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The human pathogenic bacterium Clostridium perfringens secretes an enterotoxin (CpE) that targets claudins through its C-terminal receptor-binding domain (cCpE). Isoform-specific binding by CpE causes dissociation of claudins and tight junctions (TJs), resulting in cytotoxicity and breakdown of the gut epithelial barrier. Here, we present crystal structures of human claudin-9 (hCLDN-9) in complex with cCpE at 3.2 and 3.3 Å. We show that hCLDN-9 is a high-affinity CpE receptor and that hCLDN-9-expressing cells undergo cell death when treated with CpE but not cCpE, which lacks its cytotoxic domain. Structures reveal cCpE-induced alterations to 2 epitopes known to enable claudin self-assembly and expose high-affinity interactions between hCLDN-9 and cCpE that explain isoform-specific recognition. These findings elucidate the molecular bases for hCLDN-9 selective ion permeability and binding by CpE, and provide mechanisms for how CpE disrupts gut homeostasis by dissociating claudins and TJs to affect epithelial adhesion and intercellular transport.

The genus Trimeresurus comprises a group of venomous pitvipers endemic to Southeast Asia and the Pacific Islands. Of these, Trimeresurus insularis, the White-lipped Island Pitviper, is a nocturnal, arboreal species that occurs on nearly every major island of the Lesser Sunda archipelago. In the current study, venom phenotypic characteristics of T. insularis sampled from eight Lesser Sunda Islands (Flores, Lembata, Lombok, Pantar, Sumba, Sumbawa, Timor, and Wetar) were evaluated via SDS-PAGE, enzymatic activity assays, fibrinogenolytic assays, gelatin zymography, and RP-HPLC, and the Sumbawa sample was characterized by venomic analysis. For additional comparative analyses, venoms were also examined from several species in the Trimeresurus complex, including T. borneensis, T. gramineus, T. puniceus, T. purpureomaculatus, T. stejnegeri, and Protobothrops flavoviridis. Despite the geographical isolation, T. insularis venoms from all eight islands demonstrated remarkable similarities in gel electrophoretic profiles and RP-HPLC patterns, and all populations had protein bands in the mass ranges of phosphodiesterases (PDE), l-amino acid oxidases (LAAO), P-III snake venom metalloproteinases (SVMP), serine proteases, cysteine-rich secretory proteins (CRISP), phospholipases A2 (PLA2), and C-type lectins. An exception was observed in the Lombok sample, which lacked protein bands in the mass range of serine protease and CRISP. Venomic analysis of the Sumbawa venom also identified these protein families, in addition to several proteins of lesser abundance (

Listeriolysin O (LLO) is an essential determinant of Listeria monocytogenes pathogenesis that mediates the escape of L. monocytogenes from host cell vacuoles, thereby allowing replication in the cytosol without causing appreciable cell death. As a member of the cholesterol-dependent cytolysin (CDC) family of pore-forming toxins, LLO is unique in that it is secreted by a facultative intracellular pathogen, whereas all other CDCs are produced by pathogens that are largely extracellular. Replacement of LLO with other CDCs results in strains that are extremely cytotoxic and 10,000-fold less virulent in mice. LLO has structural and regulatory features that allow it to function intracellularly without causing cell death, most of which map to a unique N-terminal region of LLO referred to as the proline, glutamic acid, serine, threonine (PEST)-like sequence. Yet, while LLO has unique properties required for its intracellular site of action, extracellular LLO, like other CDCs, affects cells in a myriad of ways. Because all CDCs form pores in cholesterol-containing membranes that lead to rapid Ca2+ influx and K+ efflux, they consequently trigger a wide range of host cell responses, including mitogen-activated protein kinase activation, histone modification, and caspase-1 activation. There is no debate that extracellular LLO, like all other CDCs, can stimulate multiple cellular activities, but the primary question we wish to address in this perspective is whether these activities contribute to L. monocytogenes pathogenesis.

Bacillus anthracis, the causative agent of anthrax, is able to exist both in environmental conditions (soil) and in the macroorganism. The manifestation of pathogenic properties of B. anthracis strains is determined by relevant plasmid composition, because the main toxin and the capsule-related virulence factors are located in bacterial plasmid. Modeling anthrax infection in vitro in macrophage culture might reveal an influence of individual B. anthracis strain characteristics on infection and development of infectious process. The aim of this study was to analyze cytokine level during infection of in vitro macrophage cell cultures with spores of anthrax microbe strains bearing varying plasmid composition. The dependence of the macrophage cell cytokine profile on the plasmid composition of B. anthracis strains was revealed while modeling anthrax infection in vitro. The presence of the toxin-producing plasmid pXO1 in anthrax microbe strains has a powerful stimulating effect on the production of macrophages J774A cell line cytokines. B. anthracis strains lacking the pXO1 plasmid virtually stimulated no production of IL-1, caused very low secretion of IL-1, IL-6, MCP-1, MIP-1, MIP-1, IL-12 (p70) and active G-CSF products. The low cytokine response of macrophage cells infected with monoplasmid strains bearing only the capsule-forming plasmid was due not only to the absence of a binary toxin, but also to disturbed regulation of capsule production associated with the absence of the atxA gene. The capsule, along with lethal and edematous toxins, belongs to the main virulence factors of B. anthracis, but strains lacking the pXO1 virulence plasmid, had its production impaired, because the main regulator of capsule synthesis is the atxA gene localized on the pXO1 plasmid being positively regulated by the acpA and acpB genes, so that strains lacking the toxin-forming plasmid, even in the presence of the encapsulation plasmid, elicit a weak cytokine response in infected cells. Diplasmid strains of B. anthracis, due to produced main virulence factors a two-component toxin and a capsule, enforce macrophages (in the experiment) to actively produce IL-1, IL-6, MCP-1, G-CSF, MIP-1; MIP-1, IL-12 (p70). Strains with moderate virulence and capable of capsulation in air virtually did not differ from highly virulent strains in terms of their effect on in vitro macrophage culture.

Made Rai Dwitya Wiradiputra,1,2 Piyatip Khuntayaporn,1,3 Krit Thirapanmethee,1,3 Mullika Traidej Chomnawang1,3 1Antimicrobial Resistance Interdisciplinary Group (AmRIG), Faculty of Pharmacy, Mahidol University, Bangkok, Thailand; 2Biopharmaceutical Sciences Program, Faculty of Pharmacy, Mahidol University, Bangkok, Thailand; 3Department of Microbiology, Faculty of Pharmacy, Mahidol University, Bangkok, ThailandCorrespondence: Mullika Traidej Chomnawang, Department of Microbiology, Faculty of Pharmacy, Mahidol University, Bangkok, 10400, Thailand, Tel +66 2 644 8692, Email mullika.tra@mahidol.ac.thAbstract: The toxin and antitoxin modules in bacteria consist of a toxin molecule that has activity to inhibit various cellular processes and its cognate antitoxin that neutralizes the toxin. This system is considered taking part in the formation of persister cells, which are a subpopulation of recalcitrant cells able to survive antimicrobial treatment without any resistance mechanisms. Importantly, persisters have been associated with long-term infections and treatment failures in healthcare settings. It is a public health concern since persisters can be involved in the evolution and dissemination of antimicrobial resistance amidst the aggravating spread of multidrug-resistant bacteria and insufficient novel antimicrobial therapy to tackle this issue. Salmonella enterica serovar Typhimurium is one of the most prevalent Salmonella serotypes in the world and is a leading cause of food-borne salmonellosis. S. Typhimurium has been known to cause persistent infection and a wealth of investigations on Salmonella persisters indicates that toxin and antitoxin modules play a role in mediating the phenotypic switch of persisters, rendering its survival ability in the presence of antimicrobial agents. In this review, we discuss findings regarding mechanisms that underly persistence in S. Typhimurium, especially the involvement of toxin and antitoxin modules.Keywords: toxin, antitoxin, antimicrobial resistance, persisters, Salmonella

Abstract Background Wasp stinging, a neglected tropical entity can have a myriad of local and systemic effects. We present a case of multi-organ injury following multiple wasp stings and a review of literature on the systemic manifestations of wasp stings. Case presentation A 48-year-old Sri Lankan male who suffered multiple wasp stings, developed an anaphylactic shock with respiratory failure, which was treated with adrenaline and mechanical ventilation. Within the next 2 days the patient developed acute fulminant hepatitis, stage III acute kidney injury, rhabdomyolysis, haemolysis and thrombocytopenia. The patient was treated in the intensive care unit with ionopressors and continuous renal replacement therapy (CRRT). Haemoadsorbant therapy was used in adjunct with CRRT. There was a gradual recovery of the organ functions over the 1st week. However, the patient succumbed to fungal sepsis on the 16th day despite treatment. We conducted a literature review to identify the various clinical manifestations of wasp stinging. Wasp venom contains enzymes, amines, peptides and other compounds. These proteins can cause type 1 hypersensitive reactions ranging from local skin irritation to anaphylactic shock. Furthermore, the toxins can cause direct organ injury or delayed hypersensitivity reactions. The commonly affected organs are the kidneys, liver, and muscles. The effect on the haematological system manifests as coagulopathy and/or cytopenia. The heart, nervous system, lungs, intestines and skin can be affected rarely. Treatment is mainly supportive. Conclusion In conclusion, wasp envenomation can result in multi-organ injury and attention should be paid in doing further research and establishing evidence-based treatment practices.

Aflatoxin-B1 (AFB1) is a common contaminant for staple foods during the storage process. Chronic exposure to AFB1 is widely known to induce the development of hepatocellular carcinoma (HCC). However, there is a lack of understanding of AFBi role in HCC mechanism. This research aims to identify protein(s) in HCC that might interact with AFB1 and to predict the pathway effected by AFB1. Analyses were performed using bioinformatics tools. SMILES notation of AFB1 was submitted into Swiss Target Prediction. Interaction among predicted proteins were analyzed by using STRING. The 3D structure of target protein was constructed by homology modeling. Reverse docking was performed, and the result was ranked based on binding affinity score. Furthermore, protein interaction network was constructed and analyzed by using Cytoscape. Results showed that three protein groups were predicted as target of AFB1, such as kinases, phosphatases, and G protein-coupled receptor with probability of 46.7%, 20%, and 6.7%, respectively. Seven proteins of kinases were strongly related to HCC, including RAF1, MAPK1, MAPK3, AKT1, EGFR, GSK3B, and mTOR. Reverse docking considered the AKT1-AFB1 as the most potential complex with the lowest affinity score -10.2 kcal.mol-1. It has hydrophobic bonds in Trp80, Val270, Tyr272, Asp292, Thr211, Leu210, Leu264, and Lys268 residues, whereas hydrogen bond in Ser205 residues. Moreover, further analysis demonstrated that interaction of AKT1-AFB1 is related to the metastasis pathway in HCC mechanism.

The Clostridium genus is composed of a large spectrum of heterogeneous bacteria. They are Gram-positive, mostly mesophilic, and anaerobic spore-forming strains. Clostridia are widely distributed in oxygen-free habitats. They are found principally in the soil and intestines of ruminants as normal flora, but also are the cause of several infections in humans. The infections produced by important species in humans include botulism, tetanus, pseudomembranous colitis, antibiotics-associated diarrhea, and gas gangrene. Immunization with toxoid or bacterin-toxoid or genetically modified or other vaccines is a protective way against clostridial infection. Several experimental or commercial vaccines have been developed worldwide. Although conventional vaccines including toxoid vaccines are very important, the new generation of vaccines is an effective alternative to conventional vaccines. Recent advances have made it possible for new vaccines to increase immunogenicity. This review discusses briefly the important species of clostridia in humans, their toxins structure, and vaccine development and usage throughout the world.

Abstract Background Multi-omics technology provides a good tool to analyze the protein toxin composition and search for the potential pathogenic factors of Solenopsis invicta, under the great harm of the accelerated invasion in southern China. Methods Species collection, functional annotation, toxin screening, and 3D modeling construction of three interested toxins were performed based on the successfully constructed transcriptome and proteome of S. invicta. Results A total of 33,231 unigenes and 721 proteins were obtained from the constructed transcriptome and proteome, of which 9,842 (29.62%) and 4,844 (14.58%) unigenes, as well as 469 (65.05%) and 71 (99.45%) proteins were annotated against the databases of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes, respectively. After comparing with the uniprot toxin database, a total of 316 unigenes and 47 proteins (calglandulin, venom allergen 3, and venom prothrombin activator hopsarin-D, etc.) were successfully screened. Conclusions The update of annotations at the transcriptome and proteome levels presents a progression in the comprehension of S. invicta in China. We also provide a protein toxin list that could be used for further exploration of toxicity as well as its antagonistic strategy by S. invicta.

Clostridioides difficile is a principal cause of hospital-acquired gastrointestinal infections, with sporulation and toxin production being key determinants in the disease pathogenesis. Although infections have been escalating and the complications can be life-threatening, the narrow pipeline of approved therapeutics has not witnessed an equivalent surge. With the unfolding of worrisome mutations and antimicrobial resistance, attention has been drawn to either discovering new therapeutics, or even better, repurposing already available ones. Consequently, this study was undertaken to assess the anti-clostridial activity of auranofin, an anti-rheumatic FDA-approved therapeutic; and baicalin, a natural flavone glycoside with reported anti-microbial potential. In comparison with vancomycin, the in vitro efficacy of auranofin and baicalin was tested against hypervirulent C. difficile (BAA-1870TM). Broth suspensions were prepared with and without the three agents and anaerobically incubated. At 24- and 48-hours post-incubation, serial dilutions were prepared and inoculated onto agar plates. Viable cell counts and viable spore counts were then quantified. Meanwhile, toxin production was assessed via ELISA. At a concentration as low as 3 μg/mL, auranofin demonstrated a potent anti-clostridial activity. Both auranofin and baicalin exhibited a remarkable reduction in C. difficile viable cell counts (P-value 0.03 for each) and spore counts (P-values 0.023 and 0.045 respectively). While auranofin and baicalin proved to be non-inferior to vancomycin as inhibitors of C. difficile growth, both drugs proved to be superior to vancomycin in decreasing the spore counts 48-hours post inoculation. Additionally, auranofin markedly reduced C. difficile toxin production (P-value 0.021); a feature that was deficient in both baicalin and vancomycin. To enrich the currently limited repertoire of anti-clostridial drugs, further research is encouraging to compare between the in vivo efficacy of auranofin and that of baicalin. Both agents represent promising approaches that could address the unfulfilled needs in controlling C. difficile infection.

Bacillus cereus is a foodborne pathogen causing food safety issues due to the formation of difficult to eliminate spores and biofilms. The objective of this study was to investigate the occurrence of B. cereus (conducted as part of monitoring in 2017-2018) and the presence of a toxin gene in strains isolated from retail products (pastries/cakes; vegetables, spices, delicatessen products) in Poland, and to determine the susceptibility of these microorganisms to different antimicrobial agents. A total of 267 B. cereus isolates from food products were examined, of which 95.51% were found positive for the presence of at least one toxin gene, with the highest frequency of the nhe gene (91.39%). The hbl and cytK genes were detected in 53.56% and 44.19% of B. cereus strains, respectively. The lowest frequency was found for the ces gene (2.62%). The susceptibility of B. cereus isolates to 16 antimicrobials was investigated. Ampicillin and penicillin resistance was the most common resistance phenotype and was identified in 100% of the B. cereus isolates. In addition, the tested isolates exhibited resistance to: amoxicillin-clavulanic acid (96.25%), cephalothin (67.79%), ceftriaxone (64.42%), rifampicin (46.82%), trimethoprim-sulfamethoxazole (5.62%), quinupristin/dalfopristin (4.87%), chloramphenicol (3.75%), clindamycin (2.62%), teicoplanin (1.87%), erythromycin (1.87%), ciprofloxacin (0.75%), imipenem (0.75%), tetracycline (0.37%), and gentamicin (0.37%). The study results contribute to characterizing the diversity of B. cereus isolated from various food products in Poland and their impact on food safety and public health. This study delivers practical information on antibiotic resistance and the frequency of toxin genes among strains isolated from food.

A recent article in the Journal of Infectious Diseases explores the potential use of loratadine, an over-the-counter antihistamine, as a treatment for Clostridioides difficile infection (CDI). The authors conducted preclinical studies using murine and hamster models, as well as human cell lines and colonic explants, to demonstrate the anti-inflammatory effects of loratadine in CDI. The findings suggest that loratadine may attenuate toxin B-mediated colonic inflammation, independent of histamine receptors but dependent on SRC and NFкB. However, the authors note that the optimal dosage of loratadine for CDI treatment is unclear, and further research is needed to assess its safety and efficacy in humans. [Extracted from the article]

IntroductionTrace elements play a key role in dental tissue development, as dental hard tissues accumulate both essential and toxic trace elements during mineralization. Characterization of the spatial accumulation pattern of trace elements may provide insight into exposure to toxic elements over time and to the nature of disease processes affecting the hard dental tissues. Here, we present the first report of the use of laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) to map the microspatial distribution of multiple trace elements, essential and toxic, across feline dental hard tissues.MethodsEleven teeth were extracted from 8 cats. Nine teeth were from 7 cats diagnosed with idiopathic tooth resorption on intraoral radiographs prior to extraction. Two teeth were included from a cadaver that had no signs of tooth resorption on intraoral radiographs. The normal dental tissue was analyzed from each sample using LA-ICP-MS to map the microspatial distribution of essential and toxic trace elements across feline enamel, dentin, and cementum.ResultsResults showed a higher accumulation of barium and strontium in coronal dentin as compared to root dentin. The timing of the accumulation mirrors nursing timelines seen in teeth from human and non-human primates, consistent with barium and strontium being sourced from maternal milk. Results also showed a higher uptake of lead in the coronal dentin, suggesting this lead exposure was likely passed from mother to offspring.DiscussionThis work characterizes a baseline for elemental distribution in feline teeth linked to early life exposure to toxic elements such as lead and provides a framework for future studies investigating long-term environmental exposures to trace elements, essential and toxic, and their involvement in feline systemic and dental diseases.

Toxin-antitoxin (TA) systems are ubiquitous in bacteria and archaea. Most are composed of two neighboring genetic elements, a stable toxin capable of inhibiting crucial cellular processes, including replication, transcription, translation, cell division and membrane integrity, and an unstable antitoxin to counteract the toxicity of the toxin. Many new discoveries regarding the biochemical properties of the toxin and antitoxin components have been made since the first TA system was reported nearly four decades ago. The physiological functions of TA systems have been hotly debated in recent decades, and it is now increasingly clear that TA systems are important immune systems in prokaryotes. In addition to being involved in biofilm formation and persister cell formation, these modules are antiphage defense systems and provide host defenses against various phage infections via abortive infection. In this review, we explore the potential applications of TA systems based on the recent progress made in elucidating TA functions. We first describe the most recent classification of TA systems and then introduce the biochemical functions of toxins and antitoxins, respectively. Finally, we primarily focus on and devote considerable space to the application of TA complexes in synthetic biology.

Intestinal bile acids play an essential role in the Clostridioides difficile lifecycle having been shown in vitro to modulate various aspects of pathogenesis, including spore germination, vegetative growth, and more recently the action of the primary virulence determinant, TcdB. Here, we investigated whether physiological levels of the total pool of intestinal bile acids in mice and humans protect against TcdB action. Small molecules extracted from the lumenal contents of the small intestine, cecum, colon, and feces were found to inhibit TcdB in accordance with the differential amounts of total bile acids in each compartment. Extracts from antibiotic-treated and germ-free mice, despite harboring dramatically altered bile acid profiles, unexpectedly also prevented TcdB-induced cell rounding to similar extents. We show that protection, however, is surmountable and can be overcome at higher doses of TcdB--typical to those seen during severe C. difficile infection--suggesting that the protective properties of intestinal bile acids are operant primarily under low to moderate toxin levels. Taken together, these findings demonstrate a role for intestinal bile acids in attenuating virulence, provide insights into asymptomatic carriage of toxigenic C. difficile, and inform strategies to manipulate bile acid levels for therapeutic benefit. [ABSTRACT FROM AUTHOR]

Botulinum neurotoxins (BoNTs) are important therapeutic agents. The in vivo median lethal dose (LD50) assay has been commonly used to measure the potency of BoNT commercial preparations. As an alternative, we developed cell-based assays for abobotulinumtoxinA in both powder (Dysport®, Azzalure®) and liquid (Alluzience®) formulations using the in vitro BoCell® system. The assays demonstrated linearity over 50–130% of the expected relative potency, with a correlation coefficient of 0.98. Mean recoveries of 90–108% of the stated potency were observed over this range. The coefficients of variation for powder and liquid formulations, respectively, were 3.6% and 4.0% for repeatability and 8.3% and 5.0% for intermediate precision. A statistically powered comparability assessment of the BoCell® and LD50 assays was performed. Equivalence was demonstrated between the assays for the liquid formulation at release and end of shelf life using a paired equivalence test with predefined equivalence margins. For the powder formulation, the assays were also shown to be equivalent for release samples and when determining loss of potency following thermal degradation. The BoCell® assay was approved for establishing the potency of abobotulinumtoxinA for both powder and liquid formulations in Europe and for the powder formulation only in the USA. [ABSTRACT FROM AUTHOR]

Clostridioides difficile infection (CDI) presents a major public health threat by causing frequently recurrent, life-threatening cases of diarrhea and intestinal inflammation. The ability of C. difficile to express antibiotic resistance and to form long-lasting spores makes the pathogen particularly challenging to eradicate from healthcare settings, raising the need for preventative measures to curb the spread of CDI. Since C. difficile utilizes the fecal–oral route of transmission, a mucosal vaccine could be a particularly promising strategy by generating strong IgA and IgG responses that prevent colonization and disease. This mini-review summarizes the progress toward mucosal vaccines against C. difficile toxins, cell–surface components, and spore proteins. By assessing the strengths and weaknesses of particular antigens, as well as methods for delivering these antigens to mucosal sites, we hope to guide future research toward an effective mucosal vaccine against CDI. [ABSTRACT FROM AUTHOR]

Context: Recent studies have shown that the sensitivity of wild house mice to zinc phosphide (ZnP) in Australia is significantly lower than previously assumed, which may account for the reported variability in efficacy of ZnP baits used for broadacre control of house mice in grain-growing regions. Under laboratory conditions ZnP-coated grains with a new higher dose (50 g ZnP/kg grain) were readily consumed but the efficacy of using grains with this higher dose under natural field conditions has not been tested. Aims: To test whether the newly derived ZnP50 (50 g ZnP/kg grain) was more effective under field conditions than the currently registered ZnP25 (25 g ZnP/kg grain) in reducing populations of house mice during a mouse population irruption. Methods: We used a before–after-control–impact (BACI) design to assess changes in mouse population size under different baiting treatments in a replicated field trial. We assessed changes in mouse abundance in recently sown paddocks with either ZnP50 (n = 3) or ZnP25 (n = 3) compared with unbaited control sites (n = 3). Key results: Baiting with ZnP50 led to a median reduction in mouse numbers of >85%. Our modelling showed that under similar circumstances, using the ZnP50 formulation should deliver >80% reduction in population size most (>90%) of the time. In contrast, the current registered bait (ZnP25) achieved approximately 70% reduction in population size, but with more variable results. We would be confident of getting an 80% reduction in population size only 20% of the time by using the currently registered ZnP25 bait under similar field conditions. Conclusions: Consistent with laboratory studies, this study demonstrated the higher probability of achieving a consistently high kill rate under field conditions with the new ZnP50 bait compared with the currently registered formulation (ZnP25). Implications: By using the new ZnP50 bait, farmers are far more likely to get good kill rates, thereby reducing the need for repeated baiting (which is costly and generally ineffective at protecting newly sown crops). Using the new bait should result in lower control costs for farmers and fewer toxic grains being spread to control mice. Recent studies have shown that the tolerance of wild house mice to zinc phosphide (ZnP) in Australia is significantly higher than previously assumed, which may account for the reported variability in efficacy of ZnP baits used for house mice control in grain-growing regions. We used a BACI design to show that a newly derived ZnP50 bait is more effective under field conditions than the is currently registered ZnP bait in reducing populations of house mice during a mouse outbreak. Photograph by Nikki Van de Weyer. [ABSTRACT FROM AUTHOR]

Tenuazonic acid (TeA) is synthesized by phytopathogenic and opportunistic fungi and is detected in a broad range of foods. This natural compound is of interest in terms of toxicity to animals, but its mechanisms of action on insects are poorly understood. We administered TeA orally at different concentrations (0.2–5.0 mg/[gram of a growth medium]) to the model insect Galleria mellonella, with subsequent estimation of physiological, histological, and immunological parameters in different tissues (midgut, fat body, and hemolymph). Susceptibility of the TeA-treated larvae to pathogenic microorganisms Beauveria bassiana and Bacillus thuringiensis was also analyzed. The feeding of TeA to the larvae led to a substation delay of larval growth, apoptosis-like changes in midgut cells, and an increase in midgut bacterial load. A decrease in activities of detoxification enzymes and downregulation of genes Nox, lysozyme, and cecropin in the midgut and/or hemocoel tissues were detected. By contrast, genes gloverin, gallerimycin, and galiomycin and phenoloxidase activity proved to be upregulated in the studied tissues. Hemocyte density did not change under the influence of TeA. TeA administration increased susceptibility of the larvae to B. bassiana but diminished their susceptibility to B. thuringiensis. The results indicate that TeA disturbs wax moth gut physiology and immunity and also exerts a systemic action on this insect. Mechanisms underlying the observed changes in wax moth susceptibility to the pathogens are discussed. [ABSTRACT FROM AUTHOR]

Some people living with HIV present painful sensory neuropathy (HIV-SN) that is pharmacoresistant, sex-associated, and a major source of morbidity. Since the specific mechanisms underlying HIV-SN are not well understood, the aim of our study was to characterize a novel model of painful HIV-SN by combining the HIV-1 gp120 protein and the antiretroviral stavudine (d4T) in mice and to investigate the pronociceptive role of the family 2 voltage-gated calcium channel (VGCC) α1 subunit (Cav2.X channels) in such a model. HIV-SN was induced in male and female C57BL/6 mice by administration of gp120 and/or d4T and detected by a battery of behavior tests and by immunohistochemistry. The role of Cav2.X channels was assessed by the treatment with selective blockers and agonists as well as by mRNA detection. Repeated administration with gp120 and/or d4T produced long-lasting touch-evoked painful-like behaviors (starting at 6 days, reaching a maximum on day 13, and lasting up to 28 days after treatment started), with a greater intensity in female mice treated with the combination of gp120 + d4T. Moreover, gp120 + d4T treatment reduced the intraepidermal nerve fibers and well-being of female mice, without altering other behaviors. Mechanistically, gp120 + d4T treatment induced Cav2.1, 2.2, and 2.3 transcriptional increases in the dorsal root ganglion and the Cav2.X agonist-induced nociception. Accordingly, intrathecal selective Cav2.2 blockade presented longer and better efficacy in reversing the hyperalgesia induced by gp120 + d4T treatment compared with Cav2.1 or Cav2.3, but also presented the worst safety (inducing side effects at effective doses). We conclude that the family 2 calcium channels (Cav2.X) exert a critical pronociceptive role in a novel mouse model of HIV-SN. [ABSTRACT FROM AUTHOR]

Malachite green (MG), an antibiotic with antifungal activity, is illegally used in aquaculture. Given that this chemical is teratogenic and mutagenic, abstinence from intake seems to be a need for public safety. The goal of this systematic review and meta-analysis was to determine the global contamination of fishes by MG and its reduced metabolite, leucomalachite green (LMG), in a number of marine and farmed fish species. For literature published prior to January 2022, several databases (Web of Science, PubMed, and Scopus) were investigated. In total, 20 publications (10 countries, 724 samples) achieved the criteria for inclusion. The overall average MG and LMG concentrations were 0.48 (95% CI: 0.47, 0.49 µg kg−1) and 0.59 (95% CI: 0.39, 0.79 µg/kg−1), respectively. Eel (M. albus) 15.50 (95% CI: (14.39, 45.39 µg kg−1) and eel (A. anguilla) 4.46 (95% CI: 1.23, 7.69 µg kg−1) had the greatest contamination of MG and LMG, according to the effect size, respectively. Warm-water fish had a concentration of 2.591 (95% CI: 2.25, 2.93 µg kg−1) while cold-water fish had a concentration of 1.55 (95% CI: 0.25, 2.84 µg kg−1). Fish containing medium-fat level of 1.86 (95% CI: 1.27, 2.44 µg kg−1) and high-fat content of 1.10 (95% CI: 0.93, 1.26 µg kg−1) had accumulate MG and LMG in their tissues, respectively. As a result, total MG observed in countries including China, Iran, and the Netherlands was higher than authorized (2 µg kg−1). The toxicity of MG and LMG demands more monitoring, especially in countries where these chemicals' residues are significant. [ABSTRACT FROM AUTHOR]

Food, like air and water, is one of the essential elements for human life. High-quality nutrition rich in protein, fat, carbohydrates, vitamins, and minerals is the basis of the body"s development, immune system, intellectual and reproductive activity. However, in addition to the amount and chemical composition of food, its microbiological indicators should also be under strict control. That is, the fali of various disease-causing agents, toxic chemical and biological substances into food products can cause mass iliness. Most acute intestinal infections - shigellosis, salmonellosis, typhoid-paratyphoid diseases, hepatitis (A) and diarrhea viruses are transmitted to humans through food products. Food products are mainly contaminated with microbes and their toxins during production, processing, canning and storage. Pathogenic and conditionaliy pathogenic, endoand exotoxin-containing bacteria, some viruses, which are widespread in nature, multipiy rapidiy in protein and creamy food products, and often cause food toxicoinfections (FTİ) that occur in groups. Although they are short-term and mild, in the initia period, they can hardiy be distingutshed from terrible somatic, surgical, gynecological diseases, poisoning mushrooms, chemicals, and acute intestinal infections. We denied it in 18 (24%) of 76 patients sent to the hospital with a diagnosis of FTİ. Botulism, the dreaded form of FTJ has a mortality rate of 20-30%. However, if FTİs are detected early, their successful treatment is possible. [ABSTRACT FROM AUTHOR]

Post-weaning diarrhoea and enterotoxaemia caused by Escherichia coli are serious threats in the pig (Sus scrofa domesticus) livestock industry and are responsible for economic losses related to mortality, morbidity and stunted growth. The aim of this study was to evaluate the effect of an engineered tobacco seeds-based edible vaccine in O138 Escherichia coli-challenged piglets throughout a multidisciplinary approach. Thirty-six weaned piglets were enrolled and randomly divided into two experimental groups, a control (C; n = 18) group and a tobacco edible vaccination group (T, n = 18), for 29 days of trial. At days 0, 1, 2, 5 and 14, piglets of the T group were fed with 10 g of the engineered tobacco seeds line expressing F18 and VT2eB antigens, while the C group received wild-type tobacco seeds. After 20 days, 6 piglets/group were orally challenged with the Escherichia coli O138 strain (creating four subgroups: UC = unchallenged control, CC = challenged control, UT = unchallenged tobacco, CT = challenged tobacco) and fed with a high protein diet for 3 consecutive days. Zootechnical, clinical, microbiological, histological and immunological parameters were assayed and registered during the 9 days of post-challenge follow up. At 29 days post-challenge, the CT group displayed a lower average of the sum of clinical scores compared to the CC group (p < 0.05), while the CC group showed a higher average sum of the faecal score (diarrhoea) (p < 0.05) than the CT group. A decreased number of days of shedding of the pathogenic strain was observed in the CT compared to the CC group (p < 0.05). Specific anti-F18 IgA molecules were significantly higher in the CT group compared to the CC group's faecal samples during the post-challenge period (p < 0.01). In conclusion, edible vaccination with engineered tobacco seeds showed a protective effect on clinical symptoms and diarrhoea incidence during the post-challenge period, characterized by a limited time of pathogenic strain shedding in faeces. [ABSTRACT FROM AUTHOR]

We have purified ledodin, a cytotoxic 22‐kDa protein from shiitake mushroom (Lentinula edodes) consisting of a 197 amino acid chain. Ledodin possessed N‐glycosylase activity on the sarcin‐ricin loop of mammalian 28S rRNA and inhibited protein synthesis. However, it was not active against insect, fungal, and bacterial ribosomes. In vitro and in silico studies suggested that ledodin exhibits a catalytic mechanism like that of DNA glycosylases and plant ribosome‐inactivating proteins. Moreover, the sequence and structure of ledodin was not related to any protein of known function, although ledodin‐homologous sequences were found in the genome of several species of fungi, some edible, belonging to different orders of the class Agaricomycetes. Therefore, ledodin could be the first of a new family of enzymes widely distributed among this class of basidiomycetes. The interest of these proteins lies both, in the fact that they can be a toxic agent of some edible mushrooms and in their application in medicine and biotechnology. [ABSTRACT FROM AUTHOR]

Crystal structures of camelid heavy-chain antibody variable domains (VHHs) bound to fragments of the combined repetitive oligopeptides domain of Clostridioides difficile toxin A (TcdA) reveal that the C-terminus of VHH A20 was located 30 Å away from the N-terminus of VHH A26. Based on this observation, we generated a biparatopic fusion protein with A20 at the N-terminus, followed by a (GS)6 linker and A26 at the C-terminus. This A20-A26 fusion protein shows an improvement in binding affinity and a dramatic increase in TcdA neutralization potency (>330-fold [IC50]; =2,700-fold [IC99]) when compared to the unfused A20 and A26 VHHs. A20-A26 also shows much higher binding affinity and neutralization potency when compared to a series of control antibody constructs that include fusions of two A20 VHHs, fusions of two A26 VHHs, a biparatopic fusion with A26 at the N-terminus and A20 at the C-terminus (A26-A20), and actoxumab. In particular, A20-A26 displays a 310-fold (IC50) to 29,000-fold (IC99) higher neutralization potency than A26-A20. Size-exclusion chromatography-multiangle light scattering (SEC-MALS) analyses further reveal that A20-A26 binds to TcdA with 1:1 stoichiometry and simultaneous engagement of both A20 and A26 epitopes as expected based on the biparatopic design inspired by the crystal structures of TcdA bound to A20 and A26. In contrast, the control constructs show varied and heterogeneous binding modes. These results highlight the importance of molecular geometric constraints in generating highly potent antibody-based reagents capable of exploiting the simultaneous binding of more than one paratope to an antigen. [ABSTRACT FROM AUTHOR]

Phylum Cnidaria represents a unique group among venomous taxa, with its delivery system organised as individual organelles, known as nematocysts, heterogeneously distributed across morphological structures rather than packaged as a specialised organ. Acontia are packed with large nematocysts that are expelled from sea anemones during aggressive encounters with predatory species and are found in a limited number of species in the superfamily Metridioidea. Little is known about this specialised structure other than the commonly accepted hypothesis of its role in defence and a rudimentary understanding of its toxin content and activity. This study utilised previously published transcriptomic data and new proteomic analyses to expand this knowledge by identifying the venom profile of acontia in Calliactis polypus. Using mass spectrometry, we found limited toxin diversity in the proteome of acontia, with an abundance of a sodium channel toxin type I, and a novel toxin with two ShK-like domains. Additionally, genomic evidence suggests that the proposed novel toxin is ubiquitous across sea anemone lineages. Overall, the venom profile of acontia in Calliactis polypus and the novel toxin identified here provide the basis for future research to define the function of acontial toxins in sea anemones. [ABSTRACT FROM AUTHOR]

Dysbiosis of the gut microbiota, caused by antibiotics, plays a key role in the establishment of Clostridioides difficile CD). Toxin-producing strains are involved in the pathogenesis of Clostridioides difficile infection (CDI), one of the most common hospital-acquired infections. We cultured a total of 84 C. difficile isolates from stool samples of patients hospitalized at Louis Pasteur University Hospital in Kosice, Slovakia, that were suspected of CDI and further characterized by molecular methods. The presence of genes encoding toxin A, toxin B, and binary toxin was assessed by toxin-specific PCR. CD ribotypes were detected using capillary-based electrophoresis ribotyping. A total of 96.4% of CD isolates carried genes encoding toxins A and B, and 54.8% of them were positive for the binary toxin. PCR ribotyping showed the presence of three major ribotypes: RT 176 (n = 40, 47.6%); RT 001 (n = 23, 27.4%); and RT 014 (n = 7, 8.3%). Ribotype 176 predominated among clinical CD isolates in our hospital. The proportion of RT 176 and RT 001 in four hospital departments with the highest incidence of CDI cases was very specific, pointing to local CDI outbreaks. Based on our data, previous use of antibiotics represents a significant risk factor for the development of CDI in patients over 65 years of age. [ABSTRACT FROM AUTHOR]

Bacterial secretion systems are nanomolecular complexes that release a diverse set of virulence factors/or proteins into its surrounding or translocate to their target host cells. Among these systems, type VI secretion system 'T6SS' is a recently discovered molecular secretion system which is widely distributed in Gram-negative (−ve) bacteria, and shares structural similarity with the puncturing device of bacteriophages. The presence of T6SS is an advantage to many bacteria as it delivers toxins to its neighbour pathogens for competitive survival, and also translocates protein effectors to the host cells, leading to disruption of lipid membranes, cell walls, and cytoskeletons etc. Recent studies have characterized both anti-prokaryotic and anti-eukaryotic effectors, where T6SS is involved in diverse cellular functions including favouring colonization, enhancing the survival, adhesive modifications, internalization, and evasion of the immune system. With the evolution of advanced genomics and proteomics tools, there has been an increase in the number of characterized T6SS effector arsenals and also more clear information about the adaptive significance of this complex system. The functions of T6SS are generally regulated at the transcription, post-transcription and post-translational levels through diverse mechanisms. In the present review, we aimed to provide information about the distribution of T6SS in diverse bacteria, any structural similarity/or dissimilarity, effectors proteins, functional significance, and regulatory mechanisms. We also tried to provide information about the diverse roles played by T6SS in its natural environments and hosts, and further any changes in the microbiome. [ABSTRACT FROM AUTHOR]

The air-film reinforced concrete spherical silo (hereinafter referred to as the air-film spherical silo) is a new type of storage structure. In order to explore the effect of the air-film spherical silo, the paper takes the first air-film spherical silo in China as a research object, and takes the wheat grain pile as the research target. The grain temperature quality, quality index, toxin and other indicators were tested and analyzed. The results showed that, during the grain storage process, the wheat grain heap in the air film spherical silo had no pests, no mildew, and all quality indicators met the suitable storage standard. The grain situation was stable and the quality was good, the air-film spherical silo is a new type of warehouse suitable for grain quality and safe storage.

Bacillus cereus is a ubiquitous foodborne pathogen commonly found in various foods. Its ability to form spores, biofilms and diarrhoeal and/or emetic toxins further exacerbates the risk of food poisoning. Violacein is a tryptophan derivative with excellent antibacterial activity. However, the knowledge on the antibacterial action of violacein against B. cereus was lacking, and thus this study aimed to investigate the antibacterial activity and mechanism. The antibacterial results demonstrated that minimum inhibitory concentration and minimum bactericidal concentration of violacein were 3.125 mg/L and 12.50 mg/L, respectively. Violacein could effectively inhibit planktonic growth, spore germination and biofilm formation of B. cereus (P < 0.001). Meanwhile, violacein significantly downregulated the expression of toxin genes, including nheA (P < 0.05), nheB (P < 0.001), bceT (P < 0.01), cytK (P < 0.001), hblC (P < 0.001) and hblD (P < 0.001). Results of extracellular alkaline phosphatase, nucleotide and protein leakage assays and scanning and transmission electron microscopy observation tests showed violacein destroyed cell walls and membranes of B. cereus. In addition, 6.25 mg/kg of violacein could significantly inhibit B. cereus in grass carp fillets (P < 0.05). These results demonstrate that violacein has great potential as an effective natural antimicrobial preservative to control food contamination and poisoning events caused by B. cereus. • Antibacterial, antispore and antibiofilm activities of violacein on B. cereus were reported. • Violacein could inhibit the virulence factors expression of B. cereus. • Violacein showed antibacterial activity against B. cereus by multiple mechanisms. • Violacein exhibited good application effect in the fish fillet preservation. [ABSTRACT FROM AUTHOR]

Barley leaf stripe is an important disease caused by Pyenophora graminea that affects barley yields in the world. Ascorbic acid (AsA) interacts with key elements of a complex network orchestrating plant defense mechanisms, thereby influencing the outcome of plant-pathogen interaction. Myo-inositol oxygenase (MIOX) is a pivotal enzyme involved in plants development and environmental stimuli. However, MIOX has described functions in plants but has not been characterized in fungi. In this study, we characterized the Pgmiox gene in P. graminea pathogenesis through annotated on the metabolic pathway of ascorbic acid aldehyde. Our analysis suggested that the Pgmiox protein had a typical conserved MIOX domain. Multiple alignment analysis indicated that the P. graminea MIOX orthologue clustered with MIOX proteins of Pyrenophora species. RNA interference successfully reduced transcript abundance of Pgmiox in six transformant lines compared to wild type, and the transformants were further less virulent on the host plant barley. Transformants of Pgmiox had significant reductions in vegetative growth and pathogenicity, which had increased resistance to tebuconazole and carbendazim. In addition, Pgmiox is associated with ionic, drought, osmotic, oxidative, and heavy metal stress tolerance in P. graminea. In conclusion, our findings reveal that Pgmiox may be widely utilized by fungi to enhance pathogenesis and holds significant potential for the development of durable P. graminea resistance through genetic modifications. • We efficaciously obtained P. graminea transformants harboring silenced types of Pgmiox. • Pgmiox is required for response to osmosensitivity and fungicide tolerance of P. graminea. • Pgmiox is not only related to cell wall integrity, but also necessary for the pathogenicity in P. graminea. [ABSTRACT FROM AUTHOR]

Abstract Different gastrointestinal pathogens cause diarrhea which is a very common problem in children aged under 5 years. Among bacterial pathogens, Shigella is one of the main causes of diarrhea among children, and it accounts for approximately 11% of all deaths among children aged under 5 years. The case-fatality rates for Shigella among the infants and children aged 1 to 4 years are 13.9% and 9.4%, respectively. Shigella uses unique effector proteins to modulate intracellular pathways. Shigella cannot invade epithelial cells on the apical site; therefore, it needs to pass epithelium through other cells rather than the epithelial cell. After passing epithelium, macrophage swallows Shigella, and the latter should prepare itself to exhibit at least two types of responses: (I) escaping phagocyte and (II) mediating invasion of and injury to the recurrent PMN. The presence of PMN and invitation to a greater degree resulted in gut membrane injuries and greater bacterial penetration. Infiltration of Shigella to the basolateral space mediates (A) cell attachment, (B) cell entry, (C) evasion of autophagy recognition, (D) vacuole formation and and vacuole rapture, (E) intracellular life, (F) Shiga toxin, and (G) immune response. In this review, an attempt is made to explain the role of each factor in Shigella infection.

Abstract Background Emerging pathogenic bacteria are an increasing threat to public health. Two recently described species of the genus Aliarcobacter, A. faecis and A. lanthieri, isolated from human or livestock feces, are closely related to Aliarcobacter zoonotic pathogens (A. cryaerophilus, A. skirrowii, and A. butzleri). In this study, comparative genomics analysis was carried out to examine the virulence-related, including virulence, antibiotic, and toxin (VAT) factors in the reference strains of A. faecis and A. lanthieri that may enable them to become potentially opportunistic zoonotic pathogens. Results Our results showed that the genomes of the reference strains of both species have flagella genes (flaA, flaB, flgG, flhA, flhB, fliI, fliP, motA and cheY1) as motility and export apparatus, as well as genes encoding the Twin-arginine translocation (Tat) (tatA, tatB and tatC), type II (pulE and pulF) and III (fliF, fliN and ylqH) secretory pathways, allowing them to secrete proteins into the periplasm and host cells. Invasion and immune evasion genes (ciaB, iamA, mviN, pldA, irgA and fur2) are found in both species, while adherence genes (cadF and cj1349) are only found in A. lanthieri. Acid (clpB), heat (clpA and clpB), osmotic (mviN), and low-iron (irgA and fur2) stress resistance genes were observed in both species, although urease genes were not found in them. In addition, arcB, gyrA and gyrB were found in both species, mutations of which may mediate the resistance to quaternary ammonium compounds (QACs). Furthermore, 11 VAT genes including six virulence (cadF, ciaB, irgA, mviN, pldA, and tlyA), two antibiotic resistance [tet(O) and tet(W)] and three cytolethal distending toxin (cdtA, cdtB, and cdtC) genes were validated with the PCR assays. A. lanthieri tested positive for all 11 VAT genes. By contrast, A. faecis showed positive for ten genes except for cdtB because no PCR assay for this gene was available for this species. Conclusions The identification of the virulence, antibiotic-resistance, and toxin genes in the genomes of A. faecis and A. lanthieri reference strains through comparative genomics analysis and PCR assays highlighted the potential zoonotic pathogenicity of these two species. However, it is necessary to extend this study to include more clinical and environmental strains to explore inter-species and strain-level genetic variations in virulence-related genes and assess their potential to be opportunistic pathogens for animals and humans.

Clostridioides difficile causes a large proportion of nosocomial colon infections by producing toxins TcdA and TcdB as key virulence factors. TcdA and TcdB have analogous domain structures with a receptor-binding domain containing C-terminal combined repetitive oligopeptides (CROPs), an attractive target for the development of therapeutic antibodies. Here, we identify and characterize two potent neutralizing single-domain camelid anti-CROPsA antibodies, C4.2 and H5.2, with distinct mechanisms of action. Peptide mapping, high-resolution crystal structures and site-directed mutagenesis revealed that C4.2 and H5.2 nanobodies target the same C-terminal epitope centered on a 2667QTIN2670 motif, yet utilize different paratopes. Only for C4.2 is the complex geometry compatible with multisite binding using QTIN-like repeats throughout the CROPsA domain, as supported by Western blotting, ELISA, and SEC-MALS analysis. H5.2 binding is stronger and more selective for the C-terminal epitope than C4.2, although both nanobodies are sufficient to neutralize TcdA individually. The described epitope does not overlap with previously described epitopes of anti-CROPs antibodies and provides new modalities for disease treatment and diagnostics. • Nanobodies against the CROPs domain of C.difficile toxin A isolated (C4.2 and H5.2). • Both nanobodies exhibit toxin A-neutralizing activity in Vero E6 cells. • Crystal structures reveal antibody:toxin A interfaces at high resolution. • C4.2 and H5.2 target a previously unreported epitope centered on a 2667QTIN2670 motif. • In contrast to H5.2, C4.2 binds several QTIN-like motifs within the CROPs domain. [ABSTRACT FROM AUTHOR]

Spider venom is a natural source of diverse biomolecules, but due to technical limitations, only a small fraction has been studied. With the advancement of omics technologies, research on spider venom has broadened, greatly promoting systematic studies of spider venom. Agelena limbata is a common spider found in vegetation, known for constructing funnel-shaped webs, and feeding on insects such as Diptera and Homoptera. However, due to its small size and the difficulty in obtaining venom, the composition of Agelena limbata venom has never been studied. In this study, a transcriptomics approach was used to analyze the toxin components in the venom of Agelena limbata , resulting in the identification of 28 novel toxin-like sequences and 24 peptidases. Based on sequence similarity and differences in cysteine motifs, the 28-novel toxin-like sequences were classified into 10 superfamilies. According to the results annotated in the database, the 24 peptidases were divided into six distinct families, with the serine protease family being the most common. A phylogenetic tree was constructed using the toxin-like sequences of Agelena limbata along with Psechrus triangulus and Hippasa lycosina. An analysis of the structural domains and motifs of Agelena limbata was also conducted. The results indicated that Agelena limbata is more distantly related to the other two species of funnel-web spiders, and that the toxin superfamily IX has a unique function compared to the other superfamilies. This study reveals the components of the Agelena limbata venom, deepening our understanding of it, and through bioinformatics analysis, has identified unique functions of the toxin superfamilies, providing a scientific basis for the development of bioactive drugs in the future. [Display omitted] • First exploration of the venom of the Agelena limbata • Twenty-eight toxin sequences and 24 enzymes were identified from the venom glands of agelena limbata. • Based on the arrangement of cysteine and sequence similarity, toxins have been categorized into 10 superfamilies. • Concurrently, according to the annotation results, enzymes have been divided into 6 families. [ABSTRACT FROM AUTHOR]

Viper venom phospholipase A2 enzymes (vvPLA2s) and phospholipase A2-like (PLA2-like) proteins are two of the principal toxins in viper venom that are responsible for the severe myotoxic and neurotoxic effects caused by snakebite envenoming, among other pathologies. As snakebite envenoming is the deadliest neglected tropical disease, a complete understanding of these proteins’ properties and their mechanisms of action is urgently needed. Therefore, we created a database comprising information on the holo-form, cofactor-bound 3D structure of 217 vvPLA2 and PLA2-like proteins in their physiologic environment, as well as 79 membrane-bound viper species from 24 genera, which we have made available to the scientific community to accelerate the development of new anti-snakebite drugs. In addition, the analysis of the sequenced, 3D structure of the database proteins reveals essential aspects of the anatomy of the proteins, their toxicity mechanisms, and the conserved binding site areas that may anchor universal interspecific inhibitors. Moreover, it pinpoints hypotheses for the molecular origin of the myotoxicity of the PLA2-like proteins. Altogether, this study provides an understanding of the diversity of these toxins and how they are conserved, and it indicates how to develop broad, interspecies, efficient small-molecule inhibitors to target the toxin’s many mechanisms of action.

Cnidarians (corals, sea anemones, and jellyfish) produce toxins that play central roles in key ecological processes, including predation, defense, and competition, being the oldest extant venomous animal lineage. Cnidaria small cysteine-rich proteins (SCRiPs) were the first family of neurotoxins detected in stony corals, one of the ocean’s most crucial foundation species. Yet, their molecular evolution remains poorly understood. Moreover, the lack of a clear classification system has hindered the establishment of an accurate and phylogenetically informed nomenclature. In this study, we extensively surveyed 117 genomes and 103 transcriptomes of cnidarians to identify orthologous SCRiP gene sequences. We annotated a total of 168 novel putative SCRiPs from over 36 species of stony corals and 12 species of sea anemones. Phylogenetic reconstruction identified four distinct SCRiP subfamilies, according to strict discrimination criteria based on well-supported monophyly with a high percentage of nucleotide and amino acids’ identity. Although there is a high prevalence of purifying selection for most SCRiP subfamilies, with few positively selected sites detected, a subset of Acroporidae sequences is influenced by diversifying positive selection, suggesting potential neofunctionalizations related to the fine-tuning of toxin potency. We propose a new nomenclature classification system relying on the phylogenetic distribution and evolution of SCRiPs across Anthozoa, which will further assist future proteomic and functional research efforts.

While the unique symbiotic relationship between anemonefishes and sea anemones is iconic, it is still not fully understood how anemonefishes can withstand and thrive within the venomous environment of their host sea anemone. In this study, we used a proteotranscriptomics approach to elucidate the proteinaceous toxin repertoire from the most common host sea anemone, Entacmaea quadricolor. Although 1251 different toxin or toxin-like RNA transcripts were expressed in E. quadricolor tentacles (0.05% of gene clusters, 1.8% of expression) and 5375 proteins were detected in milked venom, only 4% of proteins detected in venom were putative toxins (230), and they only represent on average 14% of the normalised protein expression in the milked venom samples. Thus, most proteins in milked venom do not appear to have a toxin function. This work raises the perils of defining a dominant venom phenotype based on transcriptomics data alone in sea anemones, as we found that the dominant venom phenotype differs between the transcriptome and proteome abundance data. E. quadricolor venom contains a mixture of toxin-like proteins of unknown and known function. A newly identified toxin protein family, Z3, rich in conserved cysteines of unknown function, was the most abundant at the RNA transcript and protein levels. The venom was also rich in toxins from the Protease S1, Kunitz-type and PLA2 toxin protein families and contains toxins from eight venom categories. Exploring the intricate venom toxin components in other host sea anemones will be crucial for improving our understanding of how anemonefish adapt to the venomous environment.

In recent studies, emphasis has been placed on the zonula occludens toxin (Zot) from the non-toxigenic Vibrio parahaemolyticus strain PMC53.7 as an agent inducing alterations in the actin cytoskeleton of infected Caco-2 cells and which appears as a relevant virulence factor. Universal zot primers were designed by the alignment of different types of zot gene and identification of conserved sequences to investigate the presence in diverse environmental and clinical V. parahaemolyticus isolates, in co-occurrence with virulence factors, such a hemolysins and secretion systems. The study screened a total of 390 isolates from environmental sources from Chile and Italy and 95 Chilean clinical isolates. The results revealed that around 37.2% of Chilean environmental strains and 25.9% of Italian strains, and 24.2% of clinical isolates carried the zot gene. The Zot-C2 cluster was present in 71.4% of Chilean environmental strains but absent in clinical isolates, while the Zot-C4 cluster was identified in 28.6% of environmental and 100% of clinical isolates. Understanding the role of zot in V. parahaemolyticus virulence is crucial, especially considering the risk associated with consuming diverse isolates from bivalves and the co-occurrence with virulence factors such as TDH, TRH or T3SS2.

The venoms of various sea anemones are rich in diverse toxins, which usually play a dual role in capturing prey and deterring predators. However, the complex components of such venoms have not been well known yet. Here, venomics of integrating transcriptomic and proteomic technologies was applied for the first time to identify putative protein and peptide toxins from different tissues of the representative sea anemone, Heteractis magnifica. The transcriptomic analysis of H. magnifica identified 728 putative toxin sequences, including 442 and 381 from the tentacles and the column, respectively, and they were assigned to 68 gene superfamilies. The proteomic analysis confirmed 101 protein and peptide toxins in the venom, including 91 in the tentacles and 39 in the column. The integrated venomics also confirmed that some toxins such as the ShK-like peptides and defensins are co-expressed in both the tentacles and the column. Meanwhile, a homology analysis was conducted to predict the three-dimensional structures and potential activity of seven representative toxins. Altogether, this venomics study revealed the venom complexity of H. magnifica, which will help deepen our understanding of cnidarian toxins, thereby supporting the in-depth development of valuable marine drugs.

Disulfide-rich peptides (DRPs) play diverse physiological roles and have emerged as attractive sources of pharmacological tools and drug leads. Here we describe the 3D structure of a centipede venom peptide, U-SLPTX15-Sm2a, whose family defines a unique class of one of the most widespread DRP folds known, the cystine-stabilized α/β fold (CSαβ). This class, which we have named the two-disulfide CSαβ fold (2ds-CSαβ), contains only two internal disulfide bonds as opposed to at least three in all other confirmed CSαβ peptides, and constitutes one of the major neurotoxic peptide families in centipede venoms. We show the 2ds-CSαβ is widely distributed outside centipedes and is likely an ancient fold predating the split between prokaryotes and eukaryotes. Our results provide insights into the ancient evolutionary history of a widespread DRP fold and highlight the usefulness of 3D structures as evolutionary tools.

LiTCTP is a toxin from the Translationally Controlled Tumor Protein (TCTP) family identified in Loxosceles brown spider venoms. These proteins are known as histamine-releasing factors (HRF). TCTPs participate in allergic and anaphylactic reactions, which suggest their potential role as therapeutic targets. The histaminergic effect of TCTP is related to its pro-inflammatory functions. An initial characterization of LiTCTP in animal models showed that this toxin can increase the microvascular permeability of skin vessels and induce paw edema in a dose-dependent manner. We evaluated the role of LiTCTP in vitro and in vivo in the inflammatory and allergic aspects that undergo the biological responses observed in Loxoscelism, the clinical condition after an accident with Loxosceles spiders. Our results showed LiTCTP recombinant toxin (LiRecTCTP) as an essential synergistic factor for the dermonecrotic toxin actions (LiRecDT1, known as the main toxin in the pathophysiology of Loxoscelism), revealing its contribution to the exacerbated inflammatory response clinically observed in envenomated patients.

Abstract Background Enterotoxigenic Bacteroides fragilis (ETBF) has been implicated in colorectal carcinogenesis through the actions of its toxin, B. fragilis toxin (BFT). Studies on colorectal cell lines have shown that treatment with BFT causes disruption of E-cadherin leading to increased expression of the pro-inflammatory cytokine, IL-8. Stat3 activation has also been associated with ETBF-related colitis and tumour development. However, a link between E-cadherin, IL-8 and Stat3 has not been investigated in the context of ETBF infection. Results We found that co-culture of HT-29 and HCT116 colorectal cell lines with ETBF, had a similar effect on activation of IL8 gene and protein expression as treatment with purified BFT. Inhibition of Stat3 resulted in a decrease in IL-8 gene and protein expression in response to ETBF in both cell lines. A reduction in E-cadherin expression in response to ETBF treatment was not restored by blocking Stat3. Conclusion We found that treatment of colorectal cancer cell lines with live cultures of ETBF had the equivalent effect on IL-8 expression as the use of purified toxin, and this may be a more representative model of ETBF-mediated colorectal carcinogenesis. IL-8 gene and protein expression was mediated through Stat3 in HT-29 and HCT116 cells, whereas disruption of E-cadherin appeared to be independent of Stat3 signalling.