Mass spectrometry-based Shiga toxin identification: A clinical validation.

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.

Shiga Toxin/Lipopolysaccharide Activates Caspase-4 and Gasdermin D to Trigger Mitochondrial Reactive Oxygen Species Upstream of the NLRP3 Inflammasome.

The non-canonical caspase-4 and canonical NLRP3 inflammasomes are both activated by intracellular lipopolysaccharide (LPS), but the crosstalk between these two pathways remains unclear. Shiga toxin 2 (Stx2)/LPS complex, from pathogenic enterohemorrhagic Escherichia coli, activates caspase-4, gasdermin D (GSDMD), and the NLRP3 inflammasome in human THP-1 macrophages, but not mouse macrophages that lack the Stx receptor CD77. Stx2/LPS-mediated IL-1β secretion and pyroptosis are dependent on mitochondrial reactive oxygen species (ROS) downstream of the non-canonical caspase-4 inflammasome and cleaved GSDMD, which is enriched at the mitochondria.

Mass spectrometry-based Shiga toxin identification: An optimized approach.

Unlabelled: Toxin expression is a key factor in Shiga toxin (Stx)-producing E. coli, a common pathogen involved in foodborne disease outbreaks. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) based approach has been used in this study to identify commonly reported E.

BfpI, BfpJ, and BfpK Minor Pilins Are Important for the Function and Biogenesis of Bundle-Forming Pili Expressed by Enteropathogenic Escherichia coli.

Unlabelled: Enteropathogenic Escherichia coli (EPEC) remains a significant cause of infant diarrheal illness and associated morbidity and mortality in developing countries. EPEC strains are characterized by their ability to colonize the small intestines of their hosts by a multistep program involving initial loose attachment to intestinal epithelial cells followed by an intimate adhesion phase. The initial loose interaction of typical EPEC with host intestinal cells is mediated by bundle-forming pili (BFP).

The Effects of Shiga Toxin 1, 2 and Their Subunits on Cytokine and Chemokine Expression by Human Macrophage-Like THP-1 Cells.

Infection by Shiga toxin (Stx)-producing enterohemorrhagic Escherichia coli (EHEC) results in severe diarrhea, hemorrhagic colitis, and, occasionally, hemolytic-uremic syndrome (HUS). HUS is associated with an increase in pro-inflammatory cytokines and chemokines, many of which are produced by macrophages in the kidneys, indicating that localized host innate immunity likely plays a role in renal pathogenesis. EHEC serotypes may express one or two classes of serologically defined but structurally and functionally-related Shiga toxins called Stx1 and Stx2.

Structural and evolutionary analyses show unique stabilization strategies in the type IV pili of Clostridium difficile.

Type IV pili are produced by many pathogenic Gram-negative bacteria and are important for processes as diverse as twitching motility, biofilm formation, cellular adhesion, and horizontal gene transfer. However, many Gram-positive species, including Clostridium difficile, also produce type IV pili. Here, we identify the major subunit of the type IV pili of C.

Synthesis and structure-activity relationship studies of N-terminal analogues of the antimicrobial peptide tridecaptin A(1).

Chemical synthesis was used to increase the potency of the antimicrobial lipopeptide tridecaptin A1. Lipid tail modification proved to be an ideal platform for synthesizing structurally simpler analogues that are not readily accessible by isolation. The stereochemical elements of the tridecaptin A1 lipid tail are not essential for antimicrobial activity and could be replaced with hydrophobic aliphatic or aromatic groups.

Structure of Clostridium difficile PilJ exhibits unprecedented divergence from known type IV pilins.

Type IV pili are produced by many pathogenic Gram-negative bacteria and are important for processes as diverse as twitching motility, cellular adhesion, and colonization. Recently, there has been an increased appreciation of the ability of Gram-positive species, including Clostridium difficile, to produce Type IV pili. Here we report the first three-dimensional structure of a Gram-positive Type IV pilin, PilJ, demonstrate its incorporation into Type IV pili, and offer insights into how the Type IV pili of C.

Effects of nitric oxide and reactive oxygen species on HIF-1α stabilization following clostridium difficile toxin exposure of the Caco-2 epithelial cell line.

Background/aims: Stabilization of the hypoxia-inducible factor (HIF-1α) is proposed to provide a protective host-response to C. difficile intoxication. Here, we aimed to elucidate whether nitric oxide and/or reactive oxygen species produced during C.

Virulence potential and adherence properties of Escherichia coli that produce CTX-M and NDM β-lactamases.

The success of certain sequence types such as ST131 that produce CTX-M or NDM β-lactamases, and ST405 that produce CTX-M β-lactamases, among extraintestinal Escherichia coli (ExPEC) had previously been linked to a combination of antimicrobial resistance and certain virulence factors. The adherence properties of these sequence types to gastro-intestinal epithelial cells had not been investigated. A study was therefore designed to investigate the phylogenetic groups, virulence factors and adherence properties of E.

Intrarectal instillation of Clostridium difficile toxin A triggers colonic inflammation and tissue damage: development of a novel and efficient mouse model of Clostridium difficile toxin exposure.

Clostridium difficile, a major cause of hospital-acquired diarrhea, triggers disease through the release of two toxins, toxin A (TcdA) and toxin B (TcdB). These toxins disrupt the cytoskeleton of the intestinal epithelial cell, increasing intestinal permeability and triggering the release of inflammatory mediators resulting in intestinal injury and inflammation. The most prevalent animal model to study TcdA/TcdB-induced intestinal injury involves injecting toxin into the lumen of a surgically generated "ileal loop.

Impact of the nature and size of the polymeric backbone on the ability of heterobifunctional ligands to mediate shiga toxin and serum amyloid p component ternary complex formation.

Inhibition of AB(5)-type bacterial toxins can be achieved by heterobifunctional ligands (BAITs) that mediate assembly of supramolecular complexes involving the toxin's pentameric cell membrane-binding subunit and an endogenous protein, serum amyloid P component, of the innate immune system. Effective in vivo protection from Shiga toxin Type 1 (Stx1) is achieved by polymer-bound, heterobifunctional inhibitors-adaptors (PolyBAITs), which exhibit prolonged half-life in circulation and by mediating formation of face-to-face SAP-AB(5) complexes, block receptor recognition sites and redirect toxins to the spleen and liver for degradation. Direct correlation between solid-phase activity and protective dose of PolyBAITs both in the cytotoxicity assay and in vivo indicate that the mechanism of protection from intoxication is inhibition of toxin binding to the host cell membrane.

Mutagenic analysis of the Clostridium difficile flagellar proteins, FliC and FliD, and their contribution to virulence in hamsters.

Although toxins A and B are known to be important contributors to the acute phase of Clostridium difficile infection, the role of colonization and adherence to host tissues in the overall pathogenesis of these organisms remains unclear. Consequently, we used the recently introduced intron-based ClosTron gene interruption system to eliminate the expression of two reported C. difficile colonization factors, the major flagellar structural subunit (FliC) and the flagellar cap protein (FliD), to gain greater insight into how flagella and motility contribute to C.

Lipopolysaccharide renders transgenic mice expressing human serum amyloid P component sensitive to Shiga toxin 2.

Transgenic C57BL/6 mice expressing human serum amyloid P component (HuSAP) are resistant to Shiga toxin 2 (Stx2) at dosages that are lethal in HuSAP-negative wild-type mice. However, it is well established that Stx2 initiates extra-intestinal complications such as the haemolytic-uremic syndrome despite the presence of HuSAP in human sera. We now demonstrate that co-administering purified Escherichia coli O55 lipopolysaccharide (LPS), at a dosage of 300 ng/g body weight, to HuSAP-transgenic mice increases their susceptibility to the lethal effects of Stx2.

Binding of Clostridium difficile toxins to human milk oligosaccharides.

The binding of recombinant fragments of the C-terminal cell-binding domains of the two large exotoxins, toxin A (TcdA) and toxin B (TcdB), expressed by Clostridium difficile and a library consisting of the most abundant neutral and acidic human milk oligosaccharides (HMOs) was examined quantitatively at 25°C and pH 7 using the direct electrospray ionization mass spectrometry (ES-MS) assay. The results of the ES-MS measurements indicate that both toxin fragments investigated, TcdB-B1 and TcdA-A2, which possess one and two carbohydrate binding sites, respectively, bind specifically to HMOs ranging in size from tri- to heptasaccharides. Notably, five of the HMOs tested bind to both toxins: Fuc(α1-2)Gal(β1-4)Glc, Gal(β1-3)GlcNAc(β1-3)Gal(β1-4)Glc, Fuc(α1-2)Gal(β1-3)GlcNAc(β1-3)Gal(β1-4)Glc, Gal(β1-3)[Fuc(α1-4)]GlcNAc(β1-3)Gal(β1-4)Glc and Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-3)Gal(β1-4)Glc.

Sticky situation: localized adherence of enteropathogenic Escherichia coli to the small intestine epithelium.

Enteropathogenic Escherichia coli (EPEC) primarily cause gastrointestinal illness in neonates. They accomplish this by a complex coordinated multistage strategy, whereby the organisms colonize the epithelial lining of the small intestine. This process can be divided into four stages: first, localized, nonintimate adherence; second, type III secretion-mediated injection of effector proteins, third effacement of microvilli and, finally, intimate adherence.

N-acetyllactosamine-induced retraction of bundle-forming pili regulates virulence-associated gene expression in enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) are a major cause of infant morbidity and mortality due to diarrhoea in developing countries. The pathogenesis of EPEC is dependent on a coordinated multi-step process culminating in the intimate adherence of the organisms to the host's intestinal mucosa. During the initial stages of the EPEC colonization process, the fimbrial adhesin, bundle-forming pili (BFP), plays an integral role.

The Cpx envelope stress response both facilitates and inhibits elaboration of the enteropathogenic Escherichia coli bundle-forming pilus.

The Cpx envelope stress response is induced by the misfolding of periplasmic proteins and restores envelope homeostasis by upregulating several periplasmic protein folding and degrading factors. The Cpx response also regulates the expression of a variety of envelope-spanning protein complexes, including flagella, secretion systems and pili, which play an important role in pathogenesis. In a previous study, we inactivated the Cpx response in enteropathogenic Escherichia coli (EPEC), a causative agent of infant diarrhoea, and observed decreased expression of its major adhesin, the bundle-forming pilus (BFP).

A real-time quantitative PCR assay for evaluating Clostridium difficile adherence to differentiated intestinal Caco-2 cells.

Herein we describe a real-time quantitative PCR assay for evaluating the adherence of Clostridium difficile to differentiated human intestinal Caco-2 cells. Our investigations demonstrated that the method, employing the C. difficile-specific triose-phosphate isomerase gene, is as reliable but less time-consuming than counting c.

Clostridium difficile toxin-induced inflammation and intestinal injury are mediated by the inflammasome.

Background & Aims: Clostridium difficile-associated disease (CDAD) is the leading cause of nosocomial diarrhea in the United States. C difficile toxins TcdA and TcdB breach the intestinal barrier and trigger mucosal inflammation and intestinal damage. The inflammasome is an intracellular danger sensor of the innate immune system.

Interactions of enteropathogenic Escherichia coli with pediatric and adult intestinal biopsy specimens during early adherence.

Enteropathogenic Escherichia coli (EPEC) strains cause watery diarrhea almost exclusively in young children. The basis for this age discrimination has never been determined, but it may be related to host cell receptors. During infection, EPEC strains express type IV bundle-forming pili composed of repeating subunits of the protein called bundlin.

From alpha to beta: identification of amino acids required for the N-acetyllactosamine-specific lectin-like activity of bundlin.

Bundle-forming pili (BFP) promote the adherence of typical enteropathogenic Escherichia coli (EPEC) to human intestinal epithelial cells. BFP are polymers of bundlin and nine bundlin alleles have been identified in EPEC isolated from diverse sources. These alleles are divided into two main groups, alpha and beta, based on their amino acid sequences.

Assembly and stability of the shiga toxins investigated by electrospray ionization mass spectrometry.

A systematic investigation into the assembly and stability of native and modified subunits of the Shiga toxins (Stx) in vitro is described. Analysis of the assembly of native and modified B subunits of Stx1 and Stx2 in solution, carried out using electrospray ionization mass spectrometry (ES-MS), suggests that the lower thermodynamic stability of the B subunit homopentamer of Stx2, compared to that of Stx1, is due to the presence of a repulsive interaction involving Asp70 of the Stx2 B subunit. In Stx1 B, the corresponding (spatially) residue is Arg.