RESUMEN
Enterohemorrhagic or Shiga toxin-producing Escherichia coli is a food-poisoning bacterium that grows in the intestine to produce Shiga toxin (Stx). In this study, the effects of 20 polyphenols on the cytotoxicity of Stx1 and Stx2 in Vero cells were investigated. Among these, epigallocatechin gallate, butein, isorhapontigenin, hesperetin, morin, luteolin, resveratrol, and rhapontigenin showed inhibitory effects on the cytotoxicity of Stxs at 0.4 mmol/L. Furthermore, Vero cells pre-treated with these polyphenols were resistant to Stx at 0.4 mmol/L. However, luteolin showed the most potent inhibitory and cytoprotective effect against Stxs at 0.08 mmol/L or more. This inhibitory mechanism of luteolin was determined using a cell-free protein synthesis system and quantitative reverse transcription PCR assay to detect depurination of 28S rRNA in Vero cells. Luteolin did not inhibit the cell-free protein synthesis by Stxs, suggesting that the enzymatic activity of the Stx A subunit was not inhibited by luteolin. The depurination of 28S rRNA by Stxs was also investigated in Vero cells. The 28S rRNA depurination by Stxs was suppressed in Vero cells treated with Stxs which had been pretreated with luteolin. These results suggest that luteolin inhibits the incorporation of Stxs into Vero cells. This is the first report to show that luteolin inhibits the cytotoxicity of both Stx1 and Stx2 by inhibiting the incorporation of Stxs into Vero cells.
Asunto(s)
Toxina Shiga II , Toxina Shiga , Animales , Chlorocebus aethiops , Células Vero , Toxina Shiga/toxicidad , Toxina Shiga I/toxicidad , Toxina Shiga I/metabolismo , Toxina Shiga II/toxicidad , Toxina Shiga II/metabolismo , Luteolina/farmacología , ARN Ribosómico 28SRESUMEN
Enterohaemorrhagic Escherichia coli (EHEC) produces Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2). Although stx1 and stx2 were found within the late operons of the Stx-encoding phages (Stx-phages), stx1 could mainly be transcribed from the stx1 promoter (PStx1), which represents the functional operator-binding site (Fur box) for the transcriptional regulator Fur (ferric uptake regulator), upstream of stx1. In this study, we found that the production of Stx1 by EHEC was affected by oxygen concentration. Increased Stx1 production in the presence of oxygen is dependent on Fur, which is an Fe2+-responsive transcription factor. The intracellular Fe2+ pool was lower under microaerobic conditions than under anaerobic conditions, suggesting that lower Fe2+ availability drove the formation of less Fe2+-Fur, less DNA binding to the PStx1 region, and an increase in Stx1 production.
Asunto(s)
Bacteriófagos , Escherichia coli Enterohemorrágica , Escherichia coli Enterohemorrágica/genética , Escherichia coli Enterohemorrágica/metabolismo , Oxígeno/metabolismo , Toxina Shiga I/genética , Toxina Shiga I/metabolismo , Toxina Shiga II/genética , Toxina Shiga II/metabolismoRESUMEN
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.
Asunto(s)
Antineoplásicos Hormonales/farmacología , Toxina Shiga I/metabolismo , Toxina Shiga II/metabolismo , Tamoxifeno/farmacología , Endosomas/efectos de los fármacos , Células HeLa , Humanos , Transporte de Proteínas/efectos de los fármacosRESUMEN
Human kidney epithelial cells are supposed to be directly involved in the pathogenesis of the hemolytic-uremic syndrome (HUS) caused by Shiga toxin (Stx)-producing enterohemorrhagic Escherichia coli (EHEC). The characterization of the major and minor Stx-binding glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer), respectively, of primary human renal cortical epithelial cells (pHRCEpiCs) revealed GSLs with Cer (d18:1, C16:0), Cer (d18:1, C22:0), and Cer (d18:1, C24:1/C24:0) as the dominant lipoforms. Using detergent-resistant membranes (DRMs) and non-DRMs, Gb3Cer and Gb4Cer prevailed in the DRM fractions, suggesting their association with microdomains in the liquid-ordered membrane phase. A preference of Gb3Cer and Gb4Cer endowed with C24:0 fatty acid accompanied by minor monounsaturated C24:1-harboring counterparts was observed in DRMs, whereas the C24:1 fatty acid increased in relation to the saturated equivalents in non-DRMs. A shift of the dominant phospholipid phosphatidylcholine with saturated fatty acids in the DRM to unsaturated species in the non-DRM fractions correlated with the GSL distribution. Cytotoxicity assays gave a moderate susceptibility of pHRCEpiCs to the Stx1a and Stx2a subtypes when compared to highly sensitive Vero-B4 cells. The results indicate that presence of Stx-binding GSLs per se and preferred occurrence in microdomains do not necessarily lead to a high cellular susceptibility towards Stx.
Asunto(s)
Células Epiteliales/metabolismo , Globósidos/metabolismo , Corteza Renal/metabolismo , Toxina Shiga I/toxicidad , Toxina Shiga II/toxicidad , Trihexosilceramidas/metabolismo , Animales , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , Células Epiteliales/patología , Infecciones por Escherichia coli/microbiología , Síndrome Hemolítico-Urémico/microbiología , Humanos , Corteza Renal/patología , Microdominios de Membrana/efectos de los fármacos , Microdominios de Membrana/metabolismo , Microdominios de Membrana/patología , Cultivo Primario de Células , Unión Proteica , Toxina Shiga I/metabolismo , Toxina Shiga II/metabolismo , Escherichia coli Shiga-Toxigénica/metabolismo , Escherichia coli Shiga-Toxigénica/patogenicidad , Células VeroRESUMEN
The human Gb3/CD77 synthase, encoded by the A4GALT gene, is an unusually promiscuous glycosyltransferase. It synthesizes the Galα1â4Gal linkage on two different glycosphingolipids (GSLs), producing globotriaosylceramide (Gb3, CD77, Pk) and the P1 antigen. Gb3 is the major receptor for Shiga toxins (Stxs) produced by enterohemorrhagic Escherichia coli. A single amino acid substitution (p.Q211E) ramps up the enzyme's promiscuity, rendering it able to attach Gal both to another Gal residue and to GalNAc, giving rise to NOR1 and NOR2 GSLs. Human Gb3/CD77 synthase was long believed to transfer Gal only to GSL acceptors, therefore its GSL products were, by default, considered the only human Stx receptors. Here, using soluble, recombinant human Gb3/CD77 synthase and p.Q211E mutein, we demonstrate that both enzymes can synthesize the P1 glycotope (terminal Galα1â4Galß1â4GlcNAc-R) on a complex type N-glycan and a synthetic N-glycoprotein (saposin D). Moreover, by transfection of CHO-Lec2 cells with vectors encoding human Gb3/CD77 synthase and its p.Q211E mutein, we demonstrate that both enzymes produce P1 glycotopes on N-glycoproteins, with the mutein exhibiting elevated activity. These P1-terminated N-glycoproteins are recognized by Stx1 but not Stx2 B subunits. Finally, cytotoxicity assays show that Stx1 can use P1 N-glycoproteins produced in CHO-Lec2 cells as functional receptors. We conclude that Stx1 can recognize and use P1 N-glycoproteins in addition to its canonical GSL receptors to enter and kill the cells, while Stx2 can use GSLs only. Collectively, these results may have important implications for our understanding of the Shiga toxin pathology.
Asunto(s)
Galactosiltransferasas/química , Globósidos/química , Toxina Shiga I/química , Trihexosilceramidas/química , Acetilgalactosamina/química , Acetilgalactosamina/metabolismo , Acetilglucosamina/química , Acetilglucosamina/metabolismo , Animales , Sitios de Unión , Células CHO , Secuencia de Carbohidratos , Cricetulus , Escherichia coli Enterohemorrágica/química , Escherichia coli Enterohemorrágica/patogenicidad , Galactosa/química , Galactosa/metabolismo , Galactosiltransferasas/genética , Galactosiltransferasas/metabolismo , Expresión Génica , Globósidos/biosíntesis , Globósidos/metabolismo , Glucosa/química , Glucosa/metabolismo , Humanos , Modelos Moleculares , Mutación , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Toxina Shiga I/metabolismo , Toxina Shiga II/química , Toxina Shiga II/metabolismo , Trihexosilceramidas/biosíntesisRESUMEN
Inflammatory caspase-dependent cytosolic lipopolysaccharide (LPS) sensing is a critical arm of host defense against bacteria. How pathogens overcome this pathway to establish infections is largely unknown. Enterohemorrhagic Escherichia coli (EHEC) is a clinically important human pathogen causing hemorrhagic colitis and hemolytic uremic syndrome. We found that a bacteriophage-encoded virulence factor of EHEC, Shiga toxin (Stx), suppresses caspase-11-mediated activation of the cytosolic LPS sensing pathway. Stx was essential and sufficient to inhibit pyroptosis and interleukin-1 (IL-1) responses elicited specifically by cytosolic LPS. The catalytic activity of Stx was necessary for suppression of inflammasome responses. Stx impairment of inflammasome responses to cytosolic LPS occurs at the level of gasdermin D activation. Stx also suppresses inflammasome responses in vivo after LPS challenge and bacterial infection. Overall, this study assigns a previously undescribed inflammasome-subversive function to a well-known bacterial toxin, Stx, and reveals a new phage protein-based pathogen blockade of cytosolic immune surveillance.
Asunto(s)
Escherichia coli Enterohemorrágica/patogenicidad , Infecciones por Escherichia coli/inmunología , Inflamasomas/inmunología , Toxina Shiga I/metabolismo , Toxina Shiga II/metabolismo , Animales , Bacteriófagos/inmunología , Bacteriófagos/metabolismo , Caspasas Iniciadoras/genética , Caspasas Iniciadoras/metabolismo , Chlorocebus aethiops , Citrobacter rodentium/inmunología , Citrobacter rodentium/patogenicidad , Modelos Animales de Enfermedad , Escherichia coli Enterohemorrágica/inmunología , Escherichia coli Enterohemorrágica/virología , Infecciones por Escherichia coli/microbiología , Femenino , Humanos , Vigilancia Inmunológica , Inflamasomas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lipopolisacáridos/inmunología , Masculino , Ratones , Ratones Noqueados , Proteínas de Unión a Fosfato/metabolismo , Toxina Shiga I/inmunología , Toxina Shiga II/inmunología , Células Vero , Proteínas Virales/inmunología , Proteínas Virales/metabolismo , Factores de Virulencia/inmunología , Factores de Virulencia/metabolismoRESUMEN
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.
Asunto(s)
Estrés del Retículo Endoplásmico/fisiología , Proteínas de Escherichia coli/metabolismo , Escherichia coli Shiga-Toxigénica/metabolismo , Subtilisinas/metabolismo , Animales , Apoptosis/fisiología , Retículo Endoplásmico/fisiología , Chaperón BiP del Retículo Endoplásmico , Proteínas de Escherichia coli/genética , Enfermedades Transmitidas por los Alimentos/microbiología , Humanos , Ratones , Toxina Shiga I/metabolismo , Toxina Shiga II/metabolismo , Escherichia coli Shiga-Toxigénica/genética , Subtilisinas/genética , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
Shiga toxin (Stx)-stimulated blood cells shed extracellular vesicles (EVs) which can transfer the toxin to the kidneys and lead to hemolytic uremic syndrome. The toxin can be taken up by renal cells within EVs wherein the toxin is released, ultimately leading to cell death. The mechanism by which Stx is taken up, translocated, and sequestered in EVs was addressed in this study utilizing the B-subunit that binds to the globotriaosylceramide (Gb3) receptor. We found that Stx1B was released in EVs within minutes after stimulation of HeLa cells or red blood cells, detected by live cell imaging and flow cytometry. In the presence of Retro-2.1, an inhibitor of intracellular retrograde trafficking, a continuous release of Stx-positive EVs occurred. EVs from HeLa cells possess the Gb3 receptor on their membrane, and EVs from cells that were treated with a glycosylceramide synthase inhibitor, to reduce Gb3, bound significantly less Stx1B. Stx1B was detected both on the membrane and within the shed EVs. Stx1B was incubated with EVs derived from blood cells, in the absence of cells, and was shown to bind to, and be taken up by, these EVs, as demonstrated by electron microscopy. Using a membrane translocation assay we demonstrated that Stx1B was taken up by blood cell- and HeLa-derived EVs, an effect enhanced by chloropromazine or methyl-ß-cyclodextrin, suggesting toxin transfer within the membrane. This is a novel mechanism by which EVs derived from blood cells can sequester their toxic content, possibly to evade the host response.
Asunto(s)
Vesículas Extracelulares/metabolismo , Toxina Shiga I/metabolismo , Eritrocitos/metabolismo , Vesículas Extracelulares/ultraestructura , Femenino , Células HeLa , Humanos , Subunidades de Proteína , Transporte de Proteínas , Receptores de Superficie Celular/metabolismo , Toxina Shiga I/química , Factores de Tiempo , Trihexosilceramidas/metabolismo , Neoplasias del Cuello Uterino/metabolismoRESUMEN
There are two major antigenic forms of Shiga toxin (Stx), Stx1 and Stx2, which bind the same receptor and act on the same target but nonetheless differ in potency. Stx1a is more toxic to cultured cells, but Stx2 subtypes are more potent in animal models. To understand this phenomenon in cultured cells, we used a system that combines flow cytometry with a fluorescent reporter to monitor the Stx-induced inhibition of protein synthesis in single cells. We observed that Vero cells intoxicated with Stx1a behave differently than those intoxicated with Stx2 subtypes: cells challenged with Stx1a exhibited a population-wide loss of protein synthesis, while cells exposed to Stx2a or Stx2c exhibited a dose-dependent bimodal response in which one subpopulation of cells was unaffected (i.e., no loss of protein synthesis). Cells challenged with a hybrid toxin containing the catalytic subunit of Stx1a and the cell-binding subunit of Stx2a also exhibited a bimodal response to intoxication, while cells challenged with a hybrid toxin containing the catalytic subunit of Stx2a and the cell-binding subunit of Stx1a exhibited a population-wide loss of protein synthesis. Other experiments further supported a primary role for the subtype of the B subunit in the outcome of host-Stx interactions. Our collective observations indicate that the bimodal response to Stx2 subtypes is due to relatively weak binding between Stx2 and the host cell that reduces the total functional pool of Stx2 in comparison to that of Stx1a. This explains, in part, the molecular basis for the differential cellular toxicity between Stx1a and Stx2 subtypes.
Asunto(s)
Biosíntesis de Proteínas/fisiología , Toxina Shiga I/metabolismo , Toxina Shiga II/metabolismo , Escherichia coli Shiga-Toxigénica/patogenicidad , Animales , Dominio Catalítico/genética , Línea Celular , Chlorocebus aethiops , Infecciones por Escherichia coli/patología , Citometría de Flujo , Unión Proteica/fisiología , Toxina Shiga I/inmunología , Toxina Shiga II/inmunología , Células VeroRESUMEN
Shiga toxin-producing enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is an important foodborne pathogen. Baicalein (5,6,7-trihydroxylflavone), a flavone isolated from the roots of Scutellaria baicalensis, is considered as a potential antibacterial agent to control foodborne pathogens. Among seven compounds selected by in silico screening of the natural compound database, baicalein inhibited the cytotoxicity of both Shiga toxins 1 and 2 (Stx1 and Stx2) against Vero cells after pretreatment at 0.13 mmol/L. In addition, baicalein reduced the susceptibility of Vero cells to both Stx1 and Stx2. Real-time qPCR showed that baicalein increased transcription of stx1 but not of stx2. However, baicalein had no effects on production or secretion of Stx1 or Stx2. Docking models suggested that baicalein formed a stable structure with StxB pentamer with low intramolecular energy. The results demonstrate that inhibitory activity of baicalein against the cytotoxicity of both Stx1 and Stx2 might be due to of the formation of a binding structure inside the pocket of the Stx1B and Stx2B pentamers.
Asunto(s)
Flavanonas/farmacología , Toxina Shiga I/toxicidad , Toxina Shiga II/toxicidad , Animales , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , Escherichia coli Enterohemorrágica/metabolismo , Simulación del Acoplamiento Molecular , Toxina Shiga I/química , Toxina Shiga I/metabolismo , Toxina Shiga II/química , Toxina Shiga II/metabolismo , Células VeroRESUMEN
Shiga toxin 1 (STx1) and 2 (STx2), produced by Shiga toxin-producing Escherichia coli, cause lethal untreatable disease. The toxins invade cells via retrograde trafficking. Direct early endosome-to-Golgi transport allows the toxins to evade degradative late endosomes. Blocking toxin trafficking, particularly at the early endosome-to-Golgi step, is appealing, but transport mechanisms of the more disease-relevant STx2 are unclear. Using data from a genome-wide siRNA screen, we discovered that disruption of the fusion of late endosomes, but not autophagosomes, with lysosomes blocked the early endosome-to-Golgi transport of STx2. A subsequent screen of clinically approved lysosome-targeting drugs identified tamoxifen (TAM) to be a potent inhibitor of the trafficking and toxicity of STx1 and STx2 in cells. The protective effect was independent of estrogen receptors but dependent on the weak base property of TAM, which allowed TAM to increase endolysosomal pH and alter endosomal dynamics. Importantly, TAM treatment enhanced survival of mice injected with a lethal dose of STx1 or STx2. Thus, it may be possible to repurpose TAM for treating Shiga toxin-producing E. coli infections.
Asunto(s)
Toxina Shiga I/metabolismo , Toxina Shiga II/metabolismo , Tamoxifeno/farmacología , Autofagia , Endosomas/metabolismo , Aparato de Golgi/metabolismo , Células HeLa , Síndrome Hemolítico-Urémico/tratamiento farmacológico , Síndrome Hemolítico-Urémico/metabolismo , Síndrome Hemolítico-Urémico/microbiología , Humanos , Espacio Intracelular/metabolismo , Lisosomas/metabolismo , Transporte de Proteínas/efectos de los fármacos , Transducción de SeñalRESUMEN
OBJECTIVES: Tumour-targeted gene therapy is a promising approach for effective control of gastric cancer cell proliferation. Our study aims to develop a cancer therapy which combines tumour-targeting promoters with cytotoxins. METHODS: The expression of globotriaosylceramide (Gb3), which is a Shiga-like toxin I (Stx1) receptor, was verified in gastric cancer compared with normal stomach tissues as assessed by flow cytometry and immunohistochemical analysis. We therefore constructed the recombinant pFZD7-Stx1 plasmid vectors with tumour-preferential Frizzled-7 promoter and Stx1. pFZD7-Stx1 was used to treat gastric cancer in vitro and in vivo. The gastric cancer cell proliferation and tumour growth were identified after the transfection with the pFZD7-Stx1. RESULTS: Globotriaosylceramide was obviously increased in gastric cancer compared with normal stomach. The gastric cancer cell proliferation and tumour growth decreased significantly after the transfection with the pFZD7-Stx1. CONCLUSION: Frizzled-7 promoter is preferentially active, and Gb3 is abundant in gastric cancer cells. Frizzled-7 promoter and Stx1 may be used to determine a novel and relatively specific and potent gastric cancer therapeutic strategy.
Asunto(s)
Receptores Frizzled/genética , Terapia Genética/métodos , Toxina Shiga I/genética , Toxina Shiga I/uso terapéutico , Neoplasias Gástricas/terapia , Animales , Antineoplásicos/uso terapéutico , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Receptores Frizzled/metabolismo , Humanos , Ratones , Ratones Desnudos , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/uso terapéutico , Toxina Shiga I/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Transfección , Trihexosilceramidas/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
SLC35A2 transports UDP-galactose from the cytosol to the lumen of the Golgi apparatus and endoplasmic reticulum for glycosylation. Mutations in SLC35A2 induce a congenital disorder of glycosylation. Despite the biomedical relevance, mechanisms of transport via SLC35A2 and the impact of disease-associated mutations on activity are unclear. To address these issues, we generated a predicted structure of SLC35A2 and assayed for the effects of a set of structural and disease-associated mutations. Activity assays were performed using a rescue approach in ΔSLC35A2 cells and took advantage of the fact that SLC35A2 is required for expression of the glycosphingolipid globotriaosylceramide (Gb3), the cell surface receptor for Shiga toxin 1 (STx1) and 2 (STx2). The N- and C-terminal cytoplasmic loops of SLC35A2 were dispensable for activity, but two critical glycine (Gly-202 and Gly-214) and lysine (Lys-78 and Lys-297) residues in transmembrane segments were required. Residues corresponding to Gly-202 and Gly-214 in the related transporter SLC35A1 form a substrate-translocating channel, suggesting that a similar mechanism may be involved in SLC35A2. Among the eight disease-associated mutations tested, SLC35A2 function was completely inhibited by two (S213F and G282R) and partially inhibited by three (R55L, G266V, and S304P), providing a straight-forward mechanism of disease. Interestingly, the remaining three (V331I, V258M, and Y267C) did not impact SLC35A2 function, suggesting that complexities beyond loss of transporter activity may underlie disease due to these mutations. Overall, our results provide new insights into the mechanisms of transport of SLC35A2 and improve understanding of the relationship between SLC35A2 mutations and disease.
Asunto(s)
Bioensayo , Proteínas de Transporte de Monosacáridos/metabolismo , Toxina Shiga I/metabolismo , Toxina Shiga II/metabolismo , Bacterias/química , Sitios de Unión , Humanos , Proteínas de Transporte de Monosacáridos/antagonistas & inhibidores , Proteínas de Transporte de Monosacáridos/genética , Mutación , Toxina Shiga I/genética , Toxina Shiga II/genéticaRESUMEN
Shiga toxin-producing Escherichia coli (STEC) serotype O121:H19 is one of the major non-O157:H7 serotypes associated with severe human disease. Here we examined population structure, virulence potential, and metabolic profile of environmental STEC O121 strains recovered from a major produce production region in California and performed comparative analyses with STEC O121 clinical isolates. Multilocus sequence typing revealed that sequence type (ST)-655, a common ST in clinical strains, was the predominant genotype among the environmental strains. Phylotyping placed all STEC O121 strains in B1 group, a lineage containing other major non-O157 serogroups of STEC. Genes encoding different subtypes of Shiga toxin 1 and 2 were detected in O121, including stx1a, stx1d, stx2a, and stx2e. Furthermore, genes encoding intimin (eae) and enterohemolysin (ehxA) were detected in a majority of environmental strains (83.3%), suggesting that the majority of environmental STEC O121 strains are enterohemorrhagic E. coli. The STEC O121 strains with the same genotype were clustered together based on the carbon utilization pattern. Among the 122 carbon substrates that supported the growth of STEC O121 strains, 44 and 35 exhibited lineage (ST) and strain-specific metabolic profiles, respectively. Although clinical ST-655 strains displayed higher metabolic activity than environmental ST-655 strains for several carbon substrates, including l-alaninamide, 5-keto-d-gluconic acid, 3-O-ß-d-galactopyranosyl-d-arabinose, α-ketoglutaric acid, and lactulose, a few environmental strains with the enhanced metabolic potential for the above substrates were detected. Variations in curli biogenesis and swimming motility were also observed in ST-655 strains, suggesting that phenotypic variants are widespread in STEC. Considering the ecological niches that STEC colonizes, increased metabolic potential for plant-derived carbohydrates, mucus-derived substrates, or secondary metabolites produced by the indigenous microorganisms might have been selected. Such traits would confer STEC competitive advantages and facilitate survival and adaptation of STEC population to a given niche, including infected humans.
Asunto(s)
Microbiología de Alimentos , Escherichia coli Shiga-Toxigénica/aislamiento & purificación , Verduras/microbiología , Animales , California , Humanos , Filogenia , Toxina Shiga I/genética , Toxina Shiga I/metabolismo , Toxina Shiga II/genética , Toxina Shiga II/metabolismo , Escherichia coli Shiga-Toxigénica/metabolismo , Escherichia coli Shiga-Toxigénica/patogenicidadRESUMEN
After we published our preliminary study on the use of liquid chromatography-tandem mass spectrometry (LC-MS/MS) and curated E. coli toxin databases on the identification of E. coli Shiga toxins (Stxs) in the Journal of Proteomics in year 2018, we were encouraged to further refine the method and test clinical isolates. In this study, different concentrations of mitomycin C (MMC) and ciprofloxacin (CF), two common antibiotic/chemotherapy agents capable of stimulating Stx production, were first tested and compared on three reference strains and eight clinical isolates to observe the toxin induction and subsequent identification. Notably, no differences were observed between the two agents other than the concentrations applied. Seventeen more clinical isolates were then tested using fixed MMC and CF concentrations and sample amount. This study confirms that the majority of stx2-positive E. coli strains can be stimulated to produce sufficient toxin for confident identification. This does not occur with stx1-positive E. coli isolates, however, despite the fact that both Stxs can be identified for several isolates without MMC or CF stimulation. BIOLOGICAL SIGNIFICANCE: Stxs, especially Stx2, are very important causes of severe food-borne disease, even death. This study confirms that receptor analogue-based affinity enrichment of Stxs, after MMC or CF treatment of E. coli, is useful for fast and accurate Stx2 identification through LC-MS/MS.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Proteómica , Toxina Shiga I , Toxina Shiga II , Escherichia coli Shiga-Toxigénica/metabolismo , Cromatografía Liquida , Humanos , Toxina Shiga I/análisis , Toxina Shiga I/metabolismo , Toxina Shiga II/análisis , Toxina Shiga II/metabolismo , Espectrometría de Masas en TándemRESUMEN
Shiga toxin (Stx)-producing Escherichia coli (STEC) causes foodborne outbreaks of bloody diarrhea. There are two major types of immunologically distinct Stxs: Stx1a and Stx2a. Stx1a is more cytotoxic to Vero cells than Stx2a, but Stx2a has a lower 50% lethal dose (LD50) in mice. Epidemiological data suggest that infections by STEC strains that produce only Stx2a progress more often to a life-threatening sequela of infection called hemolytic-uremic syndrome (HUS) than isolates that make Stx1a only or produce both Stx1a and Stx2a. In this study, we found that an E. coli O26:H11 strain that produces both Stx1a and Stx2a was virulent in streptomycin- and ciprofloxacin-treated mice and that mice were protected by administration of an anti-Stx2 antibody. However, we discovered that in the absence of ciprofloxacin, neutralization of Stx1a enhanced the virulence of the strain, a result that corroborated our previous finding that Stx1a reduces the toxicity of Stx2a by the oral route. We further found that intraperitoneal administration of the purified Stx1a B subunit delayed the mean time to death of mice intoxicated with Stx2a and reduced the cytotoxic effect of Stx2a on Vero cells. Taken together, our data suggest that Stx1a reduces both the pathogenicity of Stx2 in vivo and cytotoxicity in vitro.
Asunto(s)
Infecciones por Escherichia coli/microbiología , Toxina Shiga I/toxicidad , Toxina Shiga II/toxicidad , Escherichia coli Shiga-Toxigénica/metabolismo , Animales , Chlorocebus aethiops , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Toxina Shiga I/metabolismo , Toxina Shiga II/metabolismo , Escherichia coli Shiga-Toxigénica/genética , Escherichia coli Shiga-Toxigénica/patogenicidad , Células Vero , VirulenciaRESUMEN
KEY MESSAGE: A key module, secretory component (SC), was efficiently expressed in Arabidopsis thaliana. The plant-based SC and immunoglobulin A of animal or plant origin formed secretory IgA that maintains antigen-binding activity. Plant expression systems are suitable for scalable and cost-effective production of biologics. Secretory immunoglobulin A (SIgA) will be useful as a therapeutic antibody against mucosal pathogens. SIgA is equipped with a secretory component (SC), which assists the performance of SIgA on the mucosal surface. Here we produced SC using a plant expression system and formed SIgA with dimeric IgAs produced by mouse cells as well as by whole plants. To increase the expression level, an endoplasmic reticulum retention signal peptide, KDEL (Lys-Asp-Glu-Leu), was added to mouse SC (SC-KDEL). The SC-KDEL cDNA was inserted into a binary vector with a translational enhancer and an efficient terminator. The SC-KDEL transgenic Arabidopsis thaliana produced SC-KDEL at the level of 2.7% of total leaf proteins. In vitro reaction of the plant-derived SC-KDEL with mouse dimeric monoclonal IgAs resulted in the formation of SIgA. When reacted with Shiga toxin 1 (Stx1)-specific ones, the antigen-binding activity was maintained. When an A. thaliana plant expressing SC-KDEL was crossed with one expressing dimeric IgA specific for Stx1, the plant-based SIgA exhibited antigen-binding activity. Leaf extracts of the crossbred transgenic plants neutralized Stx1 cytotoxicity against Stx1-sensitive cells. These results suggest that transgenic plants expressing SC-KDEL will provide a versatile means of SIgA production.
Asunto(s)
Arabidopsis/metabolismo , Inmunoglobulina A Secretora/metabolismo , Multimerización de Proteína , Componente Secretorio/metabolismo , Toxina Shiga I/metabolismo , Animales , Arabidopsis/genética , Cruzamientos Genéticos , ADN Bacteriano/genética , Homocigoto , Ratones , Oligopéptidos , Plantas Modificadas Genéticamente , Señales de Clasificación de ProteínaRESUMEN
Shiga toxin (Stx)-mediated injury of the kidneys and the brain represent the major extraintestinal complications in humans upon infection by enterohemorrhagic Escherichia coli (EHEC). Damage of renal and cerebral endothelial cells is the key event in the pathogenesis of the life-threatening hemolytic uremic syndrome (HUS). Stxs are AB5 toxins and the B-pentamers of the two clinically important Stx subtypes Stx1a and Stx2a preferentially bind to the glycosphingolipid globotriaosylceramide (Gb3Cer, Galα4Galß4Glcß1Cer) and to less extent to globotetraosylceramide (Gb4Cer, GalNAcß3Galα4Galß4Glcß1), which are expected to reside in lipid rafts in the plasma membrane of the human endothelium. This review summarizes the current knowledge on the Stx glycosphingolipid receptors and their lipid membrane ensemble in primary human brain microvascular endothelial cells (pHBMECs) and primary human renal glomerular endothelial cells (pHRGECs). Increasing knowledge on the precise initial molecular mechanisms by which Stxs interact with cellular targets will help to develop specific therapeutics and/or preventive measures to combat EHEC-caused diseases.
Asunto(s)
Escherichia coli Enterohemorrágica/fisiología , Infecciones por Escherichia coli/metabolismo , Globósidos/metabolismo , Toxina Shiga I/metabolismo , Toxina Shiga II/metabolismo , Trihexosilceramidas/metabolismo , Encéfalo/citología , Células Endoteliales/citología , Escherichia coli Enterohemorrágica/patogenicidad , Infecciones por Escherichia coli/microbiología , Globósidos/química , Síndrome Hemolítico-Urémico/metabolismo , Síndrome Hemolítico-Urémico/microbiología , Interacciones Huésped-Patógeno/fisiología , Humanos , Riñón/citología , Cultivo Primario de Células , Toxina Shiga I/química , Toxina Shiga II/química , Trihexosilceramidas/químicaRESUMEN
The life-threatening sequela of hemorrhagic colitis induced by Shiga toxins (Stx)-producing Escherichia coli (STEC) infections in humans is hemolytic uremic syndrome (HUS), the main cause of acute renal failure in early childhood. The key step in the pathogenesis of HUS is the appearance of Stx in the blood of infected patients because these powerful virulence factors are capable of inducing severe microangiopathic lesions in the kidney. During precocious toxemia, which occurs in patients before the onset of HUS during the intestinal phase, Stx bind to several different circulating cells. An early response of these cells might include the release of proinflammatory mediators associated with the development of HUS. Here, we show that primary human monocytes stimulated with Shiga toxin 1a (Stx1a) through the glycolipid receptor globotriaosylceramide released larger amounts of proinflammatory molecules (IL-1ß, TNFα, IL-6, G-CSF, CXCL8, CCL2, CCL4) than Stx1a-treated neutrophils. The mediators (except IL-1ß) are among the top six proinflammatory mediators found in the sera from patients with HUS in different studies. The molecules appear to be involved in different pathogenetic steps of HUS, i.e. sensitization of renal endothelial cells to the toxin actions (IL-1ß, TNFα), activation of circulating monocytes and neutrophils (CXCL8, CCL2, CCL4) and increase in neutrophil counts in patients with poor prognosis (G-CSF). Hence, a role of circulating monocytes in the very early phases of the pathogenetic process culminating with HUS can be envisaged. Impairment of the events of precocious toxemia would prevent or reduce the risk of HUS in STEC-infected children.