Shiga toxins (Stxs) are a family of cytotoxic proteins that lead to the development of bloody diarrhea, hemolytic-uremic syndrome, and central nervous system complications caused by bacteria such as ...S. dysenteriae, E. coli O157:H7 and E. coli O104:H4. Increasing evidence indicates that macroautophagy (autophagy) is a key factor in the cell death induced by Stxs. However, the associated mechanisms are not yet clear. This study showed that Stx2 induces autophagic cell death in Caco-2 cells, a cultured line model of human enterocytes. Inhibition of autophagy using pharmacological inhibitors, such as 3-methyladenine and bafilomycin A
1
, or silencing of the autophagy genes ATG12 or BECN1 decreased the Stx2-induced death in Caco-2 cells. Furthermore, there were numerous instances of dilated endoplasmic reticulum (ER) in the Stx2-treated Caco-2 cells, and repression of ER stress due to the depletion of viable candidates of DDIT3 and NUPR1. These processes led to Stx2-induced autophagy and cell death. Finally, the data showed that the pseudokinase TRIB3-mediated DDIT3 expression and AKT1 dephosphorylation upon ER stress were triggered by Stx2. Thus, the data indicate that Stx2 causes autophagic cell death via the ER stress pathway in intestinal epithelial cells.
Shiga toxin (Stxs)-producing enterohaemorrhagic Escherichia coli (EHEC) and Shigella dysenteriae serotype 1 are major causative agents of severe bloody diarrhea (known as hemorrhagic colitis) and ...hemolytic uremic syndrome (HUS) associated with extraintestinal complications such as acute renal failure and neurologic impairment in infected patients under 9 years of age. Extreme nephrotoxicity of Stxs in HUS patients is associated with severe outcomes, highlighting the need to develop technologies to detect low levels of the toxin in environmental or food samples. Currently, the conventional polymerase chain reaction (PCR) or immunoassay is the most broadly used assay to detect the toxin. However, these assays are laborious, time-consuming, and costly. More recently, numerous studies have described novel, highly sensitive, and portable methods for detecting Stxs from EHEC. To contextualize newly emerging Stxs detection methods, we briefly explain the basic principles of these methods, including lateral flow assays, optical detection, and electrical detection. We subsequently describe existing and newly emerging rapid detection technologies to identify and measure Stxs.
Escherichia coli O157:H7 and Salmonella enterica are foodborne pathogens with major public health concern in the U.S. These pathogens utilize several virulence factors to initiate infections in ...humans. The antimicrobial effect of seven glucosinolate hydrolysis compounds against Salmonella and E. coli O157:H7 was investigated by the disc diffusion assay. Among the tested compounds, benzyl isothiocyanate (BIT), which exerted the highest antimicrobial activity, was evaluated for its anti-virulence properties against these pathogens. The effect of BIT on motility of Salmonella and E. coli O157:H7 and Shiga toxin production by E. coli O157:H7 was determined by the motility assay and ELISA procedure, respectively. Confocal and transmission electron microscopy (TEM) procedures were used to determine bacterial damage at the cellular level. Results revealed that sub-inhibitory concentrations (SICs) of BIT significantly inhibited the motility of both bacteria (P < 0.05). Shiga toxin production by E. coli O157:H7 was decreased by ~32% in the presence of BIT at SICs. TEM results showed the disruption of outer membrane, release of cytoplasmic contents, and cell lysis following BIT treatment. Results suggest that BIT could be potentially used to attenuate Salmonella and E. coli O157:H7 infections by reducing the virulence factors including bacterial motility and Shiga toxin production.
•BIT is the most effective compound against E. coli O157:H7 and Salmonella.•BIT kills Salmonella by disrupting bacterial cell membrane.•BIT reduces the motility of E. coli O157:H7 and Salmonella.•BIT decreases shiga toxin production by E. coli O157:H7.•BIT could be a potential anti-virulence compound against bacterial infection.
The Shiga toxigenic Escherichia coli (STEC) O113:H21 strain 98NK2, which was responsible for an outbreak of hemolytic uremic syndrome, secretes a highly potent and lethal subtilase cytotoxin that is ...unrelated to any bacterial toxin described to date. It is the prototype of a new family of AB(5) toxins, comprising a single 35-kilodalton (kD) A subunit and a pentamer of 13-kD B subunits. The A subunit is a subtilase-like serine protease distantly related to the BA_2875 gene product of Bacillus anthracis. The B subunit is related to a putative exported protein from Yersinia pestis, and binds to a mimic of the ganglioside GM2. Subtilase cytotoxin is encoded by two closely linked, cotranscribed genes (subA and subB), which, in strain 98NK2, are located on a large, conjugative virulence plasmid. Homologues of the genes are present in 32 out of 68 other STEC strains tested. Intraperitoneal injection of purified subtilase cytotoxin was fatal for mice and resulted in extensive microvascular thrombosis, as well as necrosis in the brain, kidneys, and liver. Oral challenge of mice with E. coli K-12-expressing cloned subA and subB resulted in dramatic weight loss. These findings suggest that the toxin may contribute to the pathogenesis of human disease.
The glycosphingolipid Gb
is a specific receptor of the bacterial Shiga toxin (STx). Binding of STx to Gb
is a prerequisite for its internalization into the host cells, and the ceramide's fatty acid ...of Gb
has been shown to influence STx binding. In
studies on liquid ordered (
)/liquid disordered (
) coexisting artificial membranes, Shiga toxin B (STxB) binds solely to
domains, thus harboring Gb
concomitant with an observed lipid redistribution process. These findings raise the question of how the molecular structure of the fatty acid of Gb
influences the interaction of Gb
with the different lipids preferentially either found in the
phase, namely, sphingomyelin and cholesterol, or in the
phase. We addressed this question by using a series of synthetically available and unlabeled Gb
glycosphingolipids carrying different long chain C
fatty acids (saturated, monounsaturated, and α-hydroxylated). In conjunction with surface tension experiments on Langmuir monolayers, we quantified the excess of free energy of mixing of the different Gb
species in monolayers composed of either sphingomyelin or cholesterol or composed of a fluid phase lipid (DOPC). From a calculation of the total free energy of mixing, we conclude that mixing of the saturated Gb
species with the
lipid DOPC is energetically less favorable than all other combinations, while the unsaturated species mix equally well with the
phase lipids sphingomyelin and cholesterol and the
phase lipid DOPC. Furthermore, we found that STxB partially penetrates in mixed lipid monolayers (DOPC/sphingomyelin/cholesterol) containing the Gb
sphingolipid with a saturated or a monounsaturated C
fatty acid. The maximum insertion pressure, as a measure for protein insertion, is >30 mN/m for both Gb
molecules and is not significantly different for the two Gb
species.
Shiga toxins (Stxs) produced by Shiga toxin-producing Escherichia coli (STEC) strains are major virulence factors that cause fatal systemic complications, such as hemolytic uremic syndrome and ...disruption of the central nervous system. Although numerous studies report proinflammatory responses to Stx type 1 (Stx1) or Stx type 2 (Stx2) both in vivo and in vitro, none have examined dynamic immune regulation involving cytokines and/or unknown inflammatory mediators during intoxication. Here, we showed that enzymatically active Stxs trigger the dissociation of lysyl-tRNA synthetase (KRS) from the multi-aminoacyl-tRNA synthetase complex in human macrophage-like differentiated THP-1 cells and its subsequent secretion. The secreted KRS acted to increase the production of proinflammatory cytokines and chemokines. Thus, KRS may be one of the key factors that mediate transduction of inflammatory signals in the STEC-infected host.
Detailed laboratory characterization of Escherichia coli O157 is essential to inform epidemiological investigations. This study assessed the utility of whole-genome sequencing (WGS) for outbreak ...detection and epidemiological surveillance of E. coli O157, and the data were used to identify discernible associations between genotypes and clinical outcomes. One hundred five E. coli O157 strains isolated over a 5-year period from human fecal samples in Lothian, Scotland, were sequenced with the Ion Torrent Personal Genome Machine. A total of 8,721 variable sites in the core genome were identified among the 105 isolates; 47% of the single nucleotide polymorphisms (SNPs) were attributable to six "atypical" E. coli O157 strains and included recombinant regions. Phylogenetic analyses showed that WGS correlated well with the epidemiological data. Epidemiological links existed between cases whose isolates differed by three or fewer SNPs. WGS also correlated well with multilocus variable-number tandem repeat analysis (MLVA) typing data, with only three discordant results observed, all among isolates from cases not known to be epidemiologically related. WGS produced a better-supported, higher-resolution phylogeny than MLVA, confirming that the method is more suitable for epidemiological surveillance of E. coli O157. A combination of in silico analyses (VirulenceFinder, ResFinder, and local BLAST searches) were used to determine stx subtypes, multilocus sequence types (15 loci), and the presence of virulence and acquired antimicrobial resistance genes. There was a high level of correlation between the WGS data and our routine typing methods, although some discordant results were observed, mostly related to the limitation of short sequence read assembly. The data were used to identify sublineages and clades of E. coli O157, and when they were correlated with the clinical outcome data, they showed that one clade, Ic3, was significantly associated with severe disease. Together, the results show that WGS data can provide higher resolution of the relationships between E. coli O157 isolates than that provided by MLVA. The method has the potential to streamline the laboratory workflow and provide detailed information for the clinical management of patients and public health interventions.
More than 470 serotypes of Shiga toxin-producing Escherichia coli (STEC) have been identified, but not all cause severe illness in humans. Most STEC that cause severe diseases can adhere to ...epithelial cells, produce specific stx subtypes, and belong to certain serotypes; therefore, these traits appear to be critical STEC risk factors. However, testing for these traits is labor intensive, and serotyping is inadequate because of extensive variations among E. coli O and H antigen types. In the present study, the E. coli identification microarray, which tests for over 40,000 E. coli gene targets, was examined for its potential to quickly characterize STEC strains. Analysis of 47 E. coli isolates, including 31 STEC isolates, recovered from 39 foods revealed that the microarray effectively determined the presence or absence of adherence genes and identified the specific eae allele in 3 isolates. The array identified most of the stx subtypes carried by all the isolates but had some difficulties in discerning between stx
, stx
, and stx
because of the genetic similarities of these subtypes. The array determined the O and H types of 68 and 96% of the isolates, respectively, and although most serotypes were unremarkable, a few known pathogenic serotypes were also found. These selected STEC traits provided a scientific basis for assessing the potential health risks of STEC strains and also showed the importance of H typing in determining health risks. However, the diversity of the STEC group, the complexity of virulence mechanisms, and the variation in pathotypes among strains continue to pose challenges to assessing the potential of STEC strains to cause severe illness.
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