Intestinal tissue response to Shiga toxin exposure.

Shiga toxin (Stx) is produced by some pathogenic strains of Escherichia coli. To study the impact of Stx on the human intestine, we utilized human intestinal organoids and human intestinal enteroids grown as human intestinal enteroid monolayers (HIEMs) in transwells. To establish optimal experimental conditions, HIEMs were grown with or without mesenchymal cells added to the basolateral wells to recapitulate the interactions between the intestinal epithelium and the underlying mesenchyme. Monolayer barrier integrity was determined through transepithelial electrical resistance (TEER) readings. Apical saline was used on the apical surface since growth medium caused uneven development of the TEER. The medium used for epithelial cells contains added growth factors, while the mesenchymal medium lacks these growth factors. We have shown that mesenchymal cells can maintain the epithelial monolayer in the medium lacking growth factors, suggesting they produce these factors. Furthermore, growth factors produced by mesenchymal cells need to build up in the medium over time, since daily medium changes were not as effective as medium changes performed every 3 days. We have also shown that addition of growth factors is toxic to mesenchymal cells. Epithelial cells were more resistant to Stx2 than the mesenchymal cells, and mesenchymal cells contributed to epithelial cell death. Epithelial cells tolerated luminal exposure better than basolateral exposure. These studies demonstrate the importance of understanding tissue interactions in a disease state when using in vitro and in vitro models. IMPORTANCE: These studies have cemented the need for complex cell culture models when studying host-pathogen interactions. Common animal models such as mice are resistant to E. coli O157:H7 infections and intestinal delivery of Stx2, while humans appear to be sensitive to both. It has been proposed that in humans, shiga toxin-producing E. coli-mediated intestinal damage destroys the intestinal barrier and allows basolateral access to Stx2. In mice, there is no epithelial damage; therefore, they are resistant to epithelial delivery of Stx2 while remaining sensitive to Stx2 injection. Our studies show that like mice, the human epithelial layer is quite resistant to Stx2, and it is the sensitivity of the mesenchymal cells that kills the epithelial cells. We have shown that Stx2 is transported through the intact epithelium without causing damage to the resistant epithelial layer. Understanding tissue interactions during infections is therefore critical in determining the effects of pathogens on human tissues.

A fluorescence anisotropy-based competition assay to identify inhibitors against ricin and Shiga toxin ribosome interactions.

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.

Shiga toxin down-regulates ERG protein in endothelial cells and impairs angiogenesis.

BACKGROUND: Shiga toxin (Stx) can activate inflammatory signaling, leading to vascular dysfunction and promotion of a pro-thrombotic tissue microenvironment. Stx can trigger the development of the enterohemorrhagic (childhood) hemolytic uremic syndrome (eHUS), a triad of thrombocytopenia, hemolytic anemia, and acute kidney injury, often requiring dialysis. Additional features may include damage to other organs, including the gastrointestinal tract, pancreas, brain and cardiovascular system; death occurs in 2-5 %. eHUS is a thrombotic microangiopathy; thus, endothelial cell (EC) injury and platelet fibrin thrombus formation in glomerular arterioles and in the arterioles of other affected organs are likely. To elucidate mechanisms of this microangiopathy, we examined in human ECs the regulation of the platelet adhesion proteins P-selectin and von Willebrand factor (VWF), along with the downregulation of erythroblast-transformation-specific transcription factor (ERG) a key regulator of angiogenesis and megakaryocyte development. METHODS: VWF, P-selectin, and ERG levels were determined using immunofluorescence and Western blot in human umbilical endothelial cells (HUVECs). HUVECs were treated with tumor necrosis factor-alpha (TNF-α), Stx-1 or both, versus normal controls. Capillary morphogenesis on Matrigel was performed using HUVECs treated, for 22 h with TNF-α, Stx-1, or both, or treated 4 h with Stx-1 alone or in combination with TNF-α for 22 h. RESULTS: Stx-1 significantly reduced ERG and VWF expression on HUVECs, but upregulated P-selectin expression. ERG levels decreased with Stx-1 alone or in combination with TNF-α, in the nuclear, perinuclear and cytoplasmatic regions. Stx-1 reduced capillary morphogenesis, while Stx-1-TNF-α combined treatment reduced capillary morphogenesis still further. CONCLUSIONS: In the presence of Stx-1 or TNF-α or both treatments, ECs were activated, expressing higher levels of P-selectin and lower levels of VWF. Our findings, further, provide evidence that Stx-1 downregulates ERG, repressing angiogenesis in vitro.

One problem, multiple potential targets: Where are we now in the development of small molecule inhibitors against Shiga toxin?

Shiga toxin-producing Escherichia coli (STEC) are a group of enteric pathogens which carry phage-encoded Shiga toxins (Stx). STEC infections begin with severe abdominal pain and non-bloody diarrhoea, which can progress to bloody diarrhoea after approximately 4-days post-infection. In high-risk groups such as children and the elderly, patients may develop haemolytic uremic syndrome (HUS). HUS is characterised by microangiopathic haemolytic anaemia, thrombocytopenia, and in severe disease acute renal failure. Traditional antibiotics have been linked with increased toxin production due to the activation of recA-mediated bacterial stress response, resulting in poorer patient outcomes. Therefore, treatment relies on supportive therapies. Antivirulence strategies have been explored as an alternative treatment for bacterial infections and blockers of virulence factors such as the Type III Secretion System. Recent improvements in the mechanistic understanding of the Stx pathway have led to the design of inhibitors to disrupt the pathway, leading to toxin-mediated ribosome damage. However, compounds have yet to progress beyond Phase III clinical trials successfully. This review explores the progress in developing small molecule inhibitors by collating lead compounds derived from in-silico and experimental approaches.

Phage Tail Fiber Protein as a Specific Probe for Recognition of Shiga Toxin-Producing Escherichia coli O91, O103, and O111.

The ability to quickly identify specific serotypes of Shiga toxin-producing Escherichia coli (STEC) could facilitate the monitoring and control of STEC pathogens. In this study, we identified the receptors and receptor-binding proteins (RBPs) of three novel phages (pO91, pO103, and pO111) isolated from hospital wastewater. Recombinant versions of these RBPs (pO91-ORF43, pO103-ORF42, and pO111-ORF8) fused to a fluorescent reporter protein were then constructed. Both fluorescence microscopy and transmission electron microscopy showed that all three recombinant RBPs were bound to the bacterial surface. Indirect enzyme-linked immunosorbent assay was used to verify that each recombinant RBP bound specifically to E. coli O91, O103, or O111, but not to any of the 83 strains of E. coli with different O-antigens, nor to 10 other bacterial species that were tested. The recombinant RBPs adsorbed to their respective host bacteria within 10 min of incubation. The minimum concentration of bacteria required for detection by the recombinant RBPs was 33 colony-forming units (CFU)/mL (range: 3.3 × 10 to 3.3 × 108 CFU/mL). Furthermore, each recombinant RBP was also able to detect bacteria in lettuce, chicken breast meat, and infected mice, indicating that their usage will facilitate the detection of STEC and may help to reduce the spread of STEC-related infections and diseases.

Shiga toxin targets the podocyte causing hemolytic uremic syndrome through endothelial complement activation.

BACKGROUND: Shiga toxin (Stx)-producing Escherichia coli hemolytic uremic syndrome (STEC-HUS) is the leading cause of acute kidney injury in children, with an associated mortality of up to 5%. The mechanisms underlying STEC-HUS and why the glomerular microvasculature is so susceptible to injury following systemic Stx infection are unclear. METHODS: Transgenic mice were engineered to express the Stx receptor (Gb3) exclusively in their kidney podocytes (Pod-Gb3) and challenged with systemic Stx. Human glomerular cell models and kidney biopsies from patients with STEC-HUS were also studied. FINDINGS: Stx-challenged Pod-Gb3 mice developed STEC-HUS. This was mediated by a reduction in podocyte vascular endothelial growth factor A (VEGF-A), which led to loss of glomerular endothelial cell (GEnC) glycocalyx, a reduction in GEnC inhibitory complement factor H binding, and local activation of the complement pathway. Early therapeutic inhibition of the terminal complement pathway with a C5 inhibitor rescued this podocyte-driven, Stx-induced HUS phenotype. CONCLUSIONS: This study potentially explains why systemic Stx exposure targets the glomerulus and supports the early use of terminal complement pathway inhibition in this devastating disease. FUNDING: This work was supported by the UK Medical Research Council (MRC) (grant nos. G0901987 and MR/K010492/1) and Kidney Research UK (grant nos. TF_007_20151127, RP42/2012, and SP/FSGS1/2013). The Mary Lyon Center is part of the MRC Harwell Institute and is funded by the MRC (A410).

A Shiga Toxin B-Subunit-Based Lectibody Boosts T Cell Cytotoxicity towards Gb3-Positive Cancer Cells.

Aberrant glycosylation plays a crucial role in tumour progression and invasiveness. Tumour-associated carbohydrate antigens (TACAs) represent a valuable set of targets for immunotherapeutic approaches. The poor immunogenicity of glycan structures, however, requires a more effective and well-directed way of targeting TACAs on the surface of cancer cells than antibodies. The glycosphingolipid globotriaosylceramide (Gb3) is a well-established TACA present in a multitude of cancer types. Its overexpression has been linked to metastasis, invasiveness, and multidrug resistance. In the present study, we propose to use a dimeric fragment of the Shiga toxin B-subunit (StxB) to selectively target Gb3-positive cancer cells in a StxB-scFv UCHT1 lectibody. The lectibody, comprised of a lectin and the UCHT1 antibody fragment, was produced in E. coli and purified via Ni-NTA affinity chromatography. Specificity of the lectibody towards Gb3-positive cancer cell lines and specificity towards the CD3 receptor on T cells, was assessed using flow cytometry. We evaluated the efficacy of the lectibody in redirecting T cell cytotoxicity towards Gb3-overexpressing cancer cells in luciferase-based cytotoxicity in vitro assays. The StxB-scFv UCHT1 lectibody has proven specific for Gb3 and could induce the killing of up to 80% of Gb3-overexpressing cancer cells in haemorrhagic and solid tumours. The lectibody developed in this study, therefore, highlights the potential that lectibodies and lectins in general have for usage in immunotherapeutic approaches to boost the efficacy of established cancer treatments.

Engineered Synthetic STxB for Enhanced Cytosolic Delivery.

Many molecular targets for cancer therapy are located in the cytosol. Therapeutic macromolecules are generally not able to spontaneously translocate across membranes to reach these cytosolic targets. Therefore a strong need exists for tools that enhance cytosolic delivery. Shiga toxin B-subunit (STxB) is used to deliver therapeutic principles to disease-relevant cells that express its receptor, the glycolipid Gb3. Based on its naturally existing membrane translocation capacity, STxB delivers antigens to the cytosol of Gb3-positive dendritic cells, leading to the induction of CD8+ T cells. Here, we have explored the possibility of further increasing the membrane translocation of STxB to enable other therapeutic applications. For this, our capacity to synthesize STxB chemically was exploited to introduce unnatural amino acids at different positions of the protein. These were then functionalized with hydrophobic entities to locally destabilize endosomal membranes. Intracellular trafficking of these functionalized STxB was measured by confocal microscopy and their cytosolic arrival with a recently developed highly robust, sensitive, and quantitative translocation assay. From different types of hydrophobic moieties that were linked to STxB, the most efficient configuration was determined. STxB translocation was increased by a factor of 2.5, paving the path for new biomedical opportunities.

Shiga Toxin, Stx2e, Influences the Activity of Porcine Lymphocytes In Vitro.

Oedema disease (OD) in piglets is one of the most important pathologies, as it causes significant losses due to the high mortality because of the Shiga toxin family, which produces Escherichia coli (STEC) strains. The main toxin responsible for the characteristic pathologies in pigs is Shiga toxin 2 subtype e (Stx2e). Moreover, there is growing evidence that Stx's family of toxins also targets immune cells. Therefore, this study evaluated the effect of different concentrations of Stx2e on porcine immune cells. Porcine peripheral blood mononuclear cells were pre-incubated with Stx2e, at three different concentrations (final concentrations of 10, 500, and 5000 CD50/mL) and with a negative control group. Cells were then stimulated with polyclonal mitogens: concanavalin A, phytohemagglutinin, pokeweed mitogen, or lipopolysaccharides. Cell proliferation was assessed by BrdU (or EdU) incorporation into newly created DNA. The activation of the lymphocyte subsets was assessed by the detection of CD25, using flow cytometry. The toxin significantly decreased mitogen-driven proliferation activity, and the effect was partially dose-dependent, with a significant impact on both T and B populations. The percentage of CD25+ cells was slightly lower in the presence of Stx2e in all the defined T cell subpopulations (CD4+, CD8+, and γδTCR+)-in a dose-dependent manner. B cells seemed to be the most affected populations. The negative effects of different concentrations of Stx2e on the immune cells in this study may explain the negative impact of the subclinical course of OD.

Hyperhydration to Improve Kidney Outcomes in Children with Shiga Toxin-Producing E. coli Infection: a multinational embedded cluster crossover randomized trial (the HIKO STEC trial).

BACKGROUND: Shiga toxin-producing E. coli (STEC) infections affect children and adults worldwide, and treatment remain solely supportive. Up to 15-20% of children infected by high-risk STEC (i.e., E. coli that produce Shiga toxin 2) develop hemolytic anemia, thrombocytopenia, and kidney failure (i.e., hemolytic uremic syndrome (HUS)), over half of whom require acute dialysis and 3% die. Although no therapy is widely accepted as being able to prevent the development of HUS and its complications, several observational studies suggest that intravascular volume expansion (hyperhydration) may prevent end organ damage. A randomized trial is needed to confirm or refute this hypothesis. METHODS: We will conduct a pragmatic, embedded, cluster-randomized, crossover trial in 26 pediatric institutions to determine if hyperhydration, compared to conservative fluid management, improves outcomes in 1040 children with high-risk STEC infections. The primary outcome is major adverse kidney events within 30 days (MAKE30), a composite measure that includes death, initiation of new renal replacement therapy, or persistent kidney dysfunction. Secondary outcomes include life-threatening, extrarenal complications, and development of HUS. Pathway eligible children will be treated per institutional allocation to each pathway. In the hyperhydration pathway, all eligible children are hospitalized and administered 200% maintenance balanced crystalloid fluids up to targets of 10% weight gain and 20% reduction in hematocrit. Sites in the conservative fluid management pathway manage children as in- or outpatients, based on clinician preference, with the pathway focused on close laboratory monitoring, and maintenance of euvolemia. Based on historical data, we estimate that 10% of children in our conservative fluid management pathway will experience the primary outcome. With 26 clusters enrolling a mean of 40 patients each with an intraclass correlation coefficient of 0.11, we will have 90% power to detect a 5% absolute risk reduction. DISCUSSION: HUS is a devastating illness with no treatment options. This pragmatic study will determine if hyperhydration can reduce morbidity associated with HUS in children with high-risk STEC infection. TRIAL REGISTRATION: ClinicalTrials.gov NCT05219110 . Registered on February 1, 2022.

Shiga Toxin (Stx) Type 1a and Stx2a Translocate through a Three-Layer Intestinal Model.

Shiga toxins (Stxs) produced by ingested E. coli can induce hemolytic uremic syndrome after crossing the intact intestinal barrier, entering the bloodstream, and targeting endothelial cells in the kidney. The method(s) by which the toxins reach the bloodstream are not fully defined. Here, we used two polarized cell models to evaluate Stx translocation: (i) a single-layer primary colonic epithelial cell model and (ii) a three-cell-layer model with colonic epithelial cells, myofibroblasts, and colonic endothelial cells. We traced the movement of Stx types 1a and 2a across the barrier models by measuring the toxicity of apical and basolateral media on Vero cells. We found that Stx1a and Stx2a crossed both models in either direction. However, approximately 10-fold more Stx translocated in the three-layer model as compared to the single-layer model. Overall, the percentage of toxin that translocated was about 0.01% in the epithelial-cell-only model but up to 0.09% in the three-cell-layer model. In both models, approximately 3- to 4-fold more Stx2a translocated than Stx1a. Infection of the three-cell-layer model with Stx-producing Escherichia coli (STEC) strains showed that serotype O157:H7 STEC reduced barrier function in the model and that the damage was not dependent on the presence of the eae gene. Infection of the three-layer model with O26:H11 STEC strain TW08571 (Stx1a+ and Stx2a+), however, allowed translocation of modest amounts of Stx without reducing barrier function. Deletion of stx2a from TW08571 or the use of anti-Stx1 antibody prevented translocation of toxin. Our results suggest that single-cell models may underestimate the amount of Stx translocation and that the more biomimetic three-layer model is suited for Stx translocation inhibitor studies.

Optical Manipulation of Gb3 Enriched Lipid Domains: Impact of Isomerization on Gb3 -Shiga Toxin B Interaction.

The plasma membrane is a complex assembly of proteins and lipids that can self-assemble in submicroscopic domains commonly termed "lipid rafts", which are implicated in membrane signaling and trafficking. Recently, photo-sensitive lipids were introduced to study membrane domain organization, and photo-isomerization was shown to trigger the mixing and de-mixing of liquid-ordered (lo ) domains in artificial phase-separated membranes. Here, we synthesized globotriaosylceramide (Gb3 ) glycosphingolipids that harbor an azobenzene moiety at different positions of the fatty acid to investigate light-induced membrane domain reorganization, and that serve as specific receptors for the protein Shiga toxin (STx). Using phase-separated supported lipid bilayers on mica surfaces doped with four different photo-Gb3 molecules, we found by fluorescence microscopy and atomic force microscopy that liquid disordered (ld ) domains were formed within lo domains upon trans-cis photo-isomerization. The fraction and size of these ld domains were largest for Gb3 molecules with the azobenzene group at the end of the fatty acid. We further investigated the impact of domain reorganization on the interaction of the B-subunits of STx with the photo-Gb3 . Fluorescence and atomic force micrographs clearly demonstrated that STxB binds to the lo phase if Gb3 is in the trans-configuration, whereas two STxB populations are formed if the photo-Gb3 is switched to the cis-configuration highlighting the idea of manipulating lipid-protein interactions with a light stimulus.

Apyrase decreases phage induction and Shiga toxin release from E. coli O157:H7 and has a protective effect during infection.

Shiga toxin (Stx)-producing enterohemorrhagic Escherichia coli (EHEC) cause gastrointestinal infection and, in severe cases, hemolytic uremic syndrome which may lead to death. There is, to-date, no therapy for this infection. Stx induces ATP release from host cells and ATP signaling mediates its cytotoxic effects. Apyrase cleaves and neutralizes ATP and its effect on Stx and EHEC infection was therefore investigated. Apyrase decreased bacterial RecA and dose-dependently decreased toxin release from E. coli O157:H7 in vitro, demonstrated by reduced phage DNA and protein levels. The effect was investigated in a mouse model of E. coli O157:H7 infection. BALB/c mice infected with Stx2-producing E. coli O157:H7 were treated with apyrase intraperitoneally, on days 0 and 2 post-infection, and monitored for 11 days. Apyrase-treated mice developed disease two days later than untreated mice. Untreated infected mice lost significantly more weight than those treated with apyrase. Apyrase-treated mice exhibited less colonic goblet cell depletion and apoptotic cells, as well as lower fecal ATP and Stx2, compared to untreated mice. Apyrase also decreased platelet aggregation induced by co-incubation of human platelet-rich-plasma with Stx2 and E. coli O157 lipopolysaccharide in the presence of collagen. Thus, apyrase had multiple protective effects, reducing RecA levels, stx2 and toxin release from EHEC, reducing fecal Stx2 and protecting mouse intestinal cells, as well as decreasing platelet activation, and could thereby delay the development of disease.

Cytokines expression from altered motor thalamus and behavior deficits following sublethal administration of Shiga toxin 2a involve the induction of the globotriaosylceramide receptor.

Encephalopathy associated with hemolytic uremic syndrome is produced by enterohemorrhagic E. coli (EHEC) infection, which releases the virulence factors Shiga toxin (Stx) and lipopolysaccharide (LPS). Neurological compromise is a poor prognosis and mortality factor of the disease, and the thalamus is one of the brain areas most frequently affected. We have previously demonstrated the effectiveness of anti-inflammatory drugs to ameliorate the deleterious effects of these toxins. However, the thalamic production of cytokines involved in pro-inflammatory processes has not yet been acknowledged. The aim of this work attempts to determine whether systemic sublethal Stx2a or co-administration of Stx2a with LPS are able to rise a proinflammatory profile accompanying alterations of the neurovascular unit in anterior and lateral ventral nuclei of the thalamus (VA-VL) and motor behavior in mice. After 4 days of treatment, Stx2a affected the lectin-bound microvasculature distribution while increasing the expression of GFAP in reactive astrocytes and producing aberrant NeuN distribution in degenerative neurons. In addition, increased swimming latency was observed in a motor behavioral test. All these alterations were heightened when Stx2a was co-administered with LPS. The expression of pro-inflammatory cytokines TNFα, INF-γ and IL-2 was detected in VA-VL. All these effects were concomitant with increased expression of the Stx receptor globotriaosylceramide (Gb3), which hints at receptor involvement in the neuroinflammatory process as a key finding of this study. In conclusion, Stx2a to Gb3 may be determinant in triggering a neuroinflammatory event, which may resemble clinical outcomes and should thus be considered in the development of preventive strategies.

Repurposing the Pentameric B-Subunit of Shiga Toxin for Gb3-Targeted Immunotherapy of Colorectal Cancer by Rhamnose Conjugation.

Globotriaosylceramide (Gb3 or CD77) is a tumor-associated carbohydrate antigen implicated in several types of cancer that serves as a potential cancer marker for developing target-specific diagnosis and therapy. However, the development of Gb3-targeted therapeutics has been challenging due to its carbohydrate nature. In the present work, taking advantage of its natural pentamer architecture and Gb3-specific targeting of shiga toxin B subunit (StxB), we constructed a pentameric antibody recruiting chimera by site-specifically conjugating StxB with the rhamnose hapten for immunotherapy of colorectal cancer. The Sortase A-catalyzed enzymatic tethering of rhamnose moieties to the C terminus of Stx1B and Stx2B had very moderate effect on their pentamer architectures and thus the resultant conjugates maintained the potent ability to bind to Gb3 antigen both immobilized on an assay plate and expressed on colorectal cancer cells. All StxB-rhamnose constructs were capable of efficiently mediating the binding of rhamnose antibodies onto HT29 colorectal cancer cells, which was further shown to be able to induce cancer cell lysis by eliciting potent antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in vitro. Finally, the best StxB-rhamnose conjugate, i.e. 1B-3R, was confirmed to be able to inhibit the colorectal tumor growth using a HT29-derived xenograft murine model. Taken together, our data demonstrated the potential of repurposing StxB as an excellent multivalent scaffold for developing Gb3-targeted biotherapeutics and StxB-rhamnose conjugates might be promising candidates for targeted immunotherapy of Gb3-related colorectal cancer.

Gene expression profile and injury sites in mice treated with Shiga toxin 2 and lipopolysaccharide as a Shiga toxin-associated hemolytic uremic syndrome model.

Shiga toxin 2 (Stx2) and lipopolysaccharide (LPS) contribute to the development of hemolytic uremic syndrome (HUS). Mouse models of HUS induced by LPS/Stx2 have been used for elucidating HUS pathophysiology and for therapeutic development. However, the underlying molecular mechanisms and detailed injury sites in this model remain unknown. We analyzed mouse kidneys after LPS/Stx2 administration using microarrays. Decreased urinary osmolality and urinary potassium were observed after LPS/Stx2 administration, suggestive of distal nephron disorders. A total of 1,212 and 1,016 differentially expressed genes were identified in microarrays at 6 h and 72 h after LPS/Stx2 administration, respectively, compared with those in controls. Ingenuity pathway analysis revealed activation of TNFR1/2, iNOS, and IL-6 signaling at both time points, and inhibition of pathways associated with lipid metabolism at 72 h only. The strongly downregulated genes in the 72-h group were expressed in the distal nephrons. In particular, genes associated with distal convoluted tubule (DCT) 2/connecting tubule (CNT) and principal cells of the cortical collecting duct (CCD) were downregulated to a greater extent than those associated with DCT1 and intercalated cells. Stx receptor globotriaosylceramide 3 (Gb3) revealed no colocalization with DCT1-specific PVALB and intercalated cell-specific SLC26A4 but did present colocalization with SLC12A3 (present in both DCT1 and DCT2), and AQP2 in principal cells. Gb3 localization tended to coincide with the segment in which the downregulated genes were present. Thus, the LPS/Stx2-induced kidney injury model represents damage to DCT2/CNT and principal cells in the CCD, based on molecular, biological, and physiological findings.

STxB as an Antigen Delivery Tool for Mucosal Vaccination.

Immunotherapy against cancer and infectious disease holds the promise of high efficacy with minor side effects. Mucosal vaccines to protect against tumors or infections disease agents that affect the upper airways or the lung are still lacking, however. One mucosal vaccine candidate is the B-subunit of Shiga toxin, STxB. In this review, we compare STxB to other immunotherapy vectors. STxB is a non-toxic protein that binds to a glycosylated lipid, termed globotriaosylceramide (Gb3), which is preferentially expressed by dendritic cells. We review the use of STxB for the cross-presentation of tumor or viral antigens in a MHC class I-restricted manner to induce humoral immunity against these antigens in addition to polyfunctional and persistent CD4+ and CD8+ T lymphocytes capable of protecting against viral infection or tumor growth. Other literature will be summarized that documents a powerful induction of mucosal IgA and resident memory CD8+ T cells against mucosal tumors specifically when STxB-antigen conjugates are administered via the nasal route. It will also be pointed out how STxB-based vaccines have been shown in preclinical cancer models to synergize with other therapeutic modalities (immune checkpoint inhibitors, anti-angiogenic therapy, radiotherapy). Finally, we will discuss how molecular aspects such as low immunogenicity, cross-species conservation of Gb3 expression, and lack of toxicity contribute to the competitive positioning of STxB among the different DC targeting approaches. STxB thereby appears as an original and innovative tool for the development of mucosal vaccines in infectious diseases and cancer.

Therapeutic Antibodies Against Shiga Toxins: Trends and Perspectives.

Shiga toxins (Stx) are AB5-type toxins, composed of five B subunits which bind to Gb3 host cell receptors and an active A subunit, whose action on the ribosome leads to protein synthesis suppression. The two Stx types (Stx1 and Stx2) and their subtypes can be produced by Shiga toxin-producing Escherichia coli strains and some Shigella spp. These bacteria colonize the colon and induce diarrhea that may progress to hemorrhagic colitis and in the most severe cases, to hemolytic uremic syndrome, which could lead to death. Since the use of antibiotics in these infections is a topic of great controversy, the treatment remains supportive and there are no specific therapies to ameliorate the course. Therefore, there is an open window for Stx neutralization employing antibodies, which are versatile molecules. Indeed, polyclonal, monoclonal, and recombinant antibodies have been raised and tested in vitro and in vivo assays, showing differences in their neutralizing ability against deleterious effects of Stx. These molecules are in different phases of development for which we decide to present herein an updated report of these antibody molecules, their source, advantages, and disadvantages of the promising ones, as well as the challenges faced until reaching their applicability.

Characterization of a T4-like Bacteriophage vB_EcoM-Sa45lw as a Potential Biocontrol Agent for Shiga Toxin-Producing Escherichia coli O45 Contaminated on Mung Bean Seeds.

Application of lytic bacteriophages is a promising and alternative intervention technology to relieve antibiotic resistance pressure and control bacterial pathogens in the food industry. Despite the increase of produce-associated outbreaks caused by non-O157 Shiga toxin-producing E. coli (STEC) serogroups, the information of phage application on sprouts to mitigate these pathogens is lacking. Therefore, the objective of this study was to characterize a T4-like Escherichia phage vB_EcoM-Sa45lw (or Sa45lw) for the biocontrol potential of STEC O45 on mung bean seeds. Phage Sa45lw belongs to the Tequatrovirus genus under the Myoviridae family and displays a close evolutionary relationship with a STEC O157-infecting phage AR1. Sa45lw contains a long-tail fiber gene (gp37), sharing high genetic similarity with the counterpart of Escherichia phage KIT03, and a unique tail lysozyme (gp5) to distinguish its host range (STEC O157, O45, ATCC 13706, and Salmonella Montevideo and Thompson) from phage KIT03 (O157 and Salmonella enterica). No stx, antibiotic resistance, and lysogenic genes were found in the Sa45lw genome. The phage has a latent period of 27 min with an estimated burst size of 80 PFU/CFU and is stable at a wide range of pH (pH 3 to pH 10.5) and temperatures (-80°C to 50°C). Phage Sa45lw is particularly effective in reducing E. coli O45:H16 both in vitro (MOI = 10) by 5 log and upon application (MOI = 1,000) on the contaminated mung bean seeds for 15 min by 2 log at 25°C. These findings highlight the potential of phage application against non-O157 STEC on sprout seeds. IMPORTANCE Seeds contaminated with foodborne pathogens, such as Shiga toxin-producing E. coli, are the primary sources of contamination in produce and have contributed to numerous foodborne outbreaks. Antibiotic resistance has been a long-lasting issue that poses a threat to human health and the food industry. Therefore, developing novel antimicrobial interventions, such as bacteriophage application, is pivotal to combat these pathogens. This study characterized a lytic bacteriophage Sa45lw as an alternative antimicrobial agent to control pathogenic E. coli on the contaminated mung bean seeds. The phage exhibited antimicrobial effects against both pathogenic E. coli and Salmonella without containing virulent or lysogenic genes that could compromise the safety of phage application. In addition, after 15 min of phage treatment, Sa45lw mitigated E. coli O45:H16 on the contaminated mung bean seeds by a 2-log reduction at room temperature, demonstrating the biocontrol potential of non-O157 Shiga toxin-producing E. coli on sprout seeds.

Shiga toxin (Stx) type 2-induced increase in O-linked N-acetyl glucosamine protein modification: a new therapeutic target?

Shiga toxin (Stx)-producing Escherichia coli (STEC) causes bloody diarrhea, which may progress to the potentially fatal hemolytic uremic syndrome (HUS). Development of HUS after STEC infection is dependent on Stx, and is particularly linked to Stx type 2a, Stx2a (Melton-Celsa, 2014; Scheutz, 2014). In this issue of EMBO Molecular Medicine, Lee et al report that O-linked N-acetyl glucosamine protein modification (O-GlcNAcylation) is increased in host cells after Stx exposure and the subsequent endoplasmic reticulum (ER) stress response. The elevated O-GlcNAcylation resulted in elevated inflammatory and apoptotic processes. Inhibition of O-GlcNAcylation with OSMI-1 protected cells from the Stx2a-induced damage. In mice intoxicated with Stx2a, OSMI-1 treatment reduced kidney damage and increased mouse survival.