RESUMEN
AB5 toxins of pathogenic bacteria enter host cells and utilize the retrograde trafficking pathway to translocate to the cytoplasm and exert its pathogenesis. Cholera toxin and Shiga toxin reach the endoplasmic reticulum (ER), and the A subunit undergoes redox regulation by ER proteins to become active fragments, which pass through the ER membrane and translocate to the cytoplasm. By acting on molecular targets in the cytoplasm, the normal function of host cells are disrupted, causing diseases. ER chaperone proteins such as protein disulfide isomerase (PDI) and binding immunoglobulin protein (BiP) induce conformational changes triggered by the reduction of disulfide bonds in the A subunit. This is thought to be dependent on cysteine thiol-mediated redox regulation, but the detailed mechanism remains unclear. On the other hand, subtilase cytotoxin (SubAB), produced by enterohemorrhagic Escherichia coli (EHEC), localizes to the ER without translocating to the cytoplasm and cleaves BiP as a substrate. Therefore, it is thought that ER stress-based cytotoxicity and intestinal bleeding occur without translocating to the cytoplasm. We reported that PDI is involved in BiP cleavage through SubAB localization to the ER. Like other AB5 toxins, this indicates the involvement of redox regulation via chaperone proteins in the ER, but also suggests that SubAB does not translocate to the cytoplasm because it cleaves BiP. Although there are few reports on the redox state of ER protein thiols, it is suggested that polysulfidation, which is discussed in this symposium, may be involved.
Asunto(s)
Escherichia coli Enterohemorrágica , Proteínas de Escherichia coli , Proteínas de Escherichia coli/toxicidad , Proteínas de Escherichia coli/metabolismo , Escherichia coli Enterohemorrágica/metabolismo , Chaperonas Moleculares , Retículo Endoplásmico/metabolismo , Enterotoxinas , Proteínas Portadoras/metabolismo , Proteína Disulfuro Isomerasas/metabolismo , Oxidación-Reducción , BiologíaRESUMEN
Subtilase cytotoxin (SubAB) is a major virulence factor produced by eae-negative Shiga-toxigenic Escherichia coli (STEC) that can cause fatal systemic complications. SubAB binds to target cells through multivalent interactions between its B-subunit pentamer and receptor molecules such as glycoproteins with a terminal N-glycolylneuraminic acid (Neu5Gc). We screened randomized multivalent peptide libraries synthesized on a cellulose membrane and identified a series of tetravalent peptides that efficiently bind to the receptor-binding region of the SubAB B-subunit pentamer. These peptides competitively inhibited the binding of the B-subunit to a receptor-mimic molecule containing clustered Neu5Gc (Neu5Gc-polymer). We selected the peptide with the highest inhibitory efficacy, FFP-tet, and covalently bound it to beads to synthesize FFP-tet-beads, a highly clustered SubAB absorber that displayed potency to absorb SubAB cytotoxicity through direct binding to the toxin. The efficacy of FFP-tet-beads to absorb SubAB cytotoxicity in solution was similar to that of Neu5Gc-polymer, suggesting that FFP-tet-beads might be an effective therapeutic agent against complications arising from eae-negative STEC infection.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli Shiga-Toxigénica , Proteínas Portadoras/metabolismo , Celulosa/metabolismo , Citotoxinas , Proteínas de Escherichia coli/metabolismo , Biblioteca de Péptidos , Polímeros/metabolismo , Escherichia coli Shiga-Toxigénica/genética , Escherichia coli Shiga-Toxigénica/metabolismo , Subtilisinas/toxicidad , Factores de Virulencia/metabolismoRESUMEN
AB5 protein toxins are produced by certain bacterial pathogens and are composed of an enzymatically active A-subunit and a B-subunit pentamer, the latter being responsible for cell receptor recognition, cellular uptake, and transport of the A-subunit into the cytosol of eukaryotic target cells. Two members of the AB5 toxin family were described in Shiga toxin-producing Escherichia coli (STEC), namely Shiga toxin (Stx) and subtilase cytotoxin (SubAB). The functional paradigm of AB toxins includes the B-subunit being mandatory for the uptake of the toxin into its target cells. Recent studies have shown that this paradigm cannot be maintained for SubAB, since SubA alone was demonstrated to intoxicate human epithelial cells in vitro. In the current study, we raised the hypothesis that this may also be true for the A-subunit of the most clinically relevant Stx-variant, Stx2a. After separate expression and purification, the recombinant Stx2a subunits StxA2a-His and StxB2a-His were applied either alone or in combination in a 1:5 molar ratio to Vero B4, HeLa, and HCT-116 cells. For all cell lines, a cytotoxic effect of StxA2a-His alone was detected. Competition experiments with Stx and SubAB subunits in combination revealed that the intoxication of StxA2a-His was reduced by addition of SubB1-His. This study showed that the enzymatic subunit StxA2a alone was active on different cells and might therefore play a yet unknown role in STEC disease development.
Asunto(s)
Toxina Shiga/toxicidad , Animales , Chlorocebus aethiops , Células Epiteliales/efectos de los fármacos , Células HCT116/efectos de los fármacos , Células HeLa/efectos de los fármacos , Humanos , Proteínas Recombinantes , Toxina Shiga/química , Toxina Shiga/aislamiento & purificación , Toxina Shiga II , Células Vero/efectos de los fármacosRESUMEN
Shiga toxin-producing E. coli (STEC) and enteropathogenic E. coli (EPEC) strains are food-borne pathogens associated with acute diarrhea. Haemolytic-uremic syndrome (HUS) is often a complication of STEC infection. In order to examine the occurrence, serotypes, virulence and antimicrobial-resistance profiles of STEC and EPEC in wildlife, 326 faecal E. coli strains from 304 clinically healthy animals were analyzed. For this approach stx1, stx2 and eae genes, as well as accessory virulence determinants (ehx, hlyA, saa, tia, bfp, subAB) were PCR-screened and sequenced. Serotyping was performed employing all available O (O1-O185) and H (H1-H56) antisera. Genetic diversity was analyzed by XbaI-PFGE and phylotyping. Thirteen STEC (4.3%) and 10 EPEC (3.3%) were identified among 12 deer, 3 mouflon, 6 wild boars and 2 birds. Nine STEC showed seropathotypes B (O145:[H28]) and C (O22:H8, O128:[H2]) associated with HUS, and D (O110:H28, O146:H21, O146:[H28], ONT:H8) associated with human diarrhea. Although most isolates harbored stx2b and stx1c variants, stx2a and stx1a (related with severe disease) were also detected. Additionally, the eae gene was present in one stx2a-positive O145:[H28] STEC from a deer and 11 STEC harbored subAB genes (mainly the subAB2 variant). EPEC isolates showed 7 different intimin variants (ß1, ß2, γ1, ε1, ζ1, ι1-A, κ). Interestingly, the O49:[H10] eae-κ EPEC isolated from a wild boar was bfpA-positive showing a combination of serotype/virulence profile previously detected among human clinical tEPEC. Based on present results, wild ruminants, wild boars and to a lesser extent birds would be carriers of potentially pathogenic STEC and EPEC strains.
Asunto(s)
Escherichia coli Enteropatógena/aislamiento & purificación , Infecciones por Escherichia coli/veterinaria , Escherichia coli Shiga-Toxigénica/aislamiento & purificación , Sus scrofa/microbiología , Adhesinas Bacterianas , Animales , Animales Salvajes , Biomarcadores , Escherichia coli Enteropatógena/genética , Escherichia coli Enteropatógena/patogenicidad , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli , Regulación Bacteriana de la Expresión Génica/fisiología , Serogrupo , Toxinas Shiga , Escherichia coli Shiga-Toxigénica/genética , Escherichia coli Shiga-Toxigénica/patogenicidad , España , VirulenciaRESUMEN
Subtilase cytotoxin (SubAB) is a member of the AB5 cytotoxin family and is produced by certain strains of Shiga toxigenic Escherichia coli. The toxin is known to be lethal to mice, but the pathological mechanisms that contribute to Uremic Hemolytic Syndrome (HUS) are poorly understood. In this study we show that intraperitoneal injection of a sublethal dose of SubAB in rats triggers a systemic response, with ascitic fluid accumulation, heart hypertrophy and damage to the liver, colon and kidney. SubAB treated rats presented microalbuminuria 20 days post inoculation. At this time we found disruption of the glomerular filtration barrier and alteration of the protein reabsorption mechanisms of the proximal tubule. In the kidney, SubAB also triggered an epithelial to mesenchymal transition (Wuyts et al., 1996). These findings indicate that apart from direct cytotoxic effects on renal tissues, SubAB causes significant damage to the other organs, with potential consequences for HUS pathogenesis. IMPORTANCE: Uremic Hemolytic Syndrome is an endemic disease in Argentina, with over 400 hundred new cases each year. We have previously described renal effects of Shiga Toxin and its ability to alter renal protein handling. Bearing in mind that Subtilase Cytotoxin is an emerging pathogenic factor, that it is not routinely searched for in patients with HUS, and that to the date its systemic effects have not been fully clarified we decided to study both its systemic effects, and its renal effects to assess whether SubAB could be contributing to pathology seen in children.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli Shiga-Toxigénica/metabolismo , Subtilisinas/metabolismo , Albuminuria/inducido químicamente , Animales , Ascitis/inducido químicamente , Cardiomegalia/inducido químicamente , Colon/efectos de los fármacos , Colon/patología , Transición Epitelial-Mesenquimal/efectos de los fármacos , Proteínas de Escherichia coli/toxicidad , Síndrome Hemolítico-Urémico/etiología , Riñón/efectos de los fármacos , Riñón/metabolismo , Riñón/patología , Hígado/efectos de los fármacos , Hígado/patología , Masculino , Ratas Sprague-Dawley , Subtilisinas/toxicidadRESUMEN
The conserved oligomeric Golgi (COG) complex is a key evolutionally conserved multisubunit protein machinery that regulates tethering and fusion of intra-Golgi transport vesicles. The Golgi apparatus specifically promotes sorting and complex glycosylation of glycoconjugates. Without proper glycosylation and processing, proteins and lipids will be mislocalized and/or have impaired function. The Golgi glycosylation machinery is kept in homeostasis by a careful balance of anterograde and retrograde trafficking to ensure proper localization of the glycosylation enzymes and their substrates. This balance, like other steps of membrane trafficking, is maintained by vesicle trafficking machinery that includes COPI vesicular coat proteins, SNAREs, Rabs, and both coiled-coil and multi-subunit vesicular tethers. The COG complex interacts with other membrane trafficking components and is essential for proper localization of Golgi glycosylation machinery. Here we describe using CRISPR-mediated gene editing coupled with a phenotype-based selection strategy directly linked to the COG complex's role in glycosylation homeostasis to obtain COG complex subunit knockouts (KOs). This has resulted in clonal KOs for each COG subunit in HEK293T cells and gives the ability to further probe the role of the COG complex in Golgi homeostasis.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Proteína Coat de Complejo I , Edición Génica/métodos , Técnicas de Inactivación de Genes/métodos , Aparato de Golgi , Proteínas SNARE , Proteína Coat de Complejo I/genética , Proteína Coat de Complejo I/metabolismo , Glicosilación , Aparato de Golgi/genética , Aparato de Golgi/metabolismo , Células HEK293 , Humanos , Proteínas SNARE/genética , Proteínas SNARE/metabolismoRESUMEN
Subtilase cytotoxin (SubAB) is an important virulence factor of eae-negative Shiga toxin-producing Escherichia coli (STEC). Three variants of SubAB-encoding genes have been reported in the literature; however, the newly described subAB variant (subAB2-2) was found only in STEC strains from deer meat, sheep, and some wild animals. In this study, subAB variants were detected by PCR and DNA sequencing in 5 out of 12 (41.6%) eae-negative STEC strains isolated from patients. Most subAB-positive STEC strains (80%) harbored the subAB1 gene. The subAB2-2 gene was detected for the first time in the clinical STEC O128:H2 strain. Other virulence genes including stx1a, stx1c, stx2b, ehxA, and tia were also detected in this strain. The DNA sequence analyses of the subAB1 and subAB2-2 genes of the clinical STEC strains showed 99% and 100% identity to those of the reference strains 98NK2 and LM27558stx2, respectively. This is the first report on the detection of the subAB2-2 gene in a clinical STEC isolate.
Asunto(s)
Citotoxinas/genética , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/veterinaria , Proteínas de Escherichia coli/genética , Carne/microbiología , Escherichia coli Shiga-Toxigénica/enzimología , Subtilisinas/genética , Animales , Citotoxinas/metabolismo , Ciervos , Proteínas de Escherichia coli/metabolismo , Humanos , Reacción en Cadena de la Polimerasa , Ovinos , Escherichia coli Shiga-Toxigénica/clasificación , Escherichia coli Shiga-Toxigénica/genética , Escherichia coli Shiga-Toxigénica/aislamiento & purificación , Subtilisinas/metabolismo , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
Subtilase cytotoxin (SubAB) is an AB5 toxin produced by Shiga toxin (Stx)-producing Escherichia coli (STEC) strains usually lacking the eae gene product intimin. Three allelic variants of SubAB encoding genes have been described: subAB1, located on a plasmid, subAB2-1, located on the pathogenicity island SE-PAI and subAB2-2 located in an outer membrane efflux protein (OEP) region. SubAB is becoming increasingly recognized as a toxin potentially involved in human pathogenesis. Ruminants and cattle have been identified as reservoirs of subAB-positive STEC. The presence of the three subAB allelic variants was investigated by PCR for 152 STEC strains originating from chamois, ibex, red deer, roe deer, cattle, sheep and pigs. Overall, subAB genes were detected in 45.5% of the strains. Prevalence was highest for STEC originating from ibex (100%), chamois (92%) and sheep (65%). None of the STEC of bovine or of porcine origin tested positive for subAB. None of the strains tested positive for subAB1. The allelic variant subAB2-2 was detected the most commonly, with 51.4% possessing subAb2-1 together with subAB2-2. STEC of ovine origin, serotypes O91:H- and O128:H2, the saa gene, which encodes for the autoagglutinating adhesin and stx2b were significantly associated with subAB-positive STEC. Our results suggest that subAB2-1 and subAB2-2 is widespread among STEC from wild ruminants and sheep and may be important as virulence markers in STEC pathogenic to humans.
Asunto(s)
Infecciones por Escherichia coli/veterinaria , Proteínas de Escherichia coli/genética , Variación Genética , Escherichia coli Shiga-Toxigénica/genética , Subtilisinas/genética , Alelos , Animales , ADN Bacteriano/química , ADN Bacteriano/genética , Infecciones por Escherichia coli/microbiología , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Rumiantes , Análisis de Secuencia de ADN , Escherichia coli Shiga-Toxigénica/aislamiento & purificación , PorcinosRESUMEN
Subtilase cytotoxin (SubAB) is an AB5 toxin produced by Shiga toxin (Stx)-producing Escherichia coli (STEC) strains usually lacking the eae gene product intimin. Two allelic variants of SubAB encoding genes have been described: subAB1, located on a plasmid, and subAB2, located on a pathogenicity island (PAI) together with tia gene. While subAB1 has been reported to be more frequent among bovine strains, subAB2 has been mainly associated with strains from small ruminants. We investigated the presence of the two variants of subAB among 59 eae-negative STEC from large game animals (deer and wild boar) and their meat and meat products in order to assess the role of other species in the epidemiology of subAB-positive, eae-negative STEC. For this approach, the strains were PCR-screened for the presence of subAB, including the specific detection of both allelic variants, for the presence of saa, tia and sab, and for stx subtyping. Overall, subAB genes were detected in 71.2% of the strains: 84.1% of the strains from deer and 33.3% of the strains from wild boar. Most of them (97.6%) possessed subAB2 and most of these subAB2-positive strains (92.7%) were also positive for tia and negative for saa, suggesting the presence of the subAB2-harbouring PAI. Subtype stx2b was present in most of the strains (67.8%) and a statistically significant association could be established between subAB2 and stx2b. Our results suggest that large game animals, mainly deer, may represent an important animal reservoir of subAB2-positive, eae-negative STEC, and also highlight the risk of human infection posed by the consumption of large game meat and meat products.