Heat-Labile Toxin from Enterotoxigenic Escherichia coli Causes Systemic Impairment in Zebrafish Model.

Heat-labile toxin I (LT-I), produced by strains of enterotoxigenic Escherichia coli (ETEC), causes profuse watery diarrhea in humans. Different in vitro and in vivo models have already elucidated the mechanism of action of this toxin; however, their use does not always allow for more specific studies on how the LT-I toxin acts in systemic tracts and intestinal cell lines. In the present work, zebrafish (Danio rerio) and human intestinal cells (Caco-2) were used as models to study the toxin LT-I. Caco-2 cells were used, in the 62nd passage, at different cell concentrations. LT-I was conjugated to FITC to visualize its transport in cells, as well as microinjected into the caudal vein of zebrafish larvae, in order to investigate its effects on survival, systemic traffic, and morphological formation. The internalization of LT-I was visualized in 3 × 104 Caco-2 cells, being associated with the cell membrane and nucleus. The systemic traffic of LT-I in zebrafish larvae showed its presence in the cardiac cavity, yolk, and regions of the intestine, as demonstrated by cardiac edema (100%), the absence of a swimming bladder (100%), and yolk edema (80%), in addition to growth limitation in the larvae, compared to the control group. There was a reduction in heart rate during the assessment of larval survival kinetics, demonstrating the cardiotoxic effect of LT-I. Thus, in this study, we provide essential new depictions of the features of LT-I.

Spatiotemporal analysis of mycolactone distribution in vivo reveals partial diffusion in the central nervous system.

Mycobacterium ulcerans, the causative agent of Buruli ulcer (BU) disease, is unique amongst human pathogens in its capacity to produce a lipid toxin called mycolactone. While previous studies have demonstrated that bacterially-released mycolactone diffuses beyond infection foci, the spatiotemporal distribution of mycolactone remained largely unknown. Here, we used the zebrafish model to provide the first global kinetic analysis of mycolactone's diffusion in vivo, and multicellular co-culture systems to address the critical question of the toxin's access to the brain. Zebrafish larvae were injected with a fluorescent-derivative of mycolactone to visualize the in vivo diffusion of the toxin from the peripheral circulation. A rapid, body-wide distribution of mycolactone was observed, with selective accumulation in tissues near the injection site and brain, together with an important excretion through the gastro-intestinal tract. Our conclusion that mycolactone reached the central nervous system was reinforced by an in cellulo model of human blood brain barrier and a mouse model of M. ulcerans-infection. Here we show that mycolactone has a broad but heterogenous profile of distribution in vivo. Our investigations in vitro and in vivo support the view that a fraction of bacterially-produced mycolactone gains access to the central nervous system. The relative persistence of mycolactone in the bloodstream suggests that assays of circulating mycolactone are relevant for BU disease monitoring and treatment optimization.

Glycan-dependent cell adhesion mechanism of Tc toxins.

Toxin complex (Tc) toxins are virulence factors of pathogenic bacteria. Tcs are composed of three subunits: TcA, TcB and TcC. TcA facilitates receptor-toxin interaction and membrane permeation, TcB and TcC form a toxin-encapsulating cocoon. While the mechanisms of holotoxin assembly and pore formation have been described, little is known about receptor binding of TcAs. Here, we identify heparins/heparan sulfates and Lewis antigens as receptors for different TcAs from insect and human pathogens. Glycan array screening reveals that all tested TcAs bind negatively charged heparins. Cryo-EM structures of Morganella morganii TcdA4 and Xenorhabdus nematophila XptA1 reveal that heparins/heparan sulfates unexpectedly bind to different regions of the shell domain, including receptor-binding domains. In addition, Photorhabdus luminescens TcdA1 binds to Lewis antigens with micromolar affinity. Here, the glycan interacts with the receptor-binding domain D of the toxin. Our results suggest a glycan dependent association mechanism of Tc toxins on the host cell surface.

In vitro assessment of cyanotoxins bioaccessibility in raw and cooked mussels.

The oral route by ingestion of water and food contaminated with cyanotoxins is the main route of exposure to these toxins. This study addresses for the first time the bioaccessibility of some of the most common Microcystins (MC-LR, MC-RR and MC-YR) and Cylindrospermopsin (CYN) simultaneously in raw and steamed mussels spiked at 250 ng/g fresh weight of each cyanotoxin, after an in vitro digestion, including the salivary (incubation with artificial saliva, 30s), gastric (with pepsin, 2h, pH 2), duodenal (with pancreatin and bile salts, 2h, pH 6.5) and colonic phases (with lactic-acid bacteria, 48h, pH 7.2). The results obtained suggest that the potential absorption of these cyanotoxins by consumption of contaminated mussels is lower than expected. After the total effect of cooking and digestion, the mean bioaccessibility levels recorded were 24.65% (CYN), 31.51% (MC-RR), 17.51% (MC-YR) and 13.20% (MC-LR). Moreover, toxins were transferred to the steaming waters at 3.77 ± 0.24 µg L-1 CYN, 2.29 ± 0.13 µg L-1 MC-LR, 6.60 ± 0.25 µg L-1 MC-RR and 3.83 ± 0.22 µg L-1 MC-YR. These bioaccessibility results should be considered for a more accurate risk assessment related to these cyanotoxins in mussels, including the fact that the steaming waters could also represent a risk after human consumption.

Results from an international phase 2 study of the anti-CD22 immunotoxin moxetumomab pasudotox in relapsed or refractory childhood B-lineage acute lymphoblastic leukemia.

BACKGROUND: In a multicenter phase 1 study of children with relapsed/refractory acute lymphoblastic leukemia (ALL), moxetumomab pasudotox, an anti-CD22 immunotoxin, demonstrated a manageable safety profile and preliminary evidence of clinical activity. A phase 2 study further evaluated efficacy. PROCEDURE: This international, multicenter, phase 2 study enrolled children with relapsed/refractory B-cell precursor ALL who received moxetumomab pasudotox 40 µg/kg intravenously every other day, for six doses per 21-day cycle. The primary objective was to evaluate the complete response (CR) rate. Secondary objectives included safety, pharmacokinetics, and immunogenicity evaluations. RESULTS: Thirty-two patients (median age, 10 years) were enrolled at 16 sites; 30 received study drug and were evaluable for safety; 28 were evaluable for response. The objective response rate was 28.6%, with three patients (10.7%) achieving morphologic CR, and five patients (17.9%) achieving partial response. Disease progression occurred in 11 patients (39.3%). Ten patients (33.3%) experienced at least one treatment-related serious adverse event, including capillary leak syndrome (CLS; n = 6), hemolytic uremic syndrome (HUS; n = 4), and treatment-related death (n = 1) from pulmonary edema. No differences were observed in inflammatory markers in patients who did or did not develop CLS or HUS. CONCLUSIONS: Despite a signal for clinical activity, this phase 2 study was terminated at interim analysis for a CR rate that did not reach the stage 1 target. Preclinical data suggest enhanced efficacy of moxetumomab pasudotox via continuous infusion or in combination regimens; thus, further studies designed to optimize the efficacy and safety of moxetumomab pasudotox in pediatric ALL may be warranted.

Moxetumomab pasudotox for the treatment of relapsed and/or refractory hairy cell leukemia.

Introduction: Hairy cell leukemia is a rare indolent B-cell malignancy, characterized by pancytopenia, recurrent infections, and splenomegaly. After initial therapy with purine nucleoside analogs, up to 50% of patients relapse after several years of remission. The number of relapsed patients is increasing and, until recently, there was no approved therapy with durable responses for hairy cell leukemia patients in the relapsed setting, thus the need for new non-chemotherapy approach with significant efficacy and less myelosuppression. Areas covered: Moxetumomab pasudotox is a recombinant immunotoxin containing a Fv fragment of an anti-CD22 monoclonal antibody and truncated Pseudomonas exotoxin (PE38). The authors reviewed pre-clinical and clinical studies that led to the FDA approval of the drug in patients with relapsed and/or refractory hairy cell leukemia, who received at least two prior therapies, including at least one purine nucleoside analog. Expert opinion: Moxetumomab pasudotox demonstrated a durable complete remission rate of 30% in heavily pretreated patients with hairy cell leukemia, and MRD eradication in 85% of responding patients. Moxetumomab pasudotox got a global FDA approval in September 2018. The US prescribing information carries boxed warnings regarding the risk of capillary leak syndrome and hemolytic uremic syndrome. Long-term follow-up of the pivotal study is ongoing (NCT01829711).

Potent and specific fusion toxins consisting of a HER2­binding, ABD­derived affinity protein, fused to truncated versions of Pseudomonas exotoxin A.

Fusion toxins consisting of an affinity protein fused to toxic polypeptides derived from Pseudomonas exotoxin A (ETA) are promising agents for targeted cancer therapy. In this study, we examined whether fusion toxins consisting of an albumin binding domain­derived affinity protein (ADAPT) interacting with human epidermal growth factor receptor 2 (HER2), coupled to the ETA­derived polypeptides PE38X8 or PE25, with or without an albumin binding domain (ABD) for half­life extension, can be used for specific killing of HER2­expressing cells. The fusion toxins could easily be expressed in a soluble form in Escherichia coli and purified to homogeneity. All constructs had strong affinity for HER2 (KD 10 to 26 nM) and no tendency for aggregation could be detected. The fusion toxins including the ABD showed strong interaction with human and mouse serum albumin [equilibrium dissociation constant (KD) 1 to 3 nM and 2 to 10 nM, respectively]. The in vitro investigation of the cytotoxic potential revealed IC50­values in the picomolar range for cells expressing high levels of HER2. The specificity was also demonstrated, by showing that free HER2 receptors on the target cells are required for fusion toxin activity. In mice, the fusion toxins containing the ABD exhibited an appreciably longer time in circulation. The uptake was highest in liver and kidney. Fusion with PE25 was associated with the highest hepatic uptake. Collectively, the results suggest that fusion toxins consisting of ADAPTs and ETA­derivatives are promising agents for targeted cancer therapy.

Insect ATP-Binding Cassette (ABC) Transporters: Roles in Xenobiotic Detoxification and Bt Insecticidal Activity.

ATP-binding cassette (ABC) transporters, a large class of transmembrane proteins, are widely found in organisms and play an important role in the transport of xenobiotics. Insect ABC transporters are involved in insecticide detoxification and Bacillus thuringiensis (Bt) toxin perforation. The complete ABC transporter is composed of two hydrophobic transmembrane domains (TMDs) and two nucleotide binding domains (NBDs). Conformational changes that are needed for their action are mediated by ATP hydrolysis. According to the similarity among their sequences and organization of conserved ATP-binding cassette domains, insect ABC transporters have been divided into eight subfamilies (ABCA-ABCH). This review describes the functions and mechanisms of ABC transporters in insecticide detoxification, plant toxic secondary metabolites transport and insecticidal activity of Bt toxin. With improved understanding of the role and mechanisms of ABC transporter in resistance to insecticides and Bt toxins, we can identify valuable target sites for developing new strategies to control pests and manage resistance and achieve green pest control.

Moxetumomab pasudotox for the treatment of hairy cell leukemia.

Introduction: Cladribine and pentostatin are the drugs of choice in the treatment of hairy cell leukemia (HCL). Recently, immunotoxin moxetumomab pasudotox has been introduced to improve the prognosis in relapsed and refractory HCL. Areas covered: This review discusses the mechanism of action, safety, and efficacy of moxetumomab pasudotox in HCL patients. A literature review of the MEDLINE database for articles in English concerning immunotoxins, moxetumomab pasudotox, and hairy cell leukemia was conducted via PubMed. Publications from 2000 through December 2018 were scrutinized. The search terms used were immunotoxins and moxetumomab pasudotox in conjunction with hairy cell leukemia. Conference proceedings from the previous five years of the American Society of Hematology, European Hematology Association and American Society of Clinical Oncology were searched manually. Additional relevant publications were obtained by reviewing the references from the chosen articles. Results/conclusion: Moxetumomab pasudotox, a novel recombinant anti-CD22 immunotoxin, was well tolerated and active in the previous phase 1 and 3 studies in patients with HCL. The drug has been approved in 2018 by the FDA for the treatment of patients with relapsed/refractory HCL who had at least two prior systemic therapies including at least one purine nucleoside analog. Expert opinion: The use of moxetumomab pasudotox is a promising new strategy for the treatment of HCL.

Specific targeting of a pore-forming toxin (listeriolysin O) to LHRH-positive cancer cells using LHRH targeting peptide.

Conventional drug delivery systems have many limitations including cytotoxicity and affecting non-specific cells. Cell-targeting peptides (CTPs) as a potential class of targeting moiety have some advantages over previous targeting moieties such as monoclonal antibodies, offer additional benefits to design systems using CTPs. Here we have engineered listeriolysin O (LLO) pore-forming toxin by adding a luteinizing hormone-releasing hormone (LHRH) targeting peptide to its N-terminus. Two versions of the toxin, with and without targeting peptide, were sub-cloned into a bacterial expression plasmid. BL21 DE3 cells were used for induction of expression and recombinant proteins were purified using nickel-immobilized metal affinity chromatography column. In order to treat MDA-MB-231 and SKOV3 cell lines as LHRH receptor positive and negative cells, two mentioned LLO toxins were used to evaluate their cytotoxicity and specificity. Our results reveal that the IC50 of LLO toxin on MDA-MB-231 and SKOV3 cells was 0.32 and 0.41 µg/ml respectively. Furthermore, IC50 of fusion LHRH-LLO toxin on the cells was 0.88 and 19.55 µg/ml. Cytotoxicity of engineered LHRH-LLO toxin on negative cells was significantly 48-fold lower than wild-type LLO toxin. But this difference has been lowered to only 2.7-fold less cytotoxicity in positive cells. To the best of our knowledge, the current work as the first study regarding engineered toxin revealed that CDC family members could be used to target the specific cell-type.

Concentrations of cylindrospermopsin toxin in water and tilapia fish of tropical fishponds in Egypt, and assessing their potential risk to human health.

Unlike microcystin, cylindrospermospin (CYN) concentrations in fishpond water and their accumulation in fish tissues have been largely unexplored. This study determined CYN levels in water and tilapia fish organs from three tropical fishponds in southern Egypt. Water and fish samples were collected monthly from fishponds for 12 months (Oct 2012 to Sep 2013). The results revealed that six CYN-producing species of cyanobacteria dominated phytoplankton populations and formed blooms in these fishponds during warm months. Among these species, Anabaena affinis, Planktothrix agardhii, Cylindrospermopsis catemaco, and C. philippinensis were assigned as CYN producers for the first time in the present study. The highest cell densities of CYN-producing species in fishponds were recorded in August and September 2013, correlating with high temperature, pH and nutrient concentrations. Dissolved CYN was found in fishpond waters at levels (0.3-2.76 µg L-1) very close to those of particulate CYN (0.4-2.37 µg L-1). CYN was also estimated in tilapia fish organs at levels up to 417 ng g-1 in the intestines, 1500 ng g-1 in the livers, and 280 ng g-1in edible muscles. Compared to the recommended guideline (0.03 µg kg-1 day-1), the estimated daily intake (EDI) of CYN in our samples of edible muscles exceeded this limit by a factor of 1.3-14 during summer and autumn. This might represent a risk to human health upon consumption of such contaminated fish muscles. Therefore, fishponds worldwide should be monitored for the presence toxic cyanobacteria to protect humans from their potent toxins.

Bioaccessibility and decomposition of cylindrospermopsin in vegetables matrices after the application of an in vitro digestion model.

Research on the human exposure to Cylindrospermopsin (CYN) via consumption of contaminated food is of great interest for risk assessment purposes. The aim of this work is to evaluate for the first time the CYN bioaccessibility in contaminated vegetables (uncooked lettuce and spinach, and boiled spinach) after an in vitro digestion model, including the salivar, gastric and duodenal phases and, colonic fermentation under lactic acid bacteria. The results obtained showed that the digestion processes are able to diminish CYN levels, mainly in the colonic phase, especially in combination with the boiling treatment, decreasing CYN levels in a significant way. Moreover, the potential decomposition products in a pure CYN solution and in CYN-contaminated vegetables were evaluated using UHPLC-MS/MS Orbitrap. Under the conditions assayed, only two diastereoisomers of the same fragment with m/z 292.09617 have been detected in all the analysed samples, with the exception of digested vegetables. Therefore, in terms of risk assessment, the digestion seems to play an important role in reducing the final bioaccesibility of CYN, and the consumption of cooked vegetables (spinach) would be safer in comparison to raw vegetables.

Cytosolic Delivery of Multidomain Cargos by the N Terminus of Pasteurella multocida Toxin.

The zoonotic pathogen Pasteurella multocida produces a 146-kDa modular toxin (PMT) that enters host cells and manipulates intracellular signaling through action on its Gα protein targets. The N terminus of PMT (PMT-N) mediates cellular uptake through receptor-mediated endocytosis, followed by the delivery of the C-terminal catalytic domain from acidic endosomes into the cytosol. The putative native cargo of PMT consists of a 710-residue polypeptide with three distinct modular subdomains (C1-C2-C3), where C1 contains a membrane localization domain (MLD), C2 has an as-yet-undefined function, and C3 catalyzes the deamidation of a specific active-site glutamine residue in Gα protein targets. However, whether the three cargo subdomains are delivered intact or undergo further proteolytic processing during or after translocation from the late endosome is unclear. Here, we demonstrate that PMT-N mediates the delivery of its native C-terminal cargo as a single polypeptide, corresponding to C1-C2-C3, including the MLD, with no evidence of cleavage between subdomains. We show that PMT-N also delivers nonnative green fluorescent protein (GFP) cargo into the cytosol, further supporting that the receptor-binding and translocation functions reside within PMT-N. Our findings further show that PMT-N can deliver C1-C2 alone but that the presence of C1-C2 is important for the cytosolic delivery of the catalytic C3 subdomain by PMT-N. In addition, we further refine the minimum C3 domain required for intracellular activity as comprising residues 1105 to 1278. These findings reinforce that PMT-N serves as the cytosolic delivery vehicle for C-terminal cargo and demonstrate that its native cargo is delivered intact as C1-C2-C3.

Recombinant immunotoxins with albumin-binding domains have long half-lives and high antitumor activity.

Recombinant immunotoxins (RITs) are chimeric proteins consisting of a Fv that binds to a cancer cell and a portion of a protein toxin. One of these, Moxetumomab pasudotox, was shown to be effective in treating patients with some leukemias, where the cells are readily accessible to the RIT. However, their short half-life limits their efficacy in solid tumors, because penetration into the tumors is slow. Albumin and agents bound to albumin have a long half-life in the circulation. To increase the time tumor cells are exposed to RITs, we have produced and evaluated variants that contain either an albumin-binding domain (ABD) from Streptococcus or single-domain antibodies from Llama. We have inserted these ABDs into RITs targeting mesothelin, between the Fv and the furin cleavage site. We find that these proteins can be produced in large amounts, are very cytotoxic to mesothelin-expressing cancer cell lines, and have a high affinity for human or mouse serum albumin. In mice, the RIT containing an ABD from Streptococcus has a longer half-life and higher antitumor activity than the other two. Its half-life in the circulation of mice ranges from 113 to 194 min compared with 13 min for an RIT with no ABD. Cell uptake studies show the RIT enters the target cell bound to serum albumin. We conclude that RITs with improved half-lives and antitumor activity should be evaluated for the treatment of cancer in humans.

Cell-penetrating peptides derived from Clostridium difficile TcdB2 and a related large clostridial toxin.

Clostridium difficile TcdB (2366 amino acid residues) is an intracellular bacterial toxin that binds to cells and enters the cytosol where it glucosylates small GTPases. In the current study, we examined a putative cell entry region of TcdB (amino acid residues 1753-1851) for short sequences that function as cell-penetrating peptides (CPPs). To screen for TcdB-derived CPPs, a panel of synthetic peptides was tested for the ability to enhance transferrin (Tf) association with cells. Four candidate CPPs were discovered, and further study on one peptide (PepB2) pinpointed an asparagine residue necessary for CPP activity. PepB2 mediated the cell entry of a wide variety of molecules including dextran, streptavidin, microspheres, and lentivirus particles. Of note, this uptake was dramatically reduced in the presence of the Na+/H+ exchange blocker and micropinocytosis inhibitor amiloride, suggesting that PepB2 invokes macropinocytosis. Moreover, we found that PepB2 had more efficient cell-penetrating activity than several other well-known CPPs (TAT, penetratin, Pep-1, and TP10). Finally, Tf assay-based screening of peptides derived from two other large clostridial toxins, TcdA and TcsL, uncovered two new TcdA-derived CPPs. In conclusion, we have identified six CPPs from large clostridial toxins and have demonstrated the ability of PepB2 to promote cell association and entry of several molecules through a putative fluid-phase macropinocytotic mechanism.

In Vitro Toxicological Assessment of Cylindrospermopsin: A Review.

Cylindrospermopsin (CYN) is a cyanobacterial toxin that is gaining importance, owing to its increasing expansion worldwide and the increased frequency of its blooms. CYN mainly targets the liver, but also involves other organs. Various mechanisms have been associated with its toxicity, such as protein synthesis inhibition, oxidative stress, etc. However, its toxic effects are not yet fully elucidated and additional data for hazard characterization purposes are required. In this regard, in vitro methods can play an important role, owing to their advantages in comparison to in vivo trials. The aim of this work was to compile and evaluate the in vitro data dealing with CYN available in the scientific literature, focusing on its toxicokinetics and its main toxicity mechanisms. This analysis would be useful to identify research needs and data gaps in order to complete knowledge about the toxicity profile of CYN. For example, it has been shown that research on various aspects, such as new emerging toxicity effects, the toxicity of analogs, or the potential interaction of CYN with other cyanotoxins, among others, is still very scarce. New in vitro studies are therefore welcome.

Phase 1 study of the anti-CD22 immunotoxin moxetumomab pasudotox for childhood acute lymphoblastic leukemia.

Novel therapies are needed to overcome chemotherapy resistance for children with relapsed/refractory acute lymphoblastic leukemia (ALL). Moxetumomab pasudotox is a recombinant anti-CD22 immunotoxin. A multicenter phase 1 study was conducted to determine the maximum-tolerated cumulative dose (MTCD) and evaluate safety, activity, pharmacokinetics, and immunogenicity of moxetumomab pasudotox in children, adolescents, and young adults with ALL (N = 55). Moxetumomab pasudotox was administered as a 30-minute IV infusion at doses of 5 to 50 µg/kg every other day for 6 (cohorts A and B) or 10 (cohort C) doses in 21-day cycles. Cohorts B and C received dexamethasone prophylaxis against capillary leak syndrome (CLS). The most common treatment-related adverse events were reversible weight gain, hepatic transaminase elevation, and hypoalbuminemia. Dose-limiting CLS occurred in 2 of 4 patients receiving 30 µg/kg of moxetumomab pasudotox every other day for 6 doses. Incorporation of dexamethasone prevented further dose-limiting CLS. Six of 14 patients receiving 50 µg/kg of moxetumomab pasudotox for 10 doses developed hemolytic uremic syndrome (HUS), thrombotic microangiopathy (TMA), or HUS-like events, exceeding the MTCD. Treatment expansion at 40 µg/kg for 10 doses (n = 11) exceeded the MTCD because of 2 HUS/TMA/HUS-like events. Dose level 6B (ie, 50 µg/kg × 6 doses) was the MTCD, selected as the recommended phase 2 dose. Among 47 evaluable patients, an objective response rate of 32% was observed, including 11 (23%) composite complete responses, 5 of which were minimal residual disease negative by flow cytometry. Moxetumomab pasudotox showed a manageable safety profile and evidence of activity in relapsed or refractory childhood ALL. This trial was registered at www.clinicaltrials.gov as #NCT00659425.

Chloroquine derivatives block the translocation pores and inhibit cellular entry of Clostridium botulinum C2 toxin and Bacillus anthracis lethal toxin.

The pathogenic bacteria Clostridium botulinum and Bacillus anthracis produce the binary protein toxins C2 and lethal toxin (LT), respectively. These toxins consist of a binding/transport (B7) component that delivers the separate enzyme (A) component into the cytosol of target cells where it modifies its specific substrate and causes cell death. The B7 components of C2 toxin and LT, C2IIa and PA63, respectively, are ring-shaped heptamers that bind to their cellular receptors and form complexes with their A components C2I and lethal factor (LF), respectively. After receptor-mediated endocytosis of the toxin complexes, C2IIa and PA63 insert into the membranes of acidified endosomes and form trans-membrane pores through which C2I and LF translocate across endosomal membranes into the cytosol. C2IIa and PA63 also form channels in planar bilayer membranes, and we used this approach earlier to identify chloroquine as a potent blocker of C2IIa and PA63 pores. Here, a series of chloroquine derivatives was investigated to identify more efficient toxin inhibitors with less toxic side effects. Chloroquine, primaquine, quinacrine, and fluphenazine blocked C2IIa and PA63 pores in planar lipid bilayers and in membranes of living epithelial cells and macrophages, thereby preventing the pH-dependent membrane transport of the A components into the cytosol and protecting cells from intoxication with C2 toxin and LT. These potent inhibitors of toxin entry underline the central role of the translocation pores for cellular uptake of binary bacterial toxins and as relevant drug targets, and might be lead compounds for novel pharmacological strategies against severe enteric diseases and anthrax.

Effects of microcystin-LR and cylindrospermopsin on plant-soil systems: A review of their relevance for agricultural plant quality and public health.

Toxic cyanobacterial blooms are recognized as an emerging environmental threat worldwide. Although microcystin-LR is the most frequently documented cyanotoxin, studies on cylindrospermopsin have been increasing due to the invasive nature of cylindrospermopsin-producing cyanobacteria. The number of studies regarding the effects of cyanotoxins on agricultural plants has increased in recent years, and it has been suggested that the presence of microcystin-LR and cylindrospermopsin in irrigation water may cause toxic effects in edible plants. The uptake of these cyanotoxins by agricultural plants has been shown to induce morphological and physiological changes that lead to a potential loss of productivity. There is also evidence that edible terrestrial plants can bioaccumulate cyanotoxins in their tissues in a concentration dependent-manner. Moreover, the number of consecutive cycles of watering and planting in addition to the potential persistence of microcystin-LR and cylindrospermopsin in the environment are likely to result in groundwater contamination. The use of cyanotoxin-contaminated water for agricultural purposes may therefore represent a threat to both food security and food safety. However, the deleterious effects of cyanotoxins on agricultural plants and public health seem to be dependent on the concentrations studied, which in most cases are non-environmentally relevant. Interestingly, at ecologically relevant concentrations, the productivity and nutritional quality of some agricultural plants seem not to be impaired and may even be enhanced. However, studies assessing if the potential tolerance of agricultural plants to these concentrations can result in cyanotoxin and allergen accumulation in the edible tissues are lacking. This review combines the most current information available regarding this topic with a realistic assessment of the impact of cyanobacterial toxins on agricultural plants, groundwater quality and public health.

Evaluation of the Toxicity and Toxicokinetics of Cereulide from an Emetic Bacillus cereus Strain of Milk Origin.

Bacillus cereus is an opportunistic foodborne agent causing food poisoning and many infectious diseases. The heat-stable emetic toxin cereulide is one of the most prevalent toxins produced by pathogenic B. cereus, resulting in symptoms such as emesis and liver failure. In the present work, the toxicity and toxicokinetics of cereulide from an emetic B. cereus isolate (CAU45) of raw milk were evaluated. The production of cereulide was tested by a cytotoxicity test and enzyme immunoassay, and confirmed by the presence of the ces (cereulide synthetase) gene and the ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method. All results showed that the amount and toxicity of cereulide produced by CAU45 was 7 to 15.3 folds higher than the reference emetic B. cereus DSMZ 4312. Cereulide in plasma was collected at different time points after a single intravenous injection to evaluate its toxicokinetics in rabbits. The maximum concentration of cereulide was achieved in 2.6 ± 3.4 h after administration, with the elimination half-life of 10.8 ± 9.1 h, which expands our understanding of the toxic effects of cereulide. Together, it suggests that urgent sanitary practices are needed to eliminate emetic toxins and emetic B. cereus in raw milk.