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

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Ricin toxin (RT) is highly cytotoxic and can release a considerable amount of pro-inflammatory factors due to depurination, causing excessive inflammation that may aggravate the harm to the body. Pyroptosis, a type of gasdermin-mediated cell death, is a contributor to the exacerbation of inflammation. Accumulating evidence indicate that pyroptosis plays a significant role in the pathogen infection and tissue injury, suggesting a potential correlation between pyroptosis and RT-induced inflammation. Here, we aim to demonstrate this correlation and explore its molecular mechanisms. Results showed that RT triggers mouse alveolar macrophage MH-S cells pyroptosis by activating caspase-3 and cleaving Gasgermin E (GSDME). In contrast, inhibition of caspase-3 with Z-DEVD-FMK (inhibitor of caspase-3) or knockdown of GSDME attenuates this process, suggesting the essential role of caspase-3/GSDME-mediated pyroptosis in contributing to RT-induced inflammation. Collectively, our study enhances our understanding of a novel mechanism of ricin cytotoxicity, which may emerge as a potential target in immunotherapy to control the RT-induced inflammation.

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The Epidermal Growth Factor Receptor (EGFR) has been of high importance as it is over expressed in a wide diversity of epithelial cancers, promoting cell proliferation and survival pathways. Recombinant immunotoxins (ITs) have emerged as a promising targeted therapy for cancer treatment. In this study, we aimed to investigate the antitumor activity of a novel recombinant immunotoxin designed against EGFR. Using an in silico approach, we confirmed the stability of the RTA-scFv fusion protein. The immunotoxin was successfully cloned and expressed in the pET32a vector, and the purified protein was analyzed by electrophoresis and western blotting. In vitro evaluations were conducted to assess the biological activities of the recombinant proteins (RTA-scFv, RTA, scFv). The novel immunotoxin demonstrated significant anti-proliferative and pro-apoptotic effects against cancer cell lines. The MTT cytotoxicity assay revealed a decrease in cell viability in the treated cancer cell lines. Additionally, Annexin V/Propidium iodide staining followed by flow cytometry analysis showed a significant induction of apoptosis in the cancer cell lines, with half maximal inhibitory concentration (IC50) values of 81.71 nM for MDA-MB-468 and 145.2 nM for HCT116 cells (P < 0.05). Furthermore, the EGFR-specific immunotoxin exhibited non-allergenic properties. The recombinant protein demonstrated high affinity binding to EGFR. Overall, this study presents a promising strategy for the development of recombinant immunotoxins as potential candidates for the treatment of EGFR-expressing cancers.

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DNA-encoded monoclonal antibodies (DMAbs) and in vivo expression of antibody therapeutics presents an innovative alternative to conventional delivery methods. Therefore, in order to prevent the lethal dose of ricin toxin (RT) and to avoid human anti-mouse antibody (HAMA) reaction, we developed the human neutralizing antibody 4-4E against RT and constructed DMAb-4-4E. The human neutralizing antibody 4-4E could neutralize RT in vitro and in vivo, while the mice in RT group all died. Using intramuscular electroporation (IM EP), antibodies were rapidly expressed in vivo within 7 days and were enriched in intestine and gastrocnemius muscle mostly. Besides, we found that DMAbs have shown a broad protective efficacy of RT poisoning prophylaxis. Driven by plasmids for IgG expression, mice were survived and the blood glucose level of mice in DMAb-IgG group returned to normal at 72 h post RT challenge, and the RT group died within 48 h. Furthermore, hindrance of protein disulfide isomerase (PDI) and accumulation of RT in endosomes were found in IgG-protected cells, revealing the possible mechanism of neutralization details. These data support the further study of RT-neutralizing monoclonal antibodies (mAbs) in the development.

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Ricin toxin A chain (RTA), from Ricinus communis, is a deadly protein that inactivates ribosomes by degrading an adenine residue at position 4324 in 28S rRNA. Recently, we have demonstrated that pterin-7-carboxamides with peptide pendants were potent RTA inhibitors. Among these, N-(pterin-7-carbonyl)glycyl-L-tyrosine (7PCGY) is the most potent RTA inhibitor as a small organic molecule. However, despite this fascinating inhibitory activity, the mode of interaction of 7PCGY with RTA remains elusive. This study aimed to elucidate the factors responsible for the high RTA inhibitory activity of 7PCGY based on X-ray crystallographic analysis. Herein, we report the successfully resolved X-ray crystal structure of 7PCGY/RTA complexes, revealing that the interaction between the phenolic hydroxy group in 7PCGY and Asn78 of RTA through a hydrogen bonding and the conformational change of Tyr80 and Asn122 are responsible for the high RTA inhibitory activity of 7PCGY.

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Autophagy is a complex and highly regulated degradative process, which acts as a survival pathway in response to cellular stress, starvation and pathogen infection. Ricin toxin is a plant toxin produced by the castor bean and classified as a category B biothreat agent. Ricin toxin inhibits cellular protein synthesis by catalytically inactivating ribosomes, leading to cell death. Currently, there is no licensed treatment for patients exposed to ricin. Ricin-induced apoptosis has been extensively studied; however, whether its intoxication via protein synthesis inhibition affects autophagy is not yet resolved. In this work, we demonstrated that ricin intoxication is accompanied by its own autophagic degradation in mammalian cells. Autophagy deficiency, by knocking down ATG5, attenuates ricin degradation, thus aggravating ricin-induced cytotoxicity. Additionally, the autophagy inducer SMER28 (Small Molecule Enhancer 28) partially protects cells against ricin cytotoxicity, an effect not observed in autophagy-deficient cells. These results demonstrate that autophagic degradation acts as a survival response of cells against ricin intoxication. This suggests that stimulation of autophagic degradation may be a strategy to counteract ricin intoxication.

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BACKGROUND/AIMS: The ribosome-inactivating proteins include the biothreat agent, ricin toxin (RT). When inhaled, RT causes near complete destruction of the lung epithelium coincident with a proinflammatory response that includes TNF family cytokines, which are death-inducing ligands. We previously demonstrated that the combination of RT and TNF-related apoptosis inducing ligand (TRAIL) induces caspase-dependent apoptosis, while RT and TNF-α or RT and Fas ligand (FasL) induces cathepsin-dependent cell death in lung epithelial cells. We hypothesize that airway macrophages constitute a major source of cytokines that drive lung epithelial cell death. METHODS: Here, we show that RT-induced apoptosis of the monocytic cell line, U937, leads to the bystander killing of the lung epithelial cell line, A549. U937 cells were treated with ricin. Following this, A549 cells were treated with supernatants from U937 cells and death was measured by WST-1 viability assay. RESULTS: Upon RT-induced U937 cell death, released RT and FasL contributed to A549 cell death. U937 cells also released nuclear protein HMGB1. The release of RT, FasL, and HMGB1 triggered A549 cell necroptosis, rather than cathepsin-dependent killing observed previously with RT and FasL. Reactive oxygen species (ROS) were produced in A549 cells due to HMGB1 ligation of the receptor for advanced glycation end products (RAGE). CONCLUSION: These findings demonstrate the potential for bystander necroptosis of lung epithelial cells during RT toxicosis which may perpetuate or increase the proinflammatory response.

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Acting as microRNA (miRNA) sponges, circular RNAs (circRNAs) have been discovered to be critical modulators of inflammatory processes. Ricin Toxin (RT) is highly toxic to mammalian cells and low doses of RT can induce acute inflammation. However, current researches on the underlying mechanism and function of circRNA/miRNA network in RT-induced inflammation are limited. Previously, we found miR-221-5p was aberrant and associated with the inflammation of RT induction. In this study, based on the circRNA high-throughput sequencing (circRNA-seq), we obtained a novel circRNA termed circNLRP3 and revealed that circNLRP3 can sponge miR-221-5p, release its target mRNA A20, and further suppress NF-κB signaling pathway to alleviated RT-induced TNF-α production. Our findings elucidated a possible mechanistic link between the circNLRP3/miR-221-5p/A20 axis and RT-induced inflammatory response, which may broaden our understanding of RT poisoning.

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Ricin toxin (RT) belongs to the ribosome-inactivating protein (RIP) family of toxins and is considered to be a moderate threat by the US Center of Disease Control and Prevention (CDC). RT poses a great potential threat to the public, but there has been a lack of effective treatment options so far. Over the past few decades, researches on the prevention and treatment of RT poisoning have been investigated, among which neutralizing antibodies targeting RT specifically have always been a research hotspot. In this review, we have summarized the mechanism of action of RT, the research results and the design strategies of RT neutralizing antibodies, and discussed the key issues in the development of RT neutralizing antibody researches.

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Ricin toxin (RT) is one of the most lethal type II ribosome-inactivating proteins (RIP), and is classified as a potential bioterror agent due to its severe cytotoxicity and high availability. The toxicity of RT is dependent on both dose and route of exposure. Increasing evidence demonstrates that sub-lethal RT induces acute inflammation and increases the release of pro-inflammatory cytokines. However, current studies on mechanism of RT-induced inflammation are limited. In this study, to evaluate the relationship between miRNAs and RT-induced inflammation, RNA sequencing (RNA-Seq) was used to analyze the expression of miRNAs and mRNAs in RT-treated RAW264.7 macrophage cells. A total of 14 significantly differently expressed (DE) miRNAs and 323 miRNA-mRNA interaction pairs were predicted by bioinformatics analysis. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that majority of those interaction pairs were involved in PI3K/Akt pathway. In addition, overexpression of miR-221-5p promoted the inflammatory response by inhibiting the mRNA expression of COL4a5. This work contributes to our understanding of RT-induced inflammation and demonstrates the potential role of miRNAs in innate immunity, which may be regarded as potential targets in developing therapies for RT poisoning.

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The issues of vaccine potency and stability constitute formidable challenges associated with the development and readiness of vaccines for biodefense. In most instances, the vaccines will be stockpiled (at considerable cost) for years and used only in the rare event of a public health emergency. It is therefore imperative that there be means to readily monitor overall stability of the stockpiled vaccines, preferably using reliable in vitro assays, without the need for expensive and labor-intensive animal studies. In this chapter, we describe an in vitro monoclonal antibody-based competition ELISA known as RiCoE for assessing the potency of a ricin toxin subunit vaccine. RiCoE can be applied to drug substance and drug products adsorbed to aluminum salts adjuvant. While RiCoE is specific for ricin toxin, the general methodologies and protocols described herein are amenable to virtually any subunit or even virus-like particle-based vaccine. Ultimately, RiCoE-like assays may replace or at least reduce the need for animal studies in vaccine potency determinations.

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Increasing studies have concentrated on investigating circular RNAs (circRNAs) as pivotal regulators in the progression of numerous diseases and biological processes and abundant evidence shows that circRNAs are participated in the regulation of innate immune responses. Several studies showed that Ricin Toxin (RT) could induce inflammatory injury. There was no research on the particular functions and underlying mechanisms of circRNAs in RT-induced inflammation. In this study, RNA sequencing performed on RT-treated and normal RAW264.7 macrophage cells was used to investigated the differentially expressed circRNAs. Based on the dataset, the expression of circEpc1 (mmu_circ_0,000,842) was identified higher in RT-treated cells. Moreover, gain-and-loss function assays showed that circEpc1 function as a promoter in RT-induced inflammation in vivo and in vitro. Mechanistically, circEpc1 acted as a miR-5114 sponge to relieve the suppressive effect of miR-5114 on its target NOD2 and thereby activating NF-κB and MAPK signaling pathways. Our results illuminated a link between RT-induced inflammation and the circEpc1 regulatory loop and provided novel insight into the functions of circRNA in innate immune, which may emerge as a potential target in immunotherapy to control the RT-induced inflammatory injury.

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Ricin toxin (RT) is a ribosome-inactivating protein derived from the beans of the castor oil plant. Our previous studies have reported that RT can induce the production of inflammatory cytokines and cause inflammatory injury in RAW264.7 cells. In order to explore the various biological processes that long noncoding RNA (lncRNA), circular RNA (circRNA) and micro RNA (miRNA) as endogenous non-coding RNAs (ceRNAs) may participate in the pro-inflammatory mechanism, RT (20 ng/mL) treated and normal RAW264.7 cells were firstly sequenced by RNA-seq. By comparing the differentially expressed genes, we obtained 10 hub genes and enriched the inflammatory-related signaling pathways. Based on our results, we concluded a lncRNA/circRNA-miRNA-mRNA network. Finally, we verified the key genes and pathways by qRT-PCR, WB and ELISA. From the experiment results, an opening MAPK signaling pathway in TNF signaling pathway via TNFR2 was found involved in RT-induced inflammation. This work provides a reference for searching for ceRNA targets or therapeutic drugs in RT-induced inflammatory injury in the future.

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Ricin toxin (RT) is one of the most lethal toxins derived from the seed of castor beans. In addition to its main toxic mechanism of inhibiting the synthesis of cellular proteins, RT can induce the production of inflammatory cytokines. MicroRNAs (miRNAs) play a key role in regulating both innate and adaptive immunity. To elucidate the regulation of miRNAs in RT-induced inflammation injury, the RNA high-throughput sequencing (RNA-Seq) technology was used to analyze the expression profile of miRNAs and mRNAs in RT-treated RAW264.7 cells. Results showed that a total of 323 mRNAs and 19 miRNAs differentially expressed after RT treated. Meanwhile, 713 miRNA-mRNA interaction pairs were identified by bioinformatics analysis. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis showed that those interaction pairs were mainly involved in JAK-STAT, T cell receptor, and MAPK signaling pathways. Moreover, we further predicted and determined the targeting relationship between miR-155-3p and GAB2 through TargetScan and dual-luciferase reporter assay. Mechanically, overexpression of miR-155-3p can reduce the secretion of TNF-α in RAW264.7 cells, revealing a possible mechanism of miR-155-3p regulating RT-induced inflammatory injury. This study provides a new perspective for clarifying the mechanism of RT-induced inflammatory injury and reveals the potential role of miRNAs in innate immune regulation.

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BACKGROUND: For efficient control of the economically important fruit pest Bactrocera dorsalis, a hybrid system combining ricin toxicity and sex-related alternative splicing of the doublesex gene has been developed. This system exhibits the expected female-specific lethal effect; however, the transgenic females do not survive, making it difficult to raise stable homozygous lines. Since modification of ricin toxin A chain (RTA) through a single-residue change (Gly212 > Arg212 ) leads to cold-sensitive posttranslational repression of its toxicity, we utilized this unique property to obtain RTA-Bddsx females that survive at low temperature for line maintenance. RESULTS: In transient expression experiments using embryonic injection, two groups treated with RTAcs-derived DNA (LERQcs and RTAcs) exhibited temperature-dependent effects. The toxicity was higher at 29 °C than at 18 °C. The proportion of males was close to 50% at 18 °C in all the tested groups except LERQcs-treated flies, which exhibited a high proportion of males (over 70%) at 29 °C. The results indicate the cold-sensitive responses of RTA and further suggest a female-specific lethal effect. Subsequently, 14 putative RTAcs-Bddsx transgenic Ds-Red+ G1 males were identified, and female-specific lethal effects were observed in Ds-Red+ G2 and G3 lines under cultivation at 29 °C but not at 18 °C. The male ratio can be increased to up to 95% in G3 line 001, indicating that RTAcs functions well in B. dorsalis. CONCLUSION: The improved RTAcs-Bddsx system with conditional toxicity represents a novel and promising step toward the practical control of B. dorsalis. © 2021 Society of Chemical Industry.

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Ricin toxin binding subunit B (RTB) is a galactose-binding lectin protein derived from the beans of the castor oil plant (Ricinus communis). Our previous studies have reported a direct immunomodulatory effect of recombinant RTB, which stimulates RAW264.7 cells to produce cytokines including TNF-α. However, the role of RTB in innate immune response and its specific mechanism have not been reported in detail. In this work, the results showed that RTB treatment of macrophages significantly increased TLR4 protein levels. RTB also activated TLR4 downstream events, including MyD88, IRAK, and TRAF6, resulting in macrophage activation and TNF-α production. This process is reflected in the increase of IκB phosphorylation. TLR4 knockdown macrophages treated with RTB exhibited greatly reduced IκB phosphorylation and TNF-α secretion. Moreover, treatment with MyD88 inhibitor also suppressed TNF-α production. The docking of RT and TLR4 was simulated by computer, and the contact residues were concentrated on RTB. Our results suggest that recombinant RTB can activate mouse macrophages to secrete TNF-α through activation of NF-κB via the TLR4 signaling pathways.

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The current study explores the detoxification effect of Retro-2 on ricin toxin (RT) cytotoxicity, as well as the mechanisms underlying such effects, to provide a basis for follow-up clinical applications of Retro-2. The mouse-derived mononuclear/macrophage cell line, RAW264.7, was used to evaluate the detoxification effect of Retro-2 on RT by detecting cell viability, capacity for protein synthesis and the expression of cytokines, as well as endoplasmic reticulum stress (ERS)-related mRNA. The results indicated that many cells died when challenged with concentrations of RT ≥50ng/mL. The protein synthesis capacity of cells decreased when challenged with 200ng/mL RT for 2hours. Furthermore, the synthesis and release of many cytokines decreased, while the expression of cytokines or ERS-related mRNA increased when challenged with 200ng/mL of RT for 12 or more hours. However, cell viability, capacity for protein synthesis and release levels of many cytokines were higher, while the expression levels of cytokine, or ERS-related mRNA, were lower in cells pretreated with 20µm Retro-2 and challenged with RT, compared with those that had not been pretreated with Retro-2. In conclusion, Retro-2 retained the capacity for protein synthesis inhibited by RT, alleviated ERS induced by RT and increased the viability of cells challenged with RT. Retro-2 shows the potential for clinical applications.

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Ricin is a highly toxic type 2 ribosome-inactivating protein (RIP) which is extracted from the seeds of castor beans. Ricin is considered a potential bioterror agent and no effective antidote for ricin exists so far. In this study, by structural modification of a retrograde transport blocker Retro-2cycl, a series of novel compounds were obtained. The primary screen revealed that compound 27 has an improved anti-ricin activity compare to positive control. In vitro pre-exposure evaluation in Madin-Darby Canine Kidney (MDCK) cells demonstrated that 27 is a powerful anti-ricin compound with an EC50 of 41.05 nmol/L against one LC (lethal concentration, 5.56 ng/mL) of ricin. Further studies surprisingly indicated that 27 confers post-exposure activity against ricin intoxication. An in vivo study showed that 1 h post-exposure administration of 27 can improve the survival rate as well as delay the death of ricin-intoxicated mice. A drug combination of 27 with monoclonal antibody mAb4C13 rescued mice from one LD (lethal dose) ricin challenge and the survival rate of tested animals is 100%. These results represent, for the first time, indication that small molecule retrograde transport blocker confers both in vitro and in vivo post-exposure protection against ricin and therefore provides a promising candidate for the development of anti-ricin medicines.

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Ricin toxin (RT) is a natural plant-derived protein toxin from the seed of castor beans that belongs to a family of type II ribosome-inactivating proteins (RIPs). In addition to its main toxic mechanism of inhibiting the synthesis of cellular proteins, RT can induce the production of inflammatory cytokines and cause inflammatory injury. Macrophages play a crucial role in innate immunity and the adaptive immune response as the first line of host defense against bacterial infections and various types of invading pathogens. Upon activation, macrophages release types of cytokines to remove pathogens. However, the effect of RT on the immune response and its mechanism are not well characterized. In the current study, we investigated the activation of the TLR4-mediated signaling pathway by low-dose RT treatment and its interaction with signaling molecules in the transduction pathway. We found that low-dose RT can activate MyD88- and TRIF-dependent signaling pathways, revealing a possible mechanism by which low-dose RT-activates TLR4-mediated signaling pathways. We also confirmed that the TLR4-induced activation of the inflammatory signaling pathways was produced via its binding to RT. This study may help to identify the most important target molecules and clarify the mechanism of inflammatory injury of ricin.

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Ricin is a highly toxic type 2 ribosome-inactivating protein (RIP) which is extracted from the seeds of castor beans. Ricin is considered a potential bioterror agent and no effective antidote for ricin exists so far. In this study, by structural modification of a retrograde transport blocker Retro-2, a series of novel compounds were obtained. The primary screen revealed that compound has an improved anti-ricin activity compare to positive control. pre-exposure evaluation in Madin-Darby Canine Kidney (MDCK) cells demonstrated that is a powerful anti-ricin compound with an EC of 41.05 nmol/L against one LC (lethal concentration, 5.56 ng/mL) of ricin. Further studies surprisingly indicated that confers post-exposure activity against ricin intoxication. An study showed that 1 h post-exposure administration of can improve the survival rate as well as delay the death of ricin-intoxicated mice. A drug combination of with monoclonal antibody mAb4C13 rescued mice from one LD (lethal dose) ricin challenge and the survival rate of tested animals is 100%. These results represent, for the first time, indication that small molecule retrograde transport blocker confers both and post-exposure protection against ricin and therefore provides a promising candidate for the development of anti-ricin medicines.