an urgent need for inhibitors. Ricin toxin A subunit (RTA) and A1 subunit of Stx2a (Stx2A1) bind to the C-terminal domain (CTD) of the ribosomal P-stalk proteins to depurinate the sarcin/ricin loop. Modulation of toxin-ribosome interactions has not been explored as a strategy for inhibition. Therefore, development of assays that detect inhibitors targeting toxin-ribosome interactions remains a critical need. Here we describe a fluorescence anisotropy (FA)-based competitive binding assay using a BODIPY-TMR labeled 11-mer peptide (P11) derived from the P-stalk CTD to measure the binding affinity of peptides ranging from 3 to 11 amino acids for the P-stalk pocket of RTA and Stx2A1. Comparison of the affinity with the surface plasmon resonance (SPR) assay indicated that although the rank order was the same by both methods, the FA assay could differentiate better between peptides that show nonspecific interactions by SPR. The FA assay detects only interactions that compete with the labeled P11 and can validate inhibitor specificity and mechanism of action.
Asunto(s)
Polarización de Fluorescencia , Ribosomas , Ricina , Ricina/antagonistas & inhibidores , Ricina/metabolismo , Ricina/química , Polarización de Fluorescencia/métodos , Ribosomas/metabolismo , Resonancia por Plasmón de Superficie , Toxina Shiga/antagonistas & inhibidores , Toxina Shiga/metabolismo , Toxina Shiga/química , Unión Competitiva , Unión Proteica , Toxina Shiga II/antagonistas & inhibidores , Toxina Shiga II/metabolismo , Toxina Shiga II/químicaRESUMEN
Two patients with acute gastroenteritis tested positive for Shiga toxin-producing Escherichia coli (STEC) by polymerase chain reaction (PCR), and both strains carried the Shiga toxin 2 encoding gene. Since routine culture using CHROMagar STEC failed to recover these isolates, immunomagnetic separation (IMS) targeting the top six non-O157:H7 serotypes was used for isolate recovery. After two subsequent IMS runs, the STEC strains were isolated from trypticase soy broth with and without overnight enrichment for runs 1 and 2, respectively. Serotyping based on whole-genome sequencing revealed that both patients carried the strain O166:H15 STEC with the stx2 gene. Hence, the magnetic beads used in IMS appeared to have cross-reactivity with other E. coli serotypes. When the STEC isolates from both stools were cultured on CHROMagar STEC and sheep blood agar (BAP), two distinct colony sizes were apparent after overnight incubation. The small and large colonies were picked and separately cultured on both media, and colony growth was observed for 2 weeks at room temperature after an initial overnight incubation at 37°C. After 1 week, the colonies showed concentric ring structures with a darker center and a lighter surrounding on CHROMagar STEC and a "fried egg"-resembling structure with a raised circular center and a flat surrounding on BAP. Both colony types remained morphologically different on CHROMagar STEC throughout the 15 days. However, on BAP, their appearance was comparable by day 7. IMPORTANCE: Shiga toxin-producing E. coli (STEC) infections can lead to severe complications such as bloody diarrhea and hemolytic uremic syndrome (HUS), especially in young children and the elderly. Strains that carry the shiga toxin 2 gene (stx2), such as O157:H7, have been mostly linked with severe disease outcomes. In recent years, outbreaks caused by non-O157:H7 strains have increased. E. coli O166:H15 has been previously reported causing a gastroenteritis outbreak in 1996 as a non-STEC strain, however the O166:H15 serotype we recovered carried the stx2 gene. It was particularly challenging to isolate this strain from stools by culture. Consequently, we tested immunomagnetic separation for the STEC recovery, which was a novel approach on clinical stools. Virulence genes were included for the characterization of these isolates.
Asunto(s)
Infecciones por Escherichia coli , Heces , Gastroenteritis , Toxina Shiga II , Escherichia coli Shiga-Toxigénica , Escherichia coli Shiga-Toxigénica/genética , Escherichia coli Shiga-Toxigénica/aislamiento & purificación , Escherichia coli Shiga-Toxigénica/clasificación , Heces/microbiología , Humanos , Toxina Shiga II/genética , Infecciones por Escherichia coli/microbiología , Gastroenteritis/microbiología , Separación Inmunomagnética , Serotipificación , Masculino , Serogrupo , Femenino , Secuenciación Completa del GenomaRESUMEN
Escherichia coli O157:H7 (E. coli O157:H7) and Campylobacter jejuni (C. jejuni) are pathogenic microorganisms that can cause severe clinical symptoms in humans and are associated with bovine meat consumption. Specific monitoring for E. coli O157: H7 or C. jejuni in meat is not mandatory under Chilean regulations. In this study, we analyzed 544 samples for the detection of both microorganisms, obtained from 272 bovine carcasses (280 kg average) at two slaughterhouses in the Bio-Bío District, Chile. Sampling was carried out at post-shower of carcasses and after channel passage through the cold chamber. Eleven samples were found to be positive for E. coli O157:H7 (4.0%) using microbiological and biochemical detection techniques and were subjected to a multiplex PCR to detect fliC and rfbE genes. Six samples (2.2%) were also found to be positive for the pathogenicity genes stx1, stx2, and eaeA. Twenty-two carcasses (8.0%) were found to be positive for C. jejuni using microbiological and biochemical detection techniques, but no sample with amplified mapA gene was found.
Asunto(s)
Mataderos , Campylobacter jejuni , Escherichia coli O157 , Proteínas de Escherichia coli , Microbiología de Alimentos , Animales , Bovinos , Campylobacter jejuni/aislamiento & purificación , Campylobacter jejuni/genética , Escherichia coli O157/aislamiento & purificación , Escherichia coli O157/genética , Chile , Proteínas de Escherichia coli/genética , Flagelina/genética , Carne/microbiología , Contaminación de Alimentos/análisis , Adhesinas Bacterianas/genética , Toxina Shiga I/genética , Toxina Shiga II/genética , Reacción en Cadena de la Polimerasa Multiplex , Proteínas Bacterianas/genética , Transaminasas , Carbohidrato EpimerasasRESUMEN
Shiga toxigenic E. coli are important foodborne zoonotic pathogens. The present study was envisaged to standardize loop-mediated isothermal amplification assays targeting stx1 and stx2 genes for rapid and visual detection of STEC and compare its sensitivity with PCR. The study also assessed the effect of short enrichment on the detection limit of LAMP and PCR. The developed LAMP assays were found to be highly specific. Analytical sensitivity of LAMP was 94 fg/µLand 25.8 fg/µL for stx-1 and stx-2 while LOD of 5 CFU/g of carabeef was measured after 6-12 h enrichment. The study highlights the importance of short (6-12 h) enrichment for improving the sensitivity of LAMP. The entire detection protocol could be performed within 9 h yielding results on the same day. The developed LAMP assays proved to be a handy and cost-effective alternative for screening STEC contamination in meat.
Asunto(s)
Carne , Técnicas de Diagnóstico Molecular , Técnicas de Amplificación de Ácido Nucleico , Sensibilidad y Especificidad , Escherichia coli Shiga-Toxigénica , Escherichia coli Shiga-Toxigénica/genética , Escherichia coli Shiga-Toxigénica/aislamiento & purificación , Técnicas de Amplificación de Ácido Nucleico/métodos , Animales , Técnicas de Diagnóstico Molecular/métodos , Carne/microbiología , Microbiología de Alimentos/métodos , Toxina Shiga I/genética , Toxina Shiga II/genética , Contaminación de Alimentos/análisisRESUMEN
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.
Asunto(s)
Abrina , Toxina Shiga II , Escherichia coli , Toxina Shiga , BioensayoRESUMEN
Shiga toxin 2a (Stx2a) is the virulence factor of Escherichia coli (STEC), which is associated with hemolytic uremic syndrome, the leading cause of pediatric kidney failure. The A1 subunit of Stx2a (Stx2A1) binds to the conserved C-terminal domain (CTD) of the ribosomal P-stalk proteins to remove an adenine from the sarcin-ricin loop (SRL) in the 28S rRNA, inhibiting protein synthesis. There are no antidotes against Stx2a or any other ribosome-inactivating protein (RIP). The structural and functional details of the binding of Stx2A1 to the P-stalk CTD are not known. Here, we carry out a deletion analysis of the conserved P-stalk CTD and show that the last eight amino acids (P8) of the P-stalk proteins are the minimal sequence required for optimal affinity and maximal inhibitory activity against Stx2A1. We determined the first X-ray crystal structure of Stx2A1 alone and in complex with P8 and identified the exact binding site. The C-terminal aspartic acid of the P-stalk CTD serves as an anchor, forming key contacts with the conserved arginine residues at the P-stalk binding pocket of Stx2A1. Although the ricin A subunit (RTA) binds to the P-stalk CTD, the last aspartic acid is more critical for the interaction with Stx2A1, indicating that RIPs differ in their requirements for the P-stalk. These results demonstrate that the catalytic activity of Stx2A1 is inhibited by blocking its interactions with the P-stalk, providing evidence that P-stalk binding is an essential first step in the recruitment of Stx2A1 to the SRL for depurination.
Asunto(s)
Ricina , Toxina Shiga II , Humanos , Niño , Toxina Shiga II/análisis , Toxina Shiga II/metabolismo , Ribosomas/metabolismo , Ricina/química , Ricina/genética , Ricina/metabolismo , Ácido Aspártico , Sitios de Unión , Péptidos/metabolismo , Escherichia coli/metabolismoRESUMEN
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.
Asunto(s)
Infecciones por Escherichia coli , Escherichia coli O157 , Síndrome Hemolítico-Urémico , Insuficiencia Renal , Microangiopatías Trombóticas , Humanos , Niño , Animales , Ratones , Toxina Shiga II , Células Endoteliales , Hemólisis , Arginasa , Síndrome Hemolítico-Urémico/complicaciones , Síndrome Hemolítico-Urémico/terapia , Eritrocitos , Microangiopatías Trombóticas/complicaciones , Urea , Arginina , Ornitina , Lactato Deshidrogenasas , Infecciones por Escherichia coli/complicaciones , Infecciones por Escherichia coli/terapiaRESUMEN
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.
Asunto(s)
Síndrome Hemolítico-Urémico , Toxina Shiga II , Niño , Adulto , Humanos , Animales , Caballos , Toxina Shiga I , Voluntarios Sanos , Método Simple CiegoRESUMEN
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.
Asunto(s)
Infecciones por Escherichia coli , Síndrome Hemolítico-Urémico , Escherichia coli Shiga-Toxigénica , Humanos , Adulto , Animales , Ratones , Toxina Shiga/uso terapéutico , Infecciones por Escherichia coli/tratamiento farmacológico , Toxina Shiga II , Anticuerpos Monoclonales Humanizados/uso terapéutico , Síndrome Hemolítico-Urémico/tratamiento farmacológicoRESUMEN
BACKGROUND: Shiga toxin-producing Escherichia coli is the main cause of post-diarrheal hemolytic uremic syndrome (HUS) which produces acute kidney injury mainly in children, although it can also affect adults. The kidneys are the organs most affected by Shiga toxin type 2 (Stx2) in patients with HUS. However, previous studies in pregnant rats showed that a sublethal dose of Stx2 causes severe damage in the uteroplacental unit and induces abortion, whereas produces mild to moderate renal damage. The aim of the present work was to study the progression of renal injury caused by a sublethal dose of Stx2, as well as renal recovery, in pregnant and non-pregnant rats, and to investigate whether pregnancy physiology may affect renal damage progression mediated by Stx2. METHODS: Renal function and histopathology was evaluated in pregnant rats intraperitoneally injected with a sublethal dose of Stx2 (0.5 ng/g bwt) at the early stage of gestation (day 8 of gestation), and results in these rats were compared over time with those observed in non-pregnant female rats injected with the same Stx2 dose. Hence, progression of cell proliferation and dedifferentiation in renal tubular epithelia was also investigated. RESULTS: The sublethal dose of Stx2 induced abortion in pregnant rats as well as a significant more extended functional and histological renal injury in non-pregnant rats than in pregnant rats. Stx2 also caused decreased ability to concentrate urine in non-pregnant rats compared to their controls. However, renal water handling in pregnant rats was not altered by Stx2, and was significantly different than in non-pregnant rats. The greatest renal injury in both pregnant and non-pregnant rats was observed at 4 days post-Stx2 injection, and coincided with a significant increase in tubular epithelial proliferation. Expression of mesenchymal marker vimentin in tubular epithelia was consistent with the level of tubular damage, being higher in non-pregnant rats than in pregnant rats. Recovery from Stx2-induced kidney injury was faster in pregnant rats than in non-pregnant rats. CONCLUSIONS: Adaptive mechanisms developed during pregnancy such as changes in water handle and renal hemodynamic may contribute to lessen the Stx2-induced renal injury, perhaps at the expense of fetal loss.
Asunto(s)
Síndrome Hemolítico-Urémico , Escherichia coli Shiga-Toxigénica , Humanos , Embarazo , Niño , Adulto , Ratas , Femenino , Animales , Toxina Shiga II/toxicidad , Riñón/patología , Síndrome Hemolítico-Urémico/patología , Agua , RegeneraciónRESUMEN
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.
Asunto(s)
Infecciones por Escherichia coli , Escherichia coli Shiga-Toxigénica , Humanos , Toxina Shiga II , Toxina Shiga , Neutrófilos , Bacterias , Infecciones por Escherichia coli/microbiologíaRESUMEN
Shiga toxin (Stx) is associated with foodborne infections of some Shigella spp. and Shiga toxin-producing Escherichia coli (STEC), leading to life-threatening hemolytic uremic syndrome (HUS). Target-specific therapeutics against HUS are currently unavailable in clinical practice. Herein, we reported the construction and in vitro characterization of Gb3-coated bovine milk exosomes (Gb3-mExo) as a multivalent Shiga toxin neutralizer, utilizing the natural advantages of milk exosomes (mExo) in drug delivery and multivalent interactions between Stx and its receptor Gb3. Gb3-mExo constructs were achieved by conjugating mExo with the Gb3 derivatives containing stearic acid-derived lipid tail, which was prepared through an efficient chemoenzymatic approach. The constructs were able to potently neutralize the binding of the B subunit of Stx2 (Stx2B) to receptor Gb3 immobilized on the plate or expressed on model cells. General safety of the constructs was evidenced by the cytotoxicity analysis and hemolysis assay. In addition to the excellent stability under conventional storage and handling conditions, the construct can also retain most of its neutralization potency under gastrointestinal pH extremes, showing the potential for oral administration. Considering the natural availability and excellent biocompatibility of mExo, Gb3-mExo conjugates should prove to be a practical prophylactic and therapeutic for the Shiga toxin-related infections.
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Exosomas , Escherichia coli Shiga-Toxigénica , Animales , Toxina Shiga , Toxina Shiga II/metabolismo , Exosomas/metabolismo , Leche/metabolismo , Escherichia coli Shiga-Toxigénica/metabolismoRESUMEN
Shiga toxin producing Escherichia coli (STEC) are foodborne pathogens that release Shiga toxin (Stx), virulence factor responsible for the development of Hemolytic Uremic Syndrome (HUS). Stx causes endothelial cell damage, which leads to platelets deposition and thrombi formation within the microvasculature. It has been described that Stx activates blood cells and induces the shedding of proinflammatory and prothrombotic microvesicles (MVs) containing the toxin. In this sense, it has been postulated that MVs containing Stx2 (MVs-Stx2+) can contribute to the physiopathology of HUS, allowing Stx2 to reach the target organs while evading the immune system. In this work, we propose that circulating MVs-Stx2+ can be a potential biomarker for the diagnosis and prognosis of STEC infections and HUS progression. We developed a rat HUS model by the intraperitoneal injection of a sublethal dose of Stx2 and observed: decrease in body weight, increase of creatinine and urea levels, decrease of creatinine clearance and histological renal damages. After characterization of renal damages, we investigated circulating total MVs and MVs-Stx2+ by flow cytometry at different times after Stx2 injection. Additionally, we evaluated the correlation of biochemical parameters such as creatinine and urea in plasma with MVs-Stx2+. As a result, we found a significant circulation of MVs-Stx2+ at 72 and 96 h after Stx2 injection, nevertheless no correlation with creatinine and urea plasma levels were detected. Our results suggest that MVs-Stx2+ may be an additional biomarker for the characterization and diagnosis of HUS progression. A further analysis is required in order to validate MVs-Stx2+ as biomarker of the disease.
Asunto(s)
Infecciones por Escherichia coli , Síndrome Hemolítico-Urémico , Escherichia coli Shiga-Toxigénica , Ratas , Animales , Toxina Shiga II/toxicidad , Creatinina , Síndrome Hemolítico-Urémico/etiología , Síndrome Hemolítico-Urémico/patología , Urea , BiomarcadoresRESUMEN
Introduction: Hemolytic-uremic syndrome (HUS) can occur as a systemic complication of infection with Shiga toxin (Stx)-producing Escherichia coli (STEC). Most well-known aspects of the pathophysiology are secondary to microthrombotic kidney disease including hemolytic anemia and thrombocytopenia. However, extrarenal manifestations, such as cardiac impairment, have also been reported. We have investigated whether these cardiac abnormalities can be reproduced in a murine animal model, in which administration of Stx, the main virulence factor of STEC, is used to induce HUS. Methods: Mice received either one high or multiple low doses of Stx to simulate the (clinically well-known) different disease courses. Cardiac function was evaluated by echocardiography and analyses of biomarkers in the plasma (troponin I and brain natriuretic peptide). Results: All Stx-challenged mice showed reduced cardiac output and depletion of intravascular volume indicated by a reduced end-diastolic volume and a higher hematocrit. Some mice exhibited myocardial injury (measured as increases in cTNI levels). A subset of mice challenged with either dosage regimen showed hyperkalemia with typical electrocardiographic abnormalities. Discussion: Myocardial injury, intravascular volume depletion, reduced cardiac output, and arrhythmias as a consequence of hyperkalemia may be prognosis-relevant disease manifestations of HUS, the significance of which should be further investigated in future preclinical and clinical studies.
Asunto(s)
Infecciones por Escherichia coli , Síndrome Hemolítico-Urémico , Hiperpotasemia , Escherichia coli Shiga-Toxigénica , Animales , Ratones , Toxina Shiga II/toxicidad , Proyectos Piloto , Infecciones por Escherichia coli/complicacionesRESUMEN
The pathogenicity of Escherichia coli (E. coli) O157:H7 is predominantly associated with Shiga toxin 2 (Stx2) that poses a huge threat to human and animal intestinal health. Production of Stx2 requires expression of stx2 gene, which is located in the genome of lambdoid Stx2 prophage. Growing evidence has implicated that many commonly consumed foods participate in the regulation of prophage induction. In this study, we aimed to explore whether specific dietary functional sugars could inhibit Stx2 prophage induction in E. coli O157:H7, thereby preventing Stx2 production and promoting intestinal health. We demonstrated that Stx2 prophage induction in E. coli O157:H7 was strongly inhibited by L-arabinose both in vitro and in a mouse model. Mechanistically, L-arabinose at doses of 9, 12, or 15 mM diminished RecA protein levels, a master mediator of the SOS response, contributing to reduced Stx2-converting phage induction. L-Arabinose inhibited quorum sensing and oxidative stress response, which are known as positive regulators of the SOS response and subsequent Stx2 phage production. Furthermore, L-arabinose impaired E. coli O157:H7 arginine transport and metabolism that were involved in producing Stx2 phage. Collectively, our results suggest that L-arabinose may be exploited as a novel Stx2 prophage induction inhibitor against E. coli O157:H7 infection.
Asunto(s)
Bacteriófagos , Escherichia coli O157 , Microbioma Gastrointestinal , Humanos , Animales , Ratones , Toxina Shiga II/genética , Toxina Shiga II/metabolismo , Bacteriófagos/genética , Escherichia coli O157/genética , Arabinosa/metabolismoRESUMEN
BACKGROUND: Shiga toxin-producing Escherichia coli (STEC) is a significant cause of foodborne illness causing various gastrointestinal diseases including hemolytic uremic syndrome (HUS), the most severe form, which can lead to kidney failure or even death. OBJECTIVE: Here, we report the development of recombinase aided amplification (RAA)-exo-probe assays targeting the stx1 and stx2 genes for the rapid detection of STEC in food samples. METHODS: Primers and exo-probes were designed and optimized for the detection of stx1 and stx2 using RAA technology. The optimal STEC RAA-exo-probe assays were then tested for specificity and sensitivity, and validated in both spiked and real food samples. RESULTS: These assays were found to be 100% specific to STEC strains and were also highly sensitive with a detection limit of 1.6 × 103 CFU/mL or 32 copies/reaction. Importantly, the assays were able to successfully detect STEC in spiked and real food samples (beef, mutton, and pork), with a detection limit as low as 0.35 CFU/25g in beef samples after an overnight enrichment step. CONCLUSIONS: Overall, the RAA assay reactions completed within â¼20 min and were less dependent on expensive equipment, suggesting they can be easily adopted for in-field testing requiring only a fluorescent reader. HIGHLIGHTS: As such, we have developed two rapid, sensitive, and specific assays that can be used for the routine monitoring of STEC contamination in food samples, particularly in the field or in poorly equipped labs.