A high-throughput gut-on-chip platform to study the epithelial responses to enterotoxins.

Enterotoxins are a type of toxins that primarily affect the intestines. Understanding their harmful effects is essential for food safety and medical research. Current methods lack high-throughput, robust, and translatable models capable of characterizing toxin-specific epithelial damage. Pressing concerns regarding enterotoxin contamination of foods and emerging interest in clinical applications of enterotoxins emphasize the need for new platforms. Here, we demonstrate how Caco-2 tubules can be used to study the effect of enterotoxins on the human intestinal epithelium, reflecting toxins' distinct pathogenic mechanisms. After exposure of the model to toxins nigericin, ochratoxin A, patulin and melittin, we observed dose-dependent reductions in barrier permeability as measured by TEER, which were detected with higher sensitivity than previous studies using conventional models. Combination of LDH release assays and DRAQ7 staining allowed comprehensive evaluation of toxin cytotoxicity, which was only observed after exposure to melittin and ochratoxin A. Furthermore, the study of actin cytoskeleton allowed to assess toxin-induced changes in cell morphology, which were only caused by nigericin. Altogether, our study highlights the potential of our Caco-2 tubular model in becoming a multi-parametric and high-throughput tool to bridge the gap between current enterotoxin research and translatable in vivo models of the human intestinal epithelium.

A Streamlined Method to Obtain Biologically Active TcdA and TcdB Toxins from Clostridioides difficile.

The major virulence factors of Clostridioides difficile (C. difficile) are enterotoxins A (TcdA) and B (TcdB). The study of toxins is a crucial step in exploring the virulence of this pathogen. Currently, the toxin purification process is either laborious and time-consuming in C. difficile or performed in heterologous hosts. Therefore, we propose a streamlined method to obtain functional toxins in C. difficile. Two C. difficile strains were generated, each harboring a sequence encoding a His-tag at the 3' end of C. difficile 630∆erm tcdA or tcdB genes. Each toxin gene is expressed using the Ptet promoter, which is inducible by anhydro-tetracycline. The obtained purification yields were 0.28 mg and 0.1 mg per liter for rTcdA and rTcdB, respectively. In this study, we successfully developed a simple routine method that allows the production and purification of biologically active rTcdA and rTcdB toxins with similar activities compared to native toxins.

Protective Effects of Alginate and Chitosan Oligosaccharides against Clostridioides difficile Bacteria and Toxin.

Clostridioides difficile infection is expected to become the most common healthcare-associated infection worldwide. C. difficile-induced pathogenicity is significantly attributed to its enterotoxin, TcdA, which primarily targets Rho-GTPases involved in regulating cytoskeletal and tight junction (TJ) dynamics, thus leading to cytoskeleton breakdown and ultimately increased intestinal permeability. This study investigated whether two non-digestible oligosaccharides (NDOs), alginate (AOS) and chitosan (COS) oligosaccharides, possess antipathogenic and barrier-protective properties against C. difficile bacteria and TcdA toxin, respectively. Both NDOs significantly reduced C. difficile growth, while cell cytotoxicity assays demonstrated that neither COS nor AOS significantly attenuated the TcdA-induced cell death 24 h post-exposure. The challenge of Caco-2 monolayers with increasing TcdA concentrations increased paracellular permeability, as measured by TEER and LY flux assays. In this experimental setup, COS completely abolished, and AOS mitigated, the deleterious effects of TcdA on the monolayer's integrity. These events were not accompanied by alterations in ZO-1 and occludin protein levels; however, immunofluorescence microscopy revealed that both AOS and COS prevented the TcdA-induced occludin mislocalization. Finally, both NDOs accelerated TJ reassembly upon a calcium-switch assay. Overall, this study established the antipathogenic and barrier-protective capacity of AOS and COS against C. difficile and its toxin, TcdA, while revealing their ability to promote TJ reassembly in Caco-2 cells.

Domperidone Protects Cells from Intoxication with Clostridioides difficile Toxins by Inhibiting Hsp70-Assisted Membrane Translocation.

Clostridioides difficile infections cause severe symptoms ranging from diarrhea to pseudomembranous colitis due to the secretion of AB-toxins, TcdA and TcdB. Both toxins are taken up into cells through receptor-mediated endocytosis, autoproteolytic processing and translocation of their enzyme domains from acidified endosomes into the cytosol. The enzyme domains glucosylate small GTPases such as Rac1, thereby inhibiting processes such as actin cytoskeleton regulation. Here, we demonstrate that specific pharmacological inhibition of Hsp70 activity protected cells from TcdB intoxication. In particular, the established inhibitor VER-155008 and the antiemetic drug domperidone, which was found to be an Hsp70 inhibitor, reduced the number of cells with TcdB-induced intoxication morphology in HeLa, Vero and intestinal CaCo-2 cells. These drugs also decreased the intracellular glucosylation of Rac1 by TcdB. Domperidone did not inhibit TcdB binding to cells or enzymatic activity but did prevent membrane translocation of TcdB's glucosyltransferase domain into the cytosol. Domperidone also protected cells from intoxication with TcdA as well as CDT toxin produced by hypervirulent strains of Clostridioides difficile. Our results reveal Hsp70 requirement as a new aspect of the cellular uptake mechanism of TcdB and identified Hsp70 as a novel drug target for potential therapeutic strategies required to combat severe Clostridioides difficile infections.

Revolutionizing treatment for toxic shock syndrome with engineered super chromones to combat antibiotic-resistant Staphylococcus aureus.

OBJECTIVE: Staphylococcus aureus-induced toxic shock syndrome (TSS) is a rare, but potentially fatal disease with limited treatment options. The emergence of antibiotic-resistant strains has led to a pressing need for the development of effective therapies. This study aimed to identify and optimize potential drug candidates against toxic shock syndrome by targeting the pathogenic toxin protein using chromones as lead compounds. MATERIALS AND METHODS: In this study, 20 chromones were screened for their ability to bind to the target protein. The top compounds were further optimized through the addition of cycloheptane and amide groups, and the resulting compounds were evaluated for their drug-like properties using chemical absorption, distribution, metabolism, excretion, and toxicity (ADMET) profiling. RESULTS: Among the compounds screened, 7-Glucosyloxy-5-hydroxy-2-[2-(4-hydroxyphenyl) ethyl] chromone exhibited the highest binding affinity with a molecular weight of 341.40 g/mol and a binding energy of -10.0 kcal/mol. The optimized compound exhibited favorable drug-like properties, including high water solubility, synthetic accessibility, skin permeation, bioavailability, and gastrointestinal absorption. CONCLUSIONS: This study suggests that chromones can be engineered to develop effective drugs against TSS caused by S. aureus. The optimized compound has the potential to be a promising therapeutic agent for the treatment of TSS, providing new hope for patients suffering from this life-threatening disease of toxic shock syndrome.

A leaky human colon model reveals uncoupled apical/basal cytotoxicity in early Clostridioides difficile toxin exposure.

Clostridioides difficile (C. difficile) toxins A (TcdA) and B (TcdB) cause antibiotic-associated colitis in part by disrupting epithelial barrier function. Accurate in vitro models are necessary to detect early toxicity kinetics, investigate disease etiology, and develop preclinical models for new therapies. Properties of cancer cell lines and organoids inherently limit these efforts. We developed adult stem cell-derived monolayers of differentiated human colonic epithelium (hCE) with barrier function, investigated the impact of toxins on apical/basal aspects of monolayers, and evaluated whether a leaky epithelial barrier enhances toxicity. Single-cell RNA-sequencing (scRNAseq) mapped C. difficile-relevant genes to human lineages. Transcriptomics compared hCE to Caco-2, informed timing of in vitro stem cell differentiation, and revealed transcriptional responses to TcdA. Transepithelial electrical resistance (TEER) and fluorescent permeability assays measured cytotoxicity. Contribution of TcdB toxicity was evaluated in a diclofenac-induced leaky gut model. scRNAseq demonstrated broad and variable toxin receptor expression. Absorptive colonocytes in vivo displayed increased toxin receptor, Rho GTPase, and cell junction gene expression. Advanced TcdA toxicity generally decreased cytokine/chemokine and increased tight junction and death receptor genes. Differentiated Caco-2 cells remained immature whereas hCE monolayers were similar to mature colonocytes in vivo. Basal exposure of TcdA/B caused greater toxicity and apoptosis than apical exposure. Apical exposure to toxins was enhanced by diclofenac. Apical/basal toxicities are uncoupled with more rapid onset and increased magnitude postbasal toxin exposure. Leaky junctions enhance toxicity of apical TcdB exposure. hCE monolayers represent a physiologically relevant and sensitive system to evaluate the impact of microbial toxins on gut epithelium.NEW & NOTEWORTHY Novel human colonocyte monolayer cultures, benchmarked by transcriptomics for physiological relevance, detect early cytopathic impacts of Clostridioides difficile toxins TcdA and TcdB. A fluorescent ZO-1 reporter in primary human colonocytes is used to track epithelial barrier disruption in response to TcdA. Basal TcdA/B exposure generally caused more rapid onset and cytotoxicity than apical exposure. Transcriptomics demonstrate changes in tight junction, chemokine, and cytokine receptor gene expression post-TcdA exposure. Diclofenac-induced leaky epithelium enhanced apical exposure toxicity.

Neutralising Effects of Different Antibodies on Clostridioides difficile Toxins TcdA and TcdB in a Translational Approach.

Given the high prevalence of intestinal disease in humans and animals, there is a strong need for clinically relevant models recapitulating gastrointestinal systems, ideally replacing in vivo models in accordance with the principles of the 3R. We established a canine organoid system and analysed the neutralising effects of recombinant versus natural antibodies on Clostridioides difficile toxins A and B in this in vitro system. Sulforhodamine B cytotoxicity assays in 2D and FITC-dextran barrier integrity assays on basal-out and apical-out organoids revealed that recombinant, but not natural antibodies, effectively neutralised C. difficile toxins. Our findings emphasise that canine intestinal organoids can be used to test different components and suggest that they can be further refined to also mirror complex interactions between the intestinal epithelium and other cells.

Staphylococcal enterotoxin A induces DNA damage in hepatocytes and liver tissues.

Staphylococcal enterotoxin A (SEA) is a foodborne bacterial toxin that can cause food poisoning, but little research has been done on the DNA damage caused by SEA. The aim of this research was to investigate the action of SEA in inducing DNA damage and oxidative stress response in hepatocytes and liver tissues. After treating HL-7702 and BRL-3A cells with different concentrations of SEA (0, 300, 600 ng/mL and 0, 400, 800 ng/mL), the production of phosphorylated H2AX (γH2AX) and p53 binding protein 1 (53BP1) aggregates was detected by confocal fluorescence microscopy, and the increases in ataxia telangiectasia mutated (ATM), checkpoint kinase 2 (Chk2), p53 protein expression were assessed by Western blot analysis, while increased reactive oxygen species (ROS) content was confirmed by flow cytometry and fluorescence probe. The genotoxicity of SEA to cells was attenuated after the addition of an oxidative inhibitor, demonstrating that SEA induced intracellular DNA damage through the oxidative pathway and a dose-dependent relationship was observed between the oxidation index and SEA. These experimental results deepen our understanding of SEA damage to cells at the genetic level, and provide a new orientation for the prevention and cure of food borne diseases caused by Staphylococcal enterotoxins (SEs).

Functional and Immunological Studies Revealed a Second Superantigen Toxin in Staphylococcal Enterotoxin C Producing Staphylococcus aureus Strains.

Staphylococcus aureus is a human and animal pathogen as well as a commensal bacterium. It can be a causative agent of severe, life-threatening infections with high mortality, e.g., toxic shock syndrome, septic shock, and multi-organ failure. S. aureus strains secrete a number of toxins. Exotoxins/enterotoxins are considered important in the pathogenesis of the above-mentioned conditions. Exotoxins, e.g., superantigen toxins, cause uncontrolled and polyclonal T cell activation and unregulated activation of inflammatory cytokines. Here we show the importance of genomic analysis of infectious strains in order to identify disease-causing exotoxins. Further, we show through functional analysis of superantigenic properties of staphylococcal exotoxins that even very small amounts of a putative superantigenic contaminant can have a significant mitogenic effect. The results show expression and production of two distinct staphylococcal exotoxins, SEC and SEL, in several strains from clinical isolates. Antibodies against both toxins are required to neutralise the superantigenic activity of staphylococcal supernatants and purified staphylococcal toxins.

Discovery of a novel natural product inhibitor of Clostridioides difficile with potent activity in vitro and in vivo.

Clostridioides difficile infection is a global health threat and remains the primary cause of hospital-acquired infections worldwide. The burgeoning incidence and severity of infections coupled with high rates of recurrence have created an urgent need for novel therapeutics. Here, we report a novel natural product scaffold as a potential anticlostridial lead with antivirulence properties and potent activity both in vitro and in vivo. A whole cell phenotypic screening of 1,000 purified natural products identified 6 compounds with potent activity against C. difficile (minimum inhibitory concentration (MIC) range from 0.03 to 2 µg/ml). All these 6 compounds were non-toxic to human colorectal cells. The natural product compounds also inhibited the production of key toxins, TcdA and TcdB, the key virulence determinants of C. difficile infection pathology. Additionally, the compounds exhibited rapid bactericidal activity and were superior to the standard-of-care antibiotic vancomycin, in reducing a high inoculum of C. difficile in vitro. Furthermore, a murine model of C. difficile infection revealed that compound NP-003875 conferred 100% protection to the infected mice from clinical manifestations of the disease. Collectively, the current study lays the foundation for further investigation of the natural product NP-003875 as a potential therapeutic choice for C. difficile infection.

Regulation of Enterotoxins Associated with Bacillus cereus Sensu Lato Toxicoinfection.

Bacillus cereus sensu lato (s.l.) includes foodborne pathogens, as well as beneficial microorganisms, such as bioinsecticides. Some of the beneficial and commercially used B. cereus s.l. strains have been shown to carry enterotoxin genes, the products of which can cause toxicoinfection in humans. Furthermore, recent epidemiological reports indicated that some bioinsecticidal strains have been linked with foodborne illness outbreaks. This demonstrates the need for improved surveillance of B. cereus s.l., which includes characterization of isolates' virulence capacity. However, the prediction of virulence capacity of B. cereus s.l. strains is challenging. Genetic screening for enterotoxin gene presence has proven to be insufficient for accurate discrimination between virulent and avirulent strains, given that nearly all B. cereus s.l. strains carry at least one enterotoxin gene. Furthermore, complex regulatory networks governing the expression of enterotoxins, and potential synergistic interactions between enterotoxins and other virulence factors make the prediction of toxicoinfection based on isolates' genome sequences challenging. In this review, we summarize and synthesize the current understanding of the regulation of enterotoxins associated with the B. cereus s.l. toxicoinfection and identify gaps in the knowledge that need to be addressed to facilitate identification of genetic markers predictive of cytotoxicity and toxicoinfection.

On the Potential Significance of the Intrinsically Disordered Regions in the Clostridiodes difficile Toxins A and B.

BACKGROUND: Clostridiodes (or Clostridium) difficile is a spore-forming, Gram-positive anaerobic bacterium that may cause symptoms ranging from diarrhea to pseudomembranous colitis. During the C. difficile infection (CDI), the two primary bacterial toxins, toxin A (TcdA) or toxin B (TcdB), disrupt host cell function mainly through the inactivation of small GTPases that regulate the actin cytoskeleton. Both toxins have complex structural organization containing several functional domains. METHODS: Analytical bioinformatics tools are used to compare the extent of disorder within TcdA and TcdB proteins, and to see if the existence of structural disorder can be used to explain the difference in the functionality of these toxins. RESULTS: This paper's aim is to offer an overall review of the structural and functional differences between TcdA and TcdB. CONCLUSION: Results of our multifactorial bioinformatics analysis revealed that intrinsic disorder may play a role in the multifunctionality of C. difficile major toxins TcdA and TcdB, suggesting that intrinsic disorder may be related to their pathogenic mechanisms.

A Protective Role for Glucagon-like Peptide-2 in Heat-stable Enterotoxin b (STb)-Induced L-Cell Toxicity.

Enterotoxigenic Escherichia coli (ETEC)-derived purified heat-stable enterotoxin b (STb) is responsible for secretory diarrhea in livestock and humans. STb disrupts intestinal fluid homeostasis, epithelial barrier function, and promotes cell death. Glucagon-like peptide-2 (GLP-2) is a potent intestinotrophic hormone secreted by enteroendocrine L cells. GLP-2 enhances crypt cell proliferation, epithelial barrier function, and inhibits enterocyte apoptosis. Whether STb can affect GLP-2 producing L cells remains to be elucidated. First, secreted-His-labeled STb from transformed E coli was collected and purified. When incubated with L-cell models (GLUTag, NCI-H716, and secretin tumor cell line [STC-1]), fluorescent immunocytochemistry revealed STb was internalized and was differentially localized in the cytoplasm and nucleus. Cell viability experiments with neutral red and resazurin revealed that STb was toxic in all but the GLUTag cells. STb stimulated 2-hour GLP-2 secretion in all cell models. Interestingly, GLUTag cells produced the highest amount of GLP-2 when treated with STb, demonstrating an inverse relationship in GLP-2 secretion and cell toxicity. To demonstrate a protective role for GLP-2, GLUTag-conditioned media (rich in GLP-2) blocked STb toxicity in STC-1 cells. Confirming a protective role of GLP-2, teduglutide was able to improve cell viability in cells treated with H2O2. In conclusion, STb interacts with the L cell, stimulates secretion, and may induce toxicity if GLP-2 is not produced at high levels. GLP-2 or receptor agonists have the ability to improve cell viability in response to toxins. These results suggest that GLP-2 secretion can play a protective role during STb intoxication. This work supports future investigation into the use of GLP-2 therapies in enterotoxigenic-related diseases.

Disruption of Claudin-Made Tight Junction Barriers by Clostridium perfringens Enterotoxin: Insights from Structural Biology.

Claudins are a family of integral membrane proteins that enable epithelial cell/cell interactions by localizing to and driving the formation of tight junctions. Via claudin self-assembly within the membranes of adjoining cells, their extracellular domains interact, forming barriers to the paracellular transport of small molecules and ions. The bacterium Clostridium perfringens causes prevalent gastrointestinal disorders in mammals by employing an enterotoxin (CpE) that targets claudins. CpE binds to claudins at or near tight junctions in the gut and disrupts their barrier function, potentially by disabling their assembly or via cell signaling means-the mechanism(s) remain unclear. CpE ultimately destroys claudin-expressing cells through the formation of a cytotoxic membrane-penetrating ß-barrel pore. Structures obtained by X-ray crystallography of CpE, claudins, and claudins in complex with CpE fragments have provided the structural bases of claudin and CpE functions, revealing potential mechanisms for the CpE-mediated disruption of claudin-made tight junctions. This review highlights current progress in this space-what has been discovered and what remains unknown-toward efforts to elucidate the molecular mechanism of CpE disruption of tight junction barriers. It further underscores the key insights obtained through structure that are being applied to develop CpE-based therapeutics that combat claudin-overexpressing cancers or modulate tight junction barriers.

Glucosyltransferase-dependent and independent effects of Clostridioides difficile toxins during infection.

Clostridioides difficile infection (CDI) is the leading cause of nosocomial diarrhea and pseudomembranous colitis in the USA. In addition to these symptoms, patients with CDI can develop severe inflammation and tissue damage, resulting in life-threatening toxic megacolon. CDI is mediated by two large homologous protein toxins, TcdA and TcdB, that bind and hijack receptors to enter host cells where they use glucosyltransferase (GT) enzymes to inactivate Rho family GTPases. GT-dependent intoxication elicits cytopathic changes, cytokine production, and apoptosis. At higher concentrations TcdB induces GT-independent necrosis in cells and tissue by stimulating production of reactive oxygen species via recruitment of the NADPH oxidase complex. Although GT-independent necrosis has been observed in vitro, the relevance of this mechanism during CDI has remained an outstanding question in the field. In this study we generated novel C. difficile toxin mutants in the hypervirulent BI/NAP1/PCR-ribotype 027 R20291 strain to test the hypothesis that GT-independent epithelial damage occurs during CDI. Using the mouse model of CDI, we observed that epithelial damage occurs through a GT-independent process that does not involve immune cell influx. The GT-activity of either toxin was sufficient to cause severe edema and inflammation, yet GT activity of both toxins was necessary to produce severe watery diarrhea. These results demonstrate that both TcdA and TcdB contribute to disease pathogenesis when present. Further, while inactivating GT activity of C. difficile toxins may suppress diarrhea and deleterious GT-dependent immune responses, the potential of severe GT-independent epithelial damage merits consideration when developing toxin-based therapeutics against CDI.

Impaired intestinal stem cell activity in ETEC infection: enterotoxins, cyclic nucleotides, and Wnt signaling.

Enterotoxigenic Escherichia coli (ETEC) in humans and animals colonizes the intestine and thereafter secrets heat-stable enterotoxin (ST) with or without heat-labile enterotoxin (LT), which triggers massive fluid and electrolyte secretion into the gut lumen. The crosstalk between the cyclic nucleotide-dependent protein kinase/cystic fibrosis transmembrane conductance regulator (cAMP or cGMP/CFTR) pathway involved in ETEC-induced diarrhea channels, and the canonical Wnt/ß-catenin signaling pathway leads to changes in intestinal stem cell (ISC) fates, which are strongly associated with developmental disorders caused by diarrhea. We review how alterations in enterotoxin-activated ion channel pathways and the canonical Wnt/ß-catenin signaling pathway can explain inhibited intestinal epithelial activity, characterize alterations in the crosstalk of cyclic nucleotides, and predict harmful effects on ISCs in targeted therapy. Besides, we discuss current deficits in the understanding of enterotoxin-intestinal epithelial cell activity relationships that should be considered when interpreting sequelae of diarrhea.

Toxigenic Properties of Yersinia enterocolitica Biotype 1A.

Yersinia (Y.) enterocolitica, an etiological agent of yersiniosis, is a bacterium whose pathogenicity is determined, among other things, by its ability to produce toxins. The aim of this article was to present the most important toxins that are produced by biotype 1A strains of Y. enterocolitica, and to discuss their role in the pathogenesis of yersiniosis. Y. enterocolitica biotype 1A strains are able to synthesize variants of thermostable YST enterotoxin and play a key role in the pathogenesis of yersiniosis. Biotype 1A strains of Y. enterocolitica also produce Y. enterocolitica pore-forming toxins, YaxA and YaxB. These toxins form pores in the cell membrane of host target cells and cause osmotic lysis, which is of particular importance in systemic infections. Insecticidal toxin complex genes have been detected in some clinical biotype 1A strains of Y. enterocolitica. However, their role has not yet been fully elucidated. Strains belonging to biotype 1A have long been considered non-pathogenic. This view is beginning to change due to the emerging knowledge about the toxigenic potential of these bacteria and their ability to overcome the defense barriers of the host organism.

Ebola virus delta peptide is an enterotoxin.

During the 2013-2016 West African (WA) Ebola virus (EBOV) outbreak, severe gastrointestinal symptoms were common in patients and associated with poor outcome. Delta peptide is a conserved product of post-translational processing of the abundant EBOV soluble glycoprotein (sGP). The murine ligated ileal loop model was used to demonstrate that delta peptide is a potent enterotoxin. Dramatic intestinal fluid accumulation follows injection of biologically relevant amounts of delta peptide into ileal loops, along with gross alteration of villous architecture and loss of goblet cells. Transcriptomic analyses show that delta peptide triggers damage response and cell survival pathways and downregulates expression of transporters and exchangers. Induction of diarrhea by delta peptide occurs via cellular damage and regulation of genes that encode proteins involved in fluid secretion. While distinct differences exist between the ileal loop murine model and EBOV infection in humans, these results suggest that delta peptide may contribute to EBOV-induced gastrointestinal pathology.

Exposure to aerosolized staphylococcal enterotoxin B potentiated by lipopolysaccharide modifies lung transcriptomes and results in lung injury in the mouse model.

Lipopolysaccharide (LPS) is one of the main constituents of the cell wall in Gram-negative bacteria. Staphylococcal enterotoxin B (SEB) is produced by the Gram-positive opportunistic pathogen, Staphylococcus aureus. Emerging evidence suggests that intraperitoneal injection of LPS combined with low-dose aerosolized SEB exposure can cause severe lung injury and even death, while SEB or LPS alone cause neither mortality nor severe pulmonary symptoms in mice. However, pulmonary effects from exposure to aerosolized SEB potentiated by LPS have not been evaluated. This study investigates the global transcriptome profile of lung tissue in mice after exposure to aerosolized SEB potentiated by LPS or LPS alone. A mouse model of intratracheal exposure to LPS-potentiated aerosolized SEB is established and described through histological examination. Transcriptome analysis revealed LPS-potentiated aerosolized SEB affected mouse lungs within 72 h post-SEB inhalation, gradually causing lung injury starting from 24 h post inhalation. Hub genes leading to lung injury at 48 h post inhalation have been identified. Flow cytometry revealed that LPS potentiation of low-dose SEB produces a superantigen response that T cells expressing a particular T cell receptor Vß induces a proliferation response by 72 h post inhalation in the lungs of mice. This study represents the first research to investigate pulmonary transcriptional responses of LPS-potentiated aerosolized low-dose SEB exposure. This research helps to elucidate the molecular mechanisms underlying the process by which the two bacterial components combined to produce lung damage and provides an insight into potential treatments for alleviating inflammation of the lung when coinfection is present.

Staphylococcal Enterotoxin A Induces Intestinal Barrier Dysfunction and Activates NLRP3 Inflammasome via NF-κB/MAPK Signaling Pathways in Mice.

Staphylococcal enterotoxin A (SEA), the toxin protein secreted by Staphylococcus aureus, can cause staphylococcal food poisoning outbreaks and seriously threaten global public health. However, little is known about the pathogenesis of SEA in staphylococcal foodborne diseases. In this study, the effect of SEA on intestinal barrier injury and NLRP3 inflammasome activation was investigated by exposing BALB/c mice to SEA with increasing doses and a potential toxic mechanism was elucidated. Our findings suggested that SEA exposure provoked villi injury and suppressed the expression of ZO-1 and occludin proteins, thereby inducing intestinal barrier dysfunction and small intestinal injury in mice. Concurrently, SEA significantly up-regulated the expression of NLRP3 inflammasome-associated proteins and triggered the mitogen-activated protein kinase (MAPK) and nuclear factor kappa-B (NF-κB) signaling pathways in jejunum tissues. Notably, selective inhibitors of MAPKs and NF-κB p65 ameliorated the activation of NLRP3 inflammasome stimulated by SEA, which further indicated that SEA could activate NLRP3 inflammasome through NF-κB/MAPK pathways. In summary, SEA was first confirmed to induce intestinal barrier dysfunction and activate NLRP3 inflammasome via NF-κB/MAPK signaling pathways. These findings will contribute to a more comprehensive understanding of the pathogenesis of SEA and related drug-screening for the treatment and prevention of bacteriotoxin-caused foodborne diseases via targeting specific pathways.