An in vitro assay for toxicity testing of Clostridium perfringens type C ß-toxin.

Introduction: Veterinary vaccines against Clostridium perfringens type C need to be tested for absence of toxicity, as mandated by pharmacopoeias worldwide. This toxicity testing is required at multiple manufacturing steps and relies on outdated mouse tests that involve severe animal suffering. Clostridium perfringens type C produces several toxins of which the ß-toxin is the primary component responsible for causing disease. Here, we describe the successful development of a new cell-based in vitro assay that can address the specific toxicity of the ß-toxin. Methods: Development of the cell-based assay followed the principle of in vitro testing developed for Cl. septicum vaccines, which is based on Vero cells. We screened four cell lines and selected the THP-1 cell line, which was shown to be the most specific and sensitive for ß-toxin activity, in combination with a commercially available method to determine cell viability (MTS assay) as a readout. Results: The current animal test is estimated to detect 100 - 1000-fold dilutions of the Cl. perfringens type C non-inactivated antigen. When tested with an active Cl. perfringens type C antigen preparation, derived from a commercial vaccine manufacturing process, our THP-1 cell-based assay was able to detect toxin activity from undiluted to over 10000-fold dilution, showing a linear range between approximately 1000- and 10000-fold dilutions. Assay specificity for the ß-toxin was confirmed with neutralizing antibodies and lack of reaction to Cl. perfringens culture medium. In addition, assay parameters demonstrated good repeatability. Conclusions: Here, we have shown proof of concept for a THP-1 cell-based assay for toxicity testing of veterinary Cl. perfringens type C vaccines that is suitable for all vaccine production steps. This result represents a significant step towards the replacement of animal-based toxicity testing of this veterinary clostridial antigen. As a next step, assessment of the assay's sensitivity and repeatability and validation of the method will have to be performed in a commercial manufacturing context in order to formally implement the assay in vaccine quality control.

The Molecular Architecture and Mode of Action of Clostridium perfringens ε-Toxin.

Clostridium perfringens ε-toxin has long been associated with a severe enterotoxaemia of livestock animals, and more recently, was proposed to play a role in the etiology of multiple sclerosis in humans. The remarkable potency of the toxin has intrigued researchers for many decades, who suggested that this indicated an enzymatic mode of action. Recently, there have been major breakthroughs by finding that it is a pore-forming toxin which shows exquisite specificity for cells bearing the myelin and lymphocyte protein (MAL) receptor. This review details the molecular structures of the toxin, the evidence which identifies MAL as the receptor and the possible roles of other cell membrane components in toxin binding. The information on structure and mode of action has allowed the functions of individual amino acids to be investigated and has led to the creation of mutants with reduced toxicity that could serve as vaccines. In spite of this progress, there are still a number of key questions around the mode of action of the toxin which need to be further investigated.

Overview of Bacterial Protein Toxins from Pathogenic Bacteria: Mode of Action and Insights into Evolution.

Bacterial protein toxins are secreted by certain bacteria and are responsible for mild to severe diseases in humans and animals. They are among the most potent molecules known, which are active at very low concentrations. Bacterial protein toxins exhibit a wide diversity based on size, structure, and mode of action. Upon recognition of a cell surface receptor (protein, glycoprotein, and glycolipid), they are active either at the cell surface (signal transduction, membrane damage by pore formation, or hydrolysis of membrane compound(s)) or intracellularly. Various bacterial protein toxins have the ability to enter cells, most often using an endocytosis mechanism, and to deliver the effector domain into the cytosol, where it interacts with an intracellular target(s). According to the nature of the intracellular target(s) and type of modification, various cellular effects are induced (cell death, homeostasis modification, cytoskeleton alteration, blockade of exocytosis, etc.). The various modes of action of bacterial protein toxins are illustrated with representative examples. Insights in toxin evolution are discussed.

Cyanobacteria and Macroinvertebrate Relationships in Freshwater Benthic Communities beyond Cytotoxicity.

Cyanobacteria are harmful algae that are monitored worldwide to prevent the effects of the toxins that they can produce. Most research efforts have focused on direct or indirect effects on human populations, with a view to gain easy accurate detection and quantification methods, mainly in planktic communities, but with increasing interest shown in benthos. However, cyanobacteria have played a fundamental role from the very beginning in both the development of our planet's biodiversity and the construction of new habitats. These organisms have colonized almost every possible planktic or benthic environment on earth, including the most extreme ones, and display a vast number of adaptations. All this explains why they are the most important or the only phototrophs in some habitats. The negative effects of cyanotoxins on macroinvertebrates have been demonstrated, but usually under conditions that are far from natural, and on forms of exposure, toxin concentration, or composition. The cohabitation of cyanobacteria with most invertebrate groups is long-standing and has probably contributed to the development of detoxification means, which would explain the survival of some species inside cyanobacteria colonies. This review focuses on benthic cyanobacteria, their capacity to produce several types of toxins, and their relationships with benthic macroinvertebrates beyond toxicity.

Arginine alleviates Clostridium perfringens α toxin-induced intestinal injury in vivo and in vitro via the SLC38A9/mTORC1 pathway.

Introduction: Clostridium perfringens α toxin is a main virulence factor responsible for gut damage in animals. Arginine is a functional amino acid exhibiting significant immunoregulatory activities. However, the effects and immunoregulatory mechanisms of arginine supplementation on α toxin-induced intestinal injury remain unclear. Methods: In vivo, 256 male Arbor Acres chickens were randomly assigned to a 2×2 factorial arrangement, involving diet treatments (with or without 0.3% arginine supplementation) and immunological stress (with or without α toxin challenge). In vitro, IEC-6 cells were treated with or without arginine in the presence or absence of α toxin. Moreover, IEC-6 cells were transfected with siRNA targeting mTOR and SLC38A9 to explore the underlying mechanisms. Results and discussion: The results showed that in vivo, arginine supplementation significantly alleviated the α toxin-induced growth performance impairment, decreases in serum immunoglobulin (Ig)A and IgG levels, and intestinal morphology damage. Arginine supplementation also significantly reduced the α toxin-induced increase in jejunal proinflammatory cytokines interleukin (IL)-1ß, IL-6 and IL-17 mRNA expression. Clostridium perfringens α toxin significantly decreased jejunal mechanistic target of rapamycin (mTOR) and solute carrier family 38 member 9 (SLC38A9) mRNA expression, while arginine supplementation significantly increased mTOR and SLC38A9 mRNA expression. In vitro, arginine pretreatment mitigated the α toxin-induced decrease in cell viability and the increase in cytotoxicity and apoptosis. Arginine pretreatment also alleviated the α toxin-induced upregulation of mRNA expression of inflammation-related cytokines IL-6, C-X-C motif chemokine ligand (CXCL)10, CXCL11 and transforming growth factor-ß (TGF-ß), as well as apoptosis-related genes B-cell lymphoma-2 associated X protein (Bax), B-cell lymphoma-2 (Bcl-2), B-cell lymphoma-extra large (Bcl-XL) and cysteinyl aspartate specific proteinase 3 (Caspase-3) and the ratio of Bax to Bcl-2. Arginine pretreatment significantly increased the α toxin-induced decrease in mTOR, SLC38A9, eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4EBP1) and ribosomal protein S6 kinase (S6K) mRNA expression. Knockdown SLC38A9 and mTOR largely abrogated the positive effects of arginine pretreatment on α toxin-induced intracellular changes. Furthermore, SLC38A9 silencing abolished the increased mTOR mRNA expression caused by arginine pretreatment. In conclusion, arginine administration attenuated α toxin-induced intestinal injury in vivo and in vitro, which could be associated with the downregulation of inflammation via regulating SLC38A9/mTORC1 pathway.

Cu-doped Fe3O4 nanoparticles for efficient detoxification of epsilon toxin: Toward substituting magnetically recyclable detoxifying agent for formaldehyde.

This research presents the synthesis and characterization of Cu-doped Fe3O4 (Cu-Fe3O4) nanoparticles as a magnetically recoverable and reusable detoxifying agent for the efficient and long-lasting neutralization of bacterial toxins. The nanoparticles were synthesized using the combustion synthesis method and characterized through SEM, XRD, BET, TGA, and VSM techniques. The detoxification potential of Cu-Fe3O4 was compared with traditional formaldehyde (FA) in detoxifying epsilon toxin (ETx) from Clostridium perfringens Type D, the causative agent of enterotoxemia in ruminants. In vivo residual toxicity tests revealed that Cu-Fe3O4 could detoxify ETx at a concentration of 2.0 mg mL-1 within 4 days at room temperature (RT) and 2 days at 37 °C, outperforming FA (12 and 6 days at RT and 37 °C, respectively). Characterization studies using dynamic light scattering (DLS) and circular dichroism (CD) highlighted lower conformational changes in Cu-Fe3O4-detoxified ETx compared to FA-detoxified ETx. Moreover, Cu-Fe3O4-detoxified ETx exhibited exceptional storage stability at 4 °C and RT for 6 months, maintaining an irreversible structure with no residual toxicity. The particles demonstrated remarkable reusability, with the ability to undergo five continuous detoxification batches. This study provides valuable insights into the development of an efficient and safe detoxifying agent, enabling the production of toxoids with a native-like structure. The magnetically recoverable and reusable nature of Cu-Fe3O4 nanoparticles offers practical advantages for easy recovery and reuse in detoxification reactions.

A poisonous cocktail: interplay of cereulide toxin and its structural isomers in emetic Bacillus cereus.

Food intoxications evoked by emetic Bacillus cereus strains constitute a serious threat to public health, leading to emesis and severe organ failure. The emetic peptide toxin cereulide, assembled by the non-ribosomal peptide synthetase CesNRPS, cannot be eradicated from contaminated food by usual hygienic measures due to its molecular size and structural stability. Next to cereulide, diverse chemical variants have been described recently that are produced concurrently with cereulide by CesNRPS. However, the contribution of these isocereulides to the actual toxicity of emetic B. cereus, which produces a cocktail of these toxins in a certain ratio, is still elusive. Since cereulide isoforms have already been detected in food remnants from foodborne outbreaks, we aimed to gain insights into the composition of isocereulides and their impact on the overall toxicity of emetic B. cereus. The amounts and ratios of cereulide and isocereulides were determined in B. cereus grown under standard laboratory conditions and in a contaminated sample of fried rice balls responsible for one of the most severe food outbreaks caused by emetic B. cereus in recent years. The ratios of variants were determined as robust, produced either under laboratory or natural, food-poisoning conditions. Examination of their actual toxicity in human epithelial HEp2-cells revealed that isocereulides A-N, although accounting for only 10% of the total cereulide toxins, were responsible for about 40% of the total cytotoxicity. An this despite the fact that some of the isocereulides were less cytotoxic than cereulide when tested individually for cytotoxicity. To estimate the additive, synergistic or antagonistic effects of the single variants, each cereulide variant was mixed with cereulide in a 1:9 and 1:1 binary blend, respectively, and tested on human cells. The results showed additive and synergistic impacts of single variants, highlighting the importance of including not only cereulide but also the isocereulides in routine food and clinical diagnostics to achieve a realistic toxicity evaluation of emetic B. cereus in contaminated food as well as in patient samples linked to foodborne outbreaks. Since the individual isoforms confer different cell toxicity both alone and in association with cereulide, further investigations are needed to fully understand their cocktail effect.

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.

Consumption of toxic benthic cyanobacteria by two common demersal fish: Growth, antioxidant and liver histopathology responses.

Benthic freshwater cyanobacteria have the potential to produce toxins. Compared with more extensively studied plankton species, little is known about the impact of harmful benthic cyanobacteria on aquatic organisms. As demersal fish are usually in direct contact with benthic cyanobacteria, it is important to understand their interactive effects. This study investigated the physio-chemical responses of two demersal fish (Xenocypris davidi and Crucian carp) after exposure to benthic Oscillatoria (producing cylindrospermopsin, 2 × 106 cells/mL) for 7 days. Interestingly, benthic Oscillatoria had less adverse effects on X. davidi than C. carp. The two demersal fish effectively ingested Oscillatoria, but Oscillatoria cell sheathes could not be fully digested in C. carp intestines and led to growth inhibition. Oscillatoria consumption induced oxidative stress and triggered alterations in detoxification enzyme activities in the X. davidi liver. Superoxide dismutase (SOD) and glutathione reductase (GR) activities significantly increased in the C. carp liver, but catalase (CAT) and detoxification enzymes glutathione S-transferase (GST) and glutathione (GSH) activities were insignificantly changed. This suggested that C. carp may have a relatively weak detoxification capacity for toxic Oscillatoria. Oscillatoria ingestion led to more pronounced liver pathological changes in C. carp, including swelling, deformation, and loss of cytoskeleton structure. Simultaneously, fish consumption of Oscillatoria increased extracellular cylindrospermopsin concentration. These results provide valuable insights into the ecological risks associated with benthic cyanobacteria in aquatic ecosystems.

Special Issue "Bacterial Toxins and Cancer".

Infection is a major contributor to the development of cancer, with more than 15% of new cancer diagnoses estimated to be caused by infection [...].

Environmental Factors Impacting the Development of Toxic Cyanobacterial Proliferations in a Central Texas Reservoir.

Cyanobacterial harmful algal proliferations (cyanoHAPs) are increasingly associated with dog and livestock deaths when benthic mats break free of their substrate and float to the surface. Fatalities have been linked to neurotoxicosis from anatoxins, potent alkaloids produced by certain genera of filamentous cyanobacteria. After numerous reports of dog illnesses and deaths at a popular recreation site on Lady Bird Lake, Austin, Texas in late summer 2019, water and floating mat samples were collected from several sites along the reservoir. Water quality parameters were measured and mat samples were maintained for algal isolation and DNA identification. Samples were also analyzed for cyanobacterial toxins using LC-MS. Dihydroanatoxin-a was detected in mat materials from two of the four sites (0.6-133 ng/g wet weight) while water samples remained toxin-free over the course of the sampling period; no other cyanobacterial toxins were detected. DNA sequencing analysis of cyanobacterial isolates yielded a total of 11 genera, including Geitlerinema, Tyconema, Pseudanabaena, and Phormidium/Microcoleus, taxa known to produce anatoxins, including dihydroanatoxin, among other cyanotoxins. Analyses indicate that low daily upriver dam discharge, higher TP and NO3 concentrations, and day of the year were the main parameters associated with the presence of toxic floating cyanobacterial mats.

Cyanobacterial blooms in surface waters - Nature-based solutions, cyanotoxins and their biotransformation products.

Cyanobacterial blooms are expected to become more frequent and severe in surface water reservoirs due to climate change and ecosystem degradation. It is an emerging challenge that especially countries relying on surface water supplies will face. Nature-based solutions (NBS) like constructed wetlands and biofilters can be used for cyanotoxin remediation. Both technologies are reviewed and critically assessed for different types of water resources. The available information on cyanotoxins (bio)transformation products (TPs) is reviewed to point out the potential research gaps and to disclose the most reliable enzymatic degradation pathways. Knowledge gaps were found, such as information on the performance of the revised NBS in pilot and full scales, the removal processes covering different cyanotoxins (besides the most widely studied microcystin-LR), and the difficulties for real-world implementation of technologies proposed in the literature. Also, most studies focus on bacterial degradation processes while fungi have been completely overlooked. This review also presents an up-to-date overview of the transformation of cyanotoxins, where degradation product data was compiled in a unified library of 22 metabolites for microcystins (MCs), 7 for cylindrospermopsin (CYN) and 10 for nodularin (NOD), most of them reported only in a single study. Major gaps are the lack of environmentally relevant studies with TPs in pilot and full- scale treatment systems, information on TP's toxicity, as well as limited knowledge of environmentally relevant degradation pathways. NBS have the potential to mitigate cyanotoxins in recreational and irrigation waters, enabling the water-energy-food nexus and avoiding the degradability of the ecosystems.

An Updated View on the Cellular Uptake and Mode-of-Action of Clostridioides difficile Toxins.

Research on the human gut pathogen Clostridioides (C.) difficile and its toxins continues to attract much attention as a consequence of the threat to human health posed by hypervirulent strains. Toxin A (TcdA) and Toxin B (TcdB) are the two major virulence determinants of C. difficile. Both are single-chain proteins with a similar multidomain architecture. Certain hypervirulent C. difficile strains also produce a third toxin, namely binary toxin CDT (C. difficile transferase). C. difficile toxins are the causative agents of C. difficile-associated diseases (CDADs), such as antibiotics-associated diarrhea and pseudomembranous colitis. For that reason, considerable efforts have been expended to unravel their molecular mode-of-action and the cellular mechanisms responsible for their uptake. Many of these studies have been conducted in European laboratories. Here, we provide an update on our previous review (Papatheodorou et al. Adv Exp Med Biol, 2018) on important advances in C. difficile toxins research.

Pro-Inflammatory Cytokines Enhanced In Vitro Cytotoxic Activity of Clostridioides difficile Toxin B in Enteric Glial Cells: The Achilles Heel of Clostridioides difficile Infection?

Bacterial infections are characterized by an inflammatory response, which is essential for infection containment but is also responsible for negative effects on the host. The pathogen itself may have evolved molecular mechanisms to antagonize the antimicrobial effects of an inflammatory response and to enhance its pathogenicity using inflammatory response mediators, such as cytokines. Clostridioides difficile (C. difficile) infection (CDI) causes gastrointestinal diseases with markedly increasing global incidence and mortality rates. The main C. difficile virulence factors, toxin A and B (TcdA/TcdB), cause cytopathic/cytotoxic effects and inflammation. We previously demonstrated that TcdB induces enteric glial cell (EGC) apoptosis, which is enhanced by the pro-inflammatory cytokine tumor necrosis factor alpha plus interferon gamma (CKs). However, it is unknown whether CKs-enhanced TcdB cytotoxicity (apoptosis/necrosis) is affected by the timing of the appearance of the CKs. Thus, we simulated in vitro, in our experimental model with TcdB and EGCs, three main situations of possible interactions between TcdB and the timing of CK stimulation: before TcdB infection, concomitantly with infection, or at different times after infection and persisting over time. In these experimental conditions, which all represent situations of possible interactions between C. difficile and the timing of CK stimulation, we evaluated apoptosis, necrosis, and cell cycle phases. The CKs, in all of these conditions, enhanced TcdB cytotoxicity, which from apoptosis became necrosis when CK stimulation persisted over time, and was most relevant after 48 h of TcdB:EGCs interaction. Particularly, the enhancement of apoptosis by CKs was dependent on the TcdB dose and in a less relevant manner on the CK stimulation time, while the enhancement of necrosis occurred always independently of the TcdB dose and CK stimulation time. However, since in all conditions stimulation with CKs strongly enhanced the TcdB cytotoxicity, it always had a negative impact on C. difficile pathogenicity. This study might have important implications for the treatment of CDI.

The Chaperonin TRiC/CCT Inhibitor HSF1A Protects Cells from Intoxication with Pertussis Toxin.

Pertussis toxin (PT) is a bacterial AB5-toxin produced by Bordetella pertussis and a major molecular determinant of pertussis, also known as whooping cough, a highly contagious respiratory disease. In this study, we investigate the protective effects of the chaperonin TRiC/CCT inhibitor, HSF1A, against PT-induced cell intoxication. TRiC/CCT is a chaperonin complex that facilitates the correct folding of proteins, preventing misfolding and aggregation, and maintaining cellular protein homeostasis. Previous research has demonstrated the significance of TRiC/CCT in the functionality of the Clostridioides difficile TcdB AB-toxin. Our findings reveal that HSF1A effectively reduces the levels of ADP-ribosylated Gαi, the specific substrate of PT, in PT-treated cells, without interfering with enzyme activity in vitro or the cellular binding of PT. Additionally, our study uncovers a novel interaction between PTS1 and the chaperonin complex subunit CCT5, which correlates with reduced PTS1 signaling in cells upon HSF1A treatment. Importantly, HSF1A mitigates the adverse effects of PT on cAMP signaling in cellular systems. These results provide valuable insights into the mechanisms of PT uptake and suggest a promising starting point for the development of innovative therapeutic strategies to counteract pertussis toxin-mediated pathogenicity.

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.

Proliferation and anatoxin production of benthic cyanobacteria associated with canine mortalities along a stream-lake continuum.

Proliferations of benthic cyanobacteria are increasingly in the public eye, with rising animal deaths associated with benthic rather than planktonic blooms. In early June 2021, two dogs died after consuming material on the shore of Shubenacadie Grand Lake, Nova Scotia. Preliminary investigations indicated anatoxins produced by benthic cyanobacterial mats were responsible for the deaths. In this study, we monitored the growth of a toxic benthic cyanobacterial species (Microcoleus sp.) along a stream-lake continuum where the canine poisonings occurred. We found that the species was able to proliferate in both lentic and lotic environments, but temporal growth dynamics and the predominant sub-species were influenced by habitat type, and differed with hydrodynamic setting, nutrient and sunlight availability. Toxin concentration was greatest in cyanobacterial mats growing in the oligotrophic lakeshore environment (maximum measured total anatoxins (ATXs) >20 mg·kg-1 wet weight). This corresponded with a shift in the profile of ATX analogues, which also indicated changing sub-species dominance along the stream-lake transition.

Cyanobacteria and cyanotoxins in the environment and water intakes: Reports, diversity of congeners, detection by mass spectrometry and their impact on health.

Cyanobacteria are aquatic microorganisms of high interest for research due to the production of secondary metabolites, among which the most popular are cyanotoxins, responsible for causing severe poisoning in humans and animals through ingestion or contact with contaminated water bodies. Monitoring the number of cyanobacteria in water and concentrations of secreted cyanotoxins with the aid of sensitive and reliable methods is considered the primary action for evaluating potentially toxic blooms. There is a great diversity of methods to detect and identify these types of micro contaminants in water, differing by the degree of sophistication and information provided. Mass Spectrometry stands out for its accuracy and sensitivity in identifying toxins, making it possible to identify and characterize toxins produced by individual species of cyanobacteria, in low quantities. In this review, we seek to update some information about cyanobacterial peptides, their effects on biological systems, and the importance of the main Mass Spectrometry methods used for detection, extraction, identification and monitoring of cyanotoxins.

A Colonic Organoid Model Challenged with the Large Toxins of Clostridioides difficile TcdA and TcdB Exhibit Deregulated Tight Junction Proteins.

BACKGROUND: Clostridioides difficile toxins TcdA and TcdB are responsible for diarrhea and colitis. Lack of functional studies in organoid models of the gut prompted us to elucidate the toxin's effects on epithelial barrier function and the molecular mechanisms for diarrhea and inflammation. METHODS: Human adult colon organoids were cultured on membrane inserts. Tight junction (TJ) proteins and actin cytoskeleton were analyzed for expression via Western blotting and via confocal laser-scanning microscopy for subcellular localization. RESULTS: Polarized intestinal organoid monolayers were established from stem cell-containing colon organoids to apply toxins from the apical side and to perform functional measurements in the organoid model. The toxins caused a reduction in transepithelial electrical resistance in human colonic organoid monolayers with sublethal concentrations. Concomitantly, we detected increased paracellular permeability fluorescein and FITC-dextran-4000. Human colonic organoid monolayers exposed to the toxins exhibited redistribution of barrier-forming TJ proteins claudin-1, -4 and tricellulin, whereas channel-forming claudin-2 expression was increased. Perijunctional F-actin cytoskeleton organization was affected. CONCLUSIONS: Adult stem cell-derived human colonic organoid monolayers were applicable as a colon infection model for electrophysiological measurements. The TJ changes noted can explain the epithelial barrier dysfunction and diarrhea in patients, as well as increased entry of luminal antigens triggering inflammation.

Harmful and beneficial properties of cyanotoxins: Two sides of the same coin.

Cyanotoxins are by definition "harmful agents" produced by cyanobacteria. Their toxicity has been extensively studied and reviewed over the years. Cyanotoxins have been commonly classified, based on their poisonous effects on mammals, into three main classes, neurotoxins, hepatotoxins and dermatotoxins, and, considering their chemical features, mainly identified as peptides, alkaloids and lipopolysaccharides. Here we propose a broader subdivision of cyanotoxins into eight distinct classes, taking into account their molecular structures, biosynthesis and modes of action: alkaloids, non-ribosomal peptides, polyketides, non-protein amino acids, indole alkaloids, organophosphates, lipopeptides and lipoglycans. For each class, the structures and primary mechanisms of toxicity of the main representative cyanotoxins are reported. Despite their powerful biological activities, only recently scientists have considered the biotechnological potential of cyanotoxins, and their applications both in medical and in industrial settings, even if only a few of these have reached the biotech market. In this perspective, we discuss the potential uses of cyanotoxins as anticancer, antimicrobial, and biocidal agents, as common applications for cytotoxic compounds. Furthermore, taking into account their mechanisms of action, we describe peculiar potential bioactivities for several cyanotoxin classes, such as local anaesthetics, antithrombotics, neuroplasticity promoters, immunomodulating and antifouling agents. In this review, we aim to stimulate research on the potential beneficial roles of cyanotoxins, which require interdisciplinary cooperation to facilitate the discovery of innovative biotechnologies.