S100A9 plays a key role in Clostridium perfringens beta2 toxin-induced inflammatory damage in porcine IPEC-J2 intestinal epithelial cells.

BACKGROUND: As an important regulator of autoimmune responses and inflammation, S100A9 may serve as a therapeutic target in inflammatory diseases. However, the role of S100A9 in Clostridium perfringens type C infectious diarrhea is poorly studied. The aim of our study was to screen downstream target genes regulated by S100A9 in Clostridium perfringens beta2 (CPB2) toxin-induced IPEC-J2 cell injury. We constructed IPEC-J2 cells with S100A9 knockdown and a CPB2-induced cell injury model, screened downstream genes regulated by S100A9 using RNA-Seq technique, and performed functional enrichment analysis. The function of S100A9 was verified using molecular biology techniques. RESULTS: We identified 316 differentially expressed genes (DEGs), of which 221 were upregulated and 95 were downregulated. Functional enrichment analysis revealed that the DEGs were significantly enriched in cilium movement, negative regulation of cell differentiation, immune response, protein digestion and absorption, and complement and coagulation cascades. The key genes of immune response were TNF, CCL1, CCR7, CSF2, and CXCL9. When CPB2 toxin-induced IPEC-J2 cells overexpressed S100A9, Bax expression increased, Bcl-2 expression and mitochondrial membrane potential decreased, and SOD activity was inhibited. CONCLUSION: In conclusion, S100A9 was involved in CPB2-induced inflammatory response in IPEC-J2 cells by regulating the expression of downstream target genes, namely, TNF, CCL1, CCR7, CSF2, and CXCL9; promoting apoptosis; and aggravating oxidative cell damage. This study laid the foundation for further study on the regulatory mechanism underlying piglet diarrhea.

Toxic or Otherwise Harmful Algae and the Built Environment.

This article gives a comprehensive overview on potentially harmful algae occurring in the built environment. Man-made structures provide diverse habitats where algae can grow, mainly aerophytic in nature. Literature reveals that algae that is potentially harmful to humans do occur in the anthropogenic environment in the air, on surfaces or in water bodies. Algae may negatively affect humans in different ways: they may be toxic, allergenic and pathogenic to humans or attack human structures. Toxin-producing alga are represented in the built environment mainly by blue green algae (Cyanoprokaryota). In special occasions, other toxic algae may also be involved. Green algae (Chlorophyta) found airborne or growing on manmade surfaces may be allergenic whereas Cyanoprokaryota and other forms may not only be toxic but also allergenic. Pathogenicity is found only in a special group of algae, especially in the genus Prototheca. In addition, rare cases with infections due to algae with green chloroplasts are reported. Algal action may be involved in the biodeterioration of buildings and works of art, which is still discussed controversially. Whereas in many cases the disfigurement of surfaces and even the corrosion of materials is encountered, in other cases a protective effect on the materials is reported. A comprehensive list of 79 taxa of potentially harmful, airborne algae supplemented with their counterparts occurring in the built environment, is given. Due to global climate change, it is not unlikely that the built environment will suffer from more and higher amounts of harmful algal species in the future. Therefore, intensified research in composition, ecophysiology and development of algal growth in the built environment is indicated.

Markers of Intestinal Permeability Are Rapidly Improved by Alcohol Withdrawal in Patients with Alcohol-Related Liver Disease.

Changes in intestinal microbiome and barrier function are critical in the development of alcohol-related liver disease (ALD). Here, we determined the effects of a one-week alcohol withdrawal on parameters of intestinal barrier function in heavy drinkers with ALD in comparison to healthy non-drinkers (controls). In serum samples of 17 controls (m = 10/f = 7) and 37 age-matched ALD patients (m = 26/f = 11) undergoing a one-week alcohol withdrawal, markers of liver health and intestinal barrier function were assessed. Liver damage, e.g., fibrosis and hepatic steatosis, were assessed using FibroScan. Before alcohol withdrawal, markers of liver damage, lipopolysaccharide binding protein (LBP) and overall TLR4/TLR2 ligands in serum were significantly higher in ALD patients than in controls, whereas intestinal fatty acid binding protein (I-FABP) and zonulin protein concentrations in serum were lower. All parameters, with the exception of LBP, were significantly improved after alcohol withdrawal; however, not to the level of controls. Our data suggest that one-week of abstinence improves markers of intestinal barrier function and liver health in ALD patients.

Bisphosphonate inhibitors of squalene synthase protect cells against cholesterol-dependent cytolysins.

Certain species of pathogenic bacteria damage tissues by secreting cholesterol-dependent cytolysins, which form pores in the plasma membranes of animal cells. However, reducing cholesterol protects cells against these cytolysins. As the first committed step of cholesterol biosynthesis is catalyzed by squalene synthase, we explored whether inhibiting this enzyme protected cells against cholesterol-dependent cytolysins. We first synthesized 22 different nitrogen-containing bisphosphonate molecules that were designed to inhibit squalene synthase. Squalene synthase inhibition was quantified using a cell-free enzyme assay, and validated by computer modeling of bisphosphonate molecules binding to squalene synthase. The bisphosphonates were then screened for their ability to protect HeLa cells against the damage caused by the cholesterol-dependent cytolysin, pyolysin. The most effective bisphosphonate reduced pyolysin-induced leakage of lactate dehydrogenase into cell supernatants by >80%, and reduced pyolysin-induced cytolysis from >75% to <25%. In addition, this bisphosphonate reduced pyolysin-induced leakage of potassium from cells, limited changes in the cytoskeleton, prevented mitogen-activated protein kinases cell stress responses, and reduced cellular cholesterol. The bisphosphonate also protected cells against another cholesterol-dependent cytolysin, streptolysin O, and protected lung epithelial cells and primary dermal fibroblasts against cytolysis. Our findings imply that treatment with bisphosphonates that inhibit squalene synthase might help protect tissues against pathogenic bacteria that secrete cholesterol-dependent cytolysins.

Two Gut Microbiota-Derived Toxins Are Closely Associated with Cardiovascular Diseases: A Review.

Cardiovascular diseases (CVDs) have become a major health problem because of the associated high morbidity and mortality rates observed in affected patients. Gut microbiota has recently been implicated as a novel endocrine organ that plays critical roles in the regulation of cardiometabolic and renal functions of the host via the production of bioactive metabolites. This review investigated the evidence from several clinical and experimental studies that indicated an association between the gut microbiota-derived toxins and CVDs. We mainly focused on the pro-inflammatory gut microbiota-derived toxins, namely lipopolysaccharides, derived from Gram-negative bacteria, and trimethylamine N-oxide and described the present status of research in association with these toxins, including our previous research findings. Several clinical studies aimed at exploring the effectiveness of reducing the levels of these toxins to inhibit cardiovascular events are currently under investigation or in the planning stages. We believe that some of the methods discussed in this review to eliminate or reduce the levels of such toxins in the body could be clinically applied to prevent CVDs in the near future.

Moxetumomab pasudotox in heavily pre-treated patients with relapsed/refractory hairy cell leukemia (HCL): long-term follow-up from the pivotal trial.

BACKGROUND: Moxetumomab pasudotox is a recombinant CD22-targeting immunotoxin. Here, we present the long-term follow-up analysis of the pivotal, multicenter, open-label trial (NCT01829711) of moxetumomab pasudotox in patients with relapsed/refractory (R/R) hairy cell leukemia (HCL). METHODS: Eligible patients had received ≥ 2 prior systemic therapies, including ≥ 2 purine nucleoside analogs (PNAs), or ≥ 1 PNA followed by rituximab or a BRAF inhibitor. Patients received 40 µg/kg moxetumomab pasudotox intravenously on Days 1, 3, and 5 of each 28-day cycle for up to six cycles. Disease response and minimal residual disease (MRD) status were determined by blinded independent central review. The primary endpoint was durable complete response (CR), defined as achieving CR with hematologic remission (HR, blood counts for CR) lasting > 180 days. RESULTS: Eighty adult patients were treated with moxetumomab pasudotox and 63% completed six cycles. Patients had received a median of three lines of prior systemic therapy; 49% were PNA-refractory, and 38% were unfit for PNA retreatment. At a median follow-up of 24.6 months, the durable CR rate (CR with HR > 180 days) was 36% (29 patients; 95% confidence interval: 26-48%); CR with HR ≥ 360 days was 33%, and overall CR was 41%. Twenty-seven complete responders (82%) were MRD-negative (34% of all patients). CR lasting ≥ 60 months was 61%, and the median progression-free survival without the loss of HR was 71.7 months. Hemolytic uremic and capillary leak syndromes were each reported in ≤ 10% of patients, and ≤ 5% had grade 3-4 events; these events were generally reversible. No treatment-related deaths were reported. CONCLUSIONS: Moxetumomab pasudotox resulted in a high rate of durable responses and MRD negativity in heavily pre-treated patients with HCL, with a manageable safety profile. Thus, it represents a new and viable treatment option for patients with R/R HCL, who currently lack adequate therapy. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT01829711; first submitted: April 9, 2013. https://clinicaltrials.gov/ct2/show/NCT01829711.

Correlation Between Clostridium perfringens Alpha- and NetB-Toxin and Chick Mortality in Commercial Broiler Farms During Different Anticoccidial Control Programs.

The purpose of the present study was to determine whether a correlation existed between chick mortality and the presence of Clostridium perfringens alpha-toxin and NetB-toxin genes (cpa and netB) in C. perfringens recovered from litter in commercial broiler houses. Because coccidiosis predisposes chickens to necrotic enteritis, the concentration of Eimeria oocysts in these samples was measured, and the numbers were used in similar correlation analyses. Litter samples were collected at 0, 2, and 4 wk growout from six broiler farms (18 houses total) during an anticoccidial drug (ACD) control program and from nine broiler farms (23 houses total) during an Eimeria vaccine (VAC) control program. Of these, litter samples were collected from five farms during both ACD and VAC programs. The litter samples were processed for Eimeria oocyst and C. perfringens spore enumerations by standard parasitologic and microbiologic techniques. DNA was also extracted for C. perfringens DNA for PCR detection of genes coding for alpha- and NetB-toxin. A general trend during the ACD programs was a transient decrease in both Eimeria maxima and non-E. maxima (Eamipt) numbers at 2 wk growout. The pattern was slightly different during VAC with E. maxima and Eamipt levels increasing over time. Average concentrations of C. perfringens in litter were highest at 2 wk (∼105-106 spores/g) during ACD and at placement during VAC (∼105-106 spores/g). During the ACD program, a strong correlation was observed between 0 and 3-wk chick mortality and the presence at placement (0 wk) of netB (r = 0.42-0.48) or cpa (r = 0.55-0.67). A very strong correlation was observed in 0-5-wk chick mortality and the presence of netB at 4 wk growout (0.73-0.95). During a VAC program, a strong correlation was only observed between the presence of netB at placement and 0-1-wk chick mortality (r = 0.67).

Lectin drug conjugate therapy for colorectal cancer.

Drug resistance represents an obstacle in colorectal cancer (CRC) treatment because of its association with poor prognosis. rBC2LCN is a lectin isolated from Burkholderia that binds cell surface glycans that have fucose moieties. Because fucosylation is enhanced in many types of cancers, this lectin could be an efficient drug carrier if CRC cells specifically present such glycans. Therefore, we examined the therapeutic efficacy and toxicity of lectin drug conjugate therapy in CRC mouse xenograft models. The affinity of rBC2LCN for human CRC cell lines HT-29, LoVo, LS174T, and DLD-1 was assessed in vitro. The cytocidal efficacy of a lectin drug conjugate, rBC2LCN-38 kDa domain of pseudomonas exotoxin A (PE38) was evaluated by MTT assay. The therapeutic effects and toxicity for each CRC cell line-derived mouse xenograft model were compared between the intervention and control groups. LS174T and DLD-1 cell lines showed a strong affinity for rBC2LCN. In the xenograft model, the tumor volume in the rBC2LCN-PE38 group was significantly reduced compared with that using control treatment alone. However, the HT-29 cell line showed weak affinity and poor therapeutic efficacy. No significant toxicities or adverse responses were observed. In conclusion, we demonstrated that rBC2LCN lectin binds CRC cells and that rBC2LCN-PE38 significantly suppresses tumor growth in vivo. In addition, the efficacy of the drug conjugate correlated with its binding affinity for each CRC cell line. These results suggest that lectin drug conjugate therapy has potential as a novel targeted therapy for CRC cell surface glycans.

Structure and Function of Bovine Whey Derived Oligosaccharides Showing Synbiotic Epithelial Barrier Protective Properties.

Commensal gut microbiota and probiotics have numerous effects on the host's metabolic and protective systems, which occur primarily through the intestinal epithelial cell interface. Prebiotics, like galacto-oligosaccharides (GOS) are widely used to modulate their function and abundance. However, important structure-function relations may exist, requiring a detailed structural characterization. Here, we detailed the structural characterization of bovine whey derived oligosaccharide preparations enriched with GOS or not, dubbed GOS-enriched milk oligosaccharides (GMOS) or MOS, respectively. We explore GMOS's and MOS's potential to improve intestinal epithelial barrier function, assessed in a model based on barrier disruptive effects of the Clostridioides difficile toxin A. GMOS and MOS contain mainly GOS species composed of ß1-6- and ß1-3-linked galactoses, and 3'- and 6'-sialyllactose. Both GMOS and MOS, combined with lactobacilli, like Lactobacillus rhamnosus (LPR, NCC4007), gave synergistic epithelial barrier protection, while no such effect was observed with Bifidobacterium longum (BL NCC3001), Escherichia coli (Nissle) or fructo-oligosaccharides. Mechanistically, for barrier protection with MOS, (i) viable LPR was required, (ii) acidification of growth medium was not enough, (iii) LPR did not directly neutralize toxin A, and (iv) physical proximity of LPR with the intestinal epithelial cells was necessary. This is the first study, highlighting the importance of structure-function specificity and the necessity of the simultaneous presence of prebiotic, probiotic and host cell interactions required for a biological effect.

Oral and intestinal bacterial exotoxins: Potential linked to carcinogenesis.

Growing evidence suggests that imbalances in resident microbes (dysbiosis) can promote chronic inflammation, immune-subversion, and production of carcinogenic metabolites, thus leading to neoplasia. Yet, evidence to support a direct link of individual bacteria species to human sporadic cancer is still limited. This chapter focuses on several emerging bacterial toxins that have recently been characterized for their potential oncogenic properties toward human orodigestive cancer and the presence of which in human tissue samples has been documented. These include cytolethal distending toxins produced by various members of gamma and epsilon Proteobacteria, Dentilisin from mammalian oral Treponema, Pasteurella multocida toxin, two Fusobacterial toxins, FadA and Fap2, Bacteroides fragilis toxin, colibactin, cytotoxic necrotizing factors and α-hemolysin from Escherichia coli, and Salmonella enterica AvrA. It was clear that these bacterial toxins have biological activities to induce several hallmarks of cancer. Some toxins directly interact with DNA or chromosomes leading to their breakdowns, causing mutations and genome instability, and others modulate cell proliferation, replication and death and facilitate immune evasion and tumor invasion, prying specific oncogene and tumor suppressor pathways, such as p53 and ß-catenin/Wnt. In addition, most bacterial toxins control tumor-promoting inflammation in complex and diverse mechanisms. Despite growing laboratory evidence to support oncogenic potential of selected bacterial toxins, we need more direct evidence from human studies and mechanistic data from physiologically relevant experimental animal models, which can reflect chronic infection in vivo, as well as take bacterial-bacterial interactions among microbiome into consideration.

The pharmacological management of hairy cell leukemia.

INTRODUCTION: Hairy cell leukemia (HCL) is a B-cell lymphoid malignancy that accounts for approximately 2% of all leukemias. Treatment with purine nucleoside analogs (PNA) results in a high response rate and remains the standard of care. Long term follow-up shows that most patients relapse and require retreatment. Newer combination strategies and agents have emerged to try to reduce the relapse rate and to address cases of PNA refractoriness. AREAS COVERED: The authors reviewed the literature on the pharmacological management of HCL, including recent studies that led to new agents being incorporated into practice. EXPERT OPINION: Combination of cladribine plus rituximab produces a high rate of measurable residual disease-negative complete remission. In our center, newly diagnosed patients are offered cladribine followed by 8 weekly doses of rituximab in an ongoing phase II trial. Patients in first relapse are also offered this combination if they were initially treated with a single-agent PNA, or if the remission duration was ≥5 years after first-line cladribine plus rituximab. Patients who relapse within 5 years are offered therapy with a novel agent that may include the BRAF inhibitor vemurafenib, alone or in combination with rituximab, dabrafenib in combination with trametinib, the BTK inhibitor ibrutinib, or moxetumomab pasudotox.

Monomicrobial Fournier's Gangrene Caused by Panton-Valentine Leukocidin-negative Methicillin-susceptible Staphylococcus aureus ST8 in Japan.

Methicillin-resistant Staphylococcus aureus USA300, belonging to sequence type (ST) 8, is a rare cause of necrotizing fasciitis in the USA. We herein report a case of monomicrobial Fournier's gangrene caused by an ST8, methicillin-susceptible Staphylococcus aureus (designated ksw1). Whole-genome sequencing and analyses for virulence determinants revealed that, unlike USA300, ksw1 lacked virulence genes, such as Panton-Valentine leukocidin and SCCmec, while harboring the toxic shock syndrome toxin-1 gene. These genomic features correlate with ST8 CA-MRSA/J, which is the major genotype of ST8 in Japan.

Cytolethal distending toxin-induced release of interleukin-1ß by human macrophages is dependent upon activation of glycogen synthase kinase 3ß, spleen tyrosine kinase (Syk) and the noncanonical inflammasome.

Cytolethal distending toxins (Cdt) are a family of toxins produced by several human pathogens which infect mucocutaneous tissue and induce inflammatory disease. We have previously demonstrated that the Aggregatibacter actinomycetemcomitans Cdt induces a pro-inflammatory response from human macrophages which involves activation of the NLRP3 inflammasome. We now demonstrate that in addition to activating caspase-1 (canonical inflammasome), Cdt treatment leads to caspase-4 activation and involvement of the noncanonical inflammasome. Cdt-treated cells exhibit pyroptosis characterised by cleavage of gasdermin-D (GSDMD), release of HMGB1 at 24 hr and LDH at 48 hr. Inhibition of either the canonical (caspase-1) or noncanonical (caspase-4) inflammasome blocks both Cdt-induced release of IL-1ß and induction of pyroptosis. Analysis of upstream events indicates that Cdt induces Syk phosphorylation (activation); furthermore, blockade of Syk expression and inhibition of pSyk activity inhibit both Cdt-induced cytokine release and pyroptosis. Finally, we demonstrate that increases in pSyk are dependent upon Cdt-induced activation of GSK3ß. These studies advance our understanding of Cdt function and provide new insight into the virulence potential of Cdt in mediating the pathogenesis of disease caused by Cdt-producing organisms such as A. actinomycetemcomitans.

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

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

A novel, safe, fast and efficient treatment for Her2-positive and negative bladder cancer utilizing an EGF-anthrax toxin chimera.

Bladder cancer is the sixth most common cancer in the United States, and it exhibits an alarming 70% recurrence rate. Thus, the development of more efficient antibladder cancer approaches is a high priority. Accordingly, this work provides the basis for a transformative anticancer strategy that takes advantage of the unique characteristics of the bladder. Unlike mucin-shielded normal bladder cells, cancer cells are exposed to the bladder lumen and overexpress EGFR. Therefore, we used an EGF-conjugated anthrax toxin that after targeting EGFR was internalized and triggered apoptosis in exposed bladder cancer cells. This unique agent presented advantages over other EGF-based technologies and other toxin-derivatives. In contrast to known agents, this EGF-toxin conjugate promoted its own uptake via receptor microclustering even in the presence of Her2 and induced cell death with a LC50 < 1 nM. Furthermore, our data showed that exposures as short as ≈3 min were enough to commit human (T24), mouse (MB49) and canine (primary) bladder cancer cells to apoptosis. Exposure of tumor-free mice and dogs with the agent resulted in no toxicity. In addition, the EGF-toxin was able to eliminate cells from human patient tumor samples. Importantly, the administration of EGF-toxin to dogs with spontaneous bladder cancer, who had failed or were not eligible for other therapies, resulted in ~30% average tumor reduction after one treatment cycle. Because of its in vitro and in vivo high efficiency, fast action (reducing treatment time from hours to minutes) and safety, we propose that this EGF-anthrax toxin conjugate provides the basis for new, transformative approaches against bladder cancer.

A novel approach to assess domain specificity of anti-drug antibodies to moxetumomab pasudotox, an immunotoxin with two functional domains.

Biologics are potentially immunogenic and can elicit immune response. Complex biologics, such as bispecific antibodies or multi-domain molecules can induce anti-drug antibodies (ADA) with specificity to different domains. Domain specific ADAs may differently affect drug efficacy and safety, and thus, characterization of ADA domain specificity has become a regulatory expectation for multi-domain biologics. Unlike well-established methods for screening, confirmation, titer and neutralizing ADA detection, characterization of ADA domain specificity is an emerging field. The conventional approach for determination of ADA domain specificity is a competitive inhibition with domain-containing molecules. When developing a conventional domain specificity assay for moxetumomab pasudotox, a recombinant anti-CD22 immunotoxin, comprised of two functional domains (CD22-binding fragment and truncated Pseudomonas exotoxin A (PE38), we encountered a bioanalytical challenge. The method was able to detect immunodominant anti-PE38 (ADA-PE) but generated false negative results for low abundant CD22-binding domain ADA (ADA-BD) in a polyclonal sample. Troubleshooting experiments using control samples with varying levels of each ADA subtype demonstrated that a major factor for successful ADA identification was the ratio of the ADA signals contributed by each ADA subtype. To overcome this unique bioanalytical challenge, we developed a novel approach, which ensures detection of a domain-specific ADA subtype regardless of its relative level in a polyclonal ADA sample by evaluating signal inhibition by a respective domain-containing molecule at the condition when signals from all other ADAs are fully blocked. The method has been used for characterization of ADA domain specificity in moxetumomab pasudotox clinical trials, including study 1053, the pivotal Phase III study in hairy cell leukemia patients. It allowed for successful detection of ADA-BD in the presence of immunodominant ADA-PE, enabling accurate determination of domain specificity for moxetumomab pasudotox. The results demonstrated that the method was superior than the conventional approach. The method could be applied broadly to other biologics with two or more domains when there is a need to detect a minor ADA subtype in polyclonal samples.

Bacterial transportable toxins of the nasopharyngeal microbiota in multiple sclerosis. Nose-to-brain direct.

Intranasal administration delivers molecules directly to the brain bypassing the blood-brain barrier. Three distinct routes of access have been identified; olfactory, trigeminal and via the paranasal sub-mucosa of the posterior sinuses. Consequently, environmental toxins may access the CNS directly to induce inflammatory and degenerative disease. They may also activate bacterial species of the nasal mucosal microbiome to release both immune-deviating cell wall antigens and transportable neurotoxins with local direct access to the CNS. Evidence is reviewed that toxins of the nasal bacterial microbiota may be directly implicated in the inflammatory and degenerative pathogenesis of multiple sclerosis.

The N-end rule ubiquitin ligase UBR2 mediates NLRP1B inflammasome activation by anthrax lethal toxin.

Anthrax lethal toxin (LT) is known to induce NLRP1B inflammasome activation and pyroptotic cell death in macrophages from certain mouse strains in its metalloprotease activity-dependent manner, but the underlying mechanism is unknown. Here, we establish a simple but robust cell system bearing dual-fluorescence reporters for LT-induced ASC specks formation and pyroptotic lysis. A genome-wide siRNA screen and a CRISPR-Cas9 knockout screen were applied to this system for identifying genes involved in LT-induced inflammasome activation. UBR2, an E3 ubiquitin ligase of the N-end rule degradation pathway, was found to be required for LT-induced NLRP1B inflammasome activation. LT is known to cleave NLRP1B after Lys44. The cleaved NLRP1B, bearing an N-terminal leucine, was targeted by UBR2-mediated ubiquitination and degradation. UBR2 partnered with an E2 ubiquitin-conjugating enzyme UBE2O in this process. NLRP1B underwent constitutive autocleavage before the C-terminal CARD domain. UBR2-mediated degradation of LT-cleaved NLRP1B thus triggered release of the noncovalent-bound CARD domain for subsequent caspase-1 activation. Our study illustrates a unique mode of inflammasome activation in cytosolic defense against bacterial insults.