Search Results (32)

Clostridium perfringens (C. perfringens), a Gram-positive bacterium, produces a variety of toxins and extracellular enzymes that can lead to disease in both humans and animals. Common symptoms include abdominal swelling, diarrhea, and intestinal inflammation. Severe cases can result in complications like intestinal hemorrhage, edema, and even death. The primary toxins contributing to morbidity in C. perfringens-infected intestines are CPA, CPB, CPB2, CPE, and PFO. Amongst these, CPB, CPB2, and CPE are implicated in apoptosis development, while CPA is associated with cell death, increased intracellular ROS levels, and the release of the inflammatory factor IL-18. However, the exact mechanism by which PFO toxins exert their effects in the infected gut is still unidentified. This study demonstrates that a C. perfringens PFO toxin infection disrupts the intestinal epithelial barrier function through in vitro and in vivo models. This study emphasizes the notable influence of PFO toxins on intestinal barrier integrity in the context of C. perfringens infections. It reveals that PFO toxins increase ROS production by causing mitochondrial damage, triggering pyroptosis in IPEC-J2 cells, and consequently resulting in compromised intestinal barrier function. These results offer a scientific foundation for developing preventive and therapeutic approaches against C. perfringens infections. Full article

Streptococcus pneumoniae is the leading cause of community-acquired pneumonia. The pore-forming cholesterol-dependent cytolysin (CDC) pneumolysin (PLY) and the physiological metabolite hydrogen peroxide (H₂O₂) can greatly increase the virulence of pneumococci. Although most studies have focused on the contribution of both virulence factors to the course of pneumococcal infection, it is unknown whether or how H₂O₂ can affect PLY activity. Of note, S. pneumoniae exploits endogenous H₂O₂ as an intracellular signalling molecule to modulate the activity of several proteins. Here, we demonstrate that H₂O₂ negatively affects the haemolytic activity of PLY in a concentration-dependent manner. Prevention of cysteine-dependent sulfenylation upon substitution of the unique and highly conserved cysteine residue to serine in PLY significantly reduces the toxin’s susceptibility to H₂O₂ treatment and completely abolishes the ability of DTT to activate PLY. We also detect a clear gradual correlation between endogenous H₂O₂ generation and PLY release, with decreased H₂O₂ production causing a decline in the release of PLY. Comparative transcriptome sequencing analysis of the wild-type S. pneumoniae strain and three mutants impaired in H₂O₂ production indicates enhanced expression of several genes involved in peptidoglycan (PG) synthesis and in the production of choline-binding proteins (CPBs). One explanation for the impact of H₂O₂ on PLY release is the observed upregulation of the PG bridge formation alanyltransferases MurM and MurN, which evidentially negatively affect the PLY release. Our findings shed light on the significance of endogenous pneumococcal H₂O₂ in controlling PLY activity and release. Full article

Clostridium perfringens beta-1 toxin (CPB1) is responsible for necrotizing enteritis and enterotoxemia. However, whether the release of host inflammatory factors caused by CPB1 is related to pyroptosis, an inflammatory form of programmed cell death, has not been reported. A construct expressing recombinant Clostridium perfringens beta-1 toxin (rCPB1) was created, and the cytotoxic activity of the purified rCPB1 toxin was assessed via CCK-8 assay. The rCPB1-induced macrophage pyroptosis by assessing changes to the expression of pyroptosis-related signal molecules and the pyroptosis pathway of macrophages using quantitative real-time PCR, immunoblotting, ELISA, immunofluorescence, and electron microscopic assays. The results showed that the intact rCPB1 protein was purified from an E. coli expression system, which exhibited moderate cytotoxicity on mouse mononuclear macrophage leukemia cells (RAW264.7), normal colon mucosal epithelial cells (NCM460), and human umbilical vein endothelial cells (HUVEC). rCPB1 could induce pyroptosis in macrophages and HUVEC cells, in part through the Caspase-1-dependent pathway. The rCPB1-induced pyroptosis of RAW264.7 cells could be blocked by inflammasome inhibitor MCC950. These results demonstrated that rCPB1 treatment of macrophages promoted the assembly of NLRP3 inflammasomes and activated Caspase 1; the activated Caspase 1 caused gasdermin D to form plasma membrane pores, leading to the release of inflammatory factors IL-18 and IL-1β, resulting in macrophage pyroptosis. NLRP3 may be a potential therapeutic target for Clostridium perfringes disease. This study provided a novel insight into the pathogenesis of CPB1. Full article

The Clostridium perfringens (C. perfringen) beta2 (CPB2) toxin produced by C. perfringens type C (CpC) can cause necrotizing enteritis in piglets. Immune system activation in response to inflammation and pathogen infection is aided by long non-coding RNAs (lncRNAs). In our previous work, we revealed the differential expression of the novel lncRNA LNC_001186 in CpC-infected ileum versus healthy piglets. This implied that LNC_001186 may be a regulatory factor essential for CpC infection in piglets. Herein, we analyzed the coding ability, chromosomal location and subcellular localization of LNC_001186 and explored its regulatory role in CPB2 toxin-induced apoptosis of porcine small intestinal epithelial (IPEC-J2) cells. RT-qPCR results indicated that LNC_001186 expression was highly enriched in the intestines of healthy piglets and significantly increased in CpC-infected piglets’ ileum tissue and CPB2 toxin-treated IPEC-J2 cells. The total sequence length of LNC_001186 was 1323 bp through RACE assay. CPC and CPAT, two online databases, both confirmed that LNC_001186 had a low coding ability. It was present on pig chromosome 3. Cytoplasmic and nuclear RNA isolation and RNA-FISH assays showed that LNC_001186 was present in the nucleus and cytoplasm of IPEC-J2 cells. Furthermore, six target genes of LNC_001186 were predicted using cis and trans approaches. Meanwhile, we constructed ceRNA regulatory networks with LNC_001186 as the center. Finally, LNC_001186 overexpression inhibited IPEC-J2 cells’ apoptosis caused by CPB2 toxin and promoted cell viability. In summary, we determined the role of LNC_001186 in IPEC-J2 cells’ apoptosis caused by CPB2 toxin, which assisted us in exploring the molecular mechanism of LNC_001186 in CpC-induced diarrhea in piglets. Full article

Piglet diarrhea caused by Clostridium perfringens (C. perfringens) type C (CpC) seriously endangers the development of the pig production industry. C. perfringens beta2 (CPB2) toxin is a virulent toxin produced by CpC. Long non-coding RNAs (lncRNAs) are key regulators in the immune inflammatory response to bacterial infection. Nevertheless, the functional mechanism of lncRNAs in bacterial piglet diarrhea is unclear. Herein, a novel lncRNA lnc001776 expression was confirmed to be substantially elevated in the ileum tissue of CpC-infected diarrhea piglets and in CPB2 toxin-treated porcine small intestinal epithelial cells (IPEC-J2). lnc001776 knockdown restrained CPB2 toxin-induced apoptosis, inflammatory injury, barrier dysfunction and activation of JNK/NF-kB pathway in IPEC-J2 cells. Additionally, ssc-let-7i-5p was identified as sponge for lnc001776. Overexpression of ssc-let-7i-5p repressed CPB2-induced injury in IPEC-J2 cells. Interleukin 6 (IL-6), a target gene of ssc-let-7i-5p, was enhanced in CPB2 toxin-treated IPEC-J2 cells. Rescue experiments demonstrated that a ssc-let-7i-5p mimic reversed the effect of lnc001776 overexpression on CPB2 toxin-induced IPEC-J2 cell injury and JNK/NF-kB pathway, whereas IL-6 overexpression partially restored the impact of lnc001776. Overall, lnc001776 overexpression exacerbated CPB2 toxin-induced IPEC-J2 cell damage by sponging ssc-let-7i-5p to regulate IL-6 to activate JNK/NF-kB pathway, indicating that lnc001776 could be a key target for piglet resistance to CpC-induced diarrhea. Full article

Long non-coding RNAs (lncRNAs) modified by n6-methyladenosine (m6A) have been implicated in the development and progression of several diseases. However, the mechanism responsible for the role of m6A-modified lncRNAs in Clostridium perfringens type C piglet diarrhea has remained largely unknown. We previously developed an in vitro model of CPB2 toxin-induced piglet diarrhea in IPEC-J2 cells. In addition, we previously performed RNA immunoprecipitation sequencing (MeRIP-seq), which demonstrated lncRNA EN_42575 as one of the most regulated m6A-modified lncRNAs in CPB2 toxin-exposed IPEC-J2 cells. In this study, we used MeRIP-qPCR, FISH, EdU, and RNA pull-down assays to determine the function of lncRNA EN_42575 in CPB2 toxin-exposed IPEC-J2 cells. LncRNA EN_42575 was significantly downregulated at different time points in CPB2 toxin-treated cells. Functionally, lncRNA EN_42575 overexpression reduced cytotoxicity, promoted cell proliferation, and inhibited apoptosis and oxidative damage, whereas the knockdown of lncRNA EN_42575 reversed these results. Furthermore, the dual-luciferase analysis revealed that METTL3 regulated lncRNA EN_42575 expression in an m6A-dependent manner. In conclusion, METTL3-mediated lncRNA EN_42575 exerted a regulatory effect on IPEC-J2 cells exposed to CPB2 toxins. These findings offer novel perspectives to further investigate the function of m6A-modified lncRNAs in piglet diarrhea. Full article

Epsilon toxin (ETX), produced by type B and D strains of Clostridium perfringens, can cause fatal enterotoxaemia in ruminant animals, particularly sheep, cattle, and goats. Previous studies show that the cytotoxicity of ETX is dependent on the integrity of lipid rafts, the maintenance of which is ensured by cholesterol. Zaragozic acid (ZA) is a statin drug that reduces the synthesis of squalene, which is responsible for cholesterol synthesis. In this study, ZA significantly reduced the toxicity of ETX in Madin–Darby canine kidney (MDCK) cells. We show that ZA does not affect the binding of ETX to MDCK cells, but propidium iodide staining (PI) and Western blotting confirmed that ZA significantly disrupts the ability of ETX to form pores or oligomers in MDCK cells. Additionally, ZA decreased the phosphatidylserine exposure on the plasma membrane and increased the Ca²⁺ influx of the cells. Results of density gradient centrifugation suggest that ZA decreased the number of lipid rafts in MDCK membranes, which probably contributed to the attenuation of pore-formation. Moreover, ZA protected mice against ETX in vivo. All mice pre-treated with ZA for 48 h before exposure to an absolute lethal dose of ETX (6400 ng/kg) survived. In summary, these findings provide an innovative method to prevent ETX intoxication. Considering many pore-forming toxins require lipid rafts, we tested and found ZA also inhibited the toxicity of other toxins such as Clostridium perfringens Net B and β-toxin (CPB) and Staphylococcus aureus α-hemolysin (Hla). We expect ZA can thus be developed as a broad-spectrum medicine for the treatment of multiple toxins. In addition, other statins, such as lovastatin (LO), also reduced the toxicity of ETX. These findings indicate that statin medicines are potential candidates for preventing and treating multiple toxin-induced diseases. Full article

Clostridium perfringens beta2 (CPB2) toxin is one of the main pathogenic toxins produced by Clostridium perfringens, which causes intestinal diseases in animals and humans. The N6-methyladenosine (m6A) modification is the most common reversible modification in eukaryotic disease processes. Methyltransferase-like 3 (METTL3) regulates immunity and inflammatory responses induced by the bacterial infections in animals. However, METTL3′s involvement in CPB2-treated intestinal porcine epithelial cell line-J2 (IPEC-J2) remains unclear. In the current study, we used methylated RNA immunoprecipitation-quantitative polymerase chain reaction, Western blotting and immunofluorescence assay to determine the role of METTL3 in CPB2-exposed IPEC-J2 cells. The findings revealed that m6A and METTL3 levels were increased in CPB2 treated IPEC-J2 cells. Functionally, METTL3 overexpression promoted the release of inflammatory factors, increased cytotoxicity, decreased cell viability and disrupted tight junctions between cells, while the knockdown of METTL3 reversed these results. Furthermore, METTL3 was involved in the inflammatory response of IPEC-J2 cells by activating the TLR2/NF-κB signaling pathway through regulating TLR2 m6A levels. In conclusion, METTL3 overexpression triggered the TLR2/NF-κB signaling pathway and promoted CPB2-induced inflammatory responses in IPEC-J2 cells. These findings may provide a new strategy for the prevention and treatment of diarrhea caused by Clostridium perfringens. Full article

A direct sandwich enzyme-linked immunosorbent assay (sELISA) was developed for the detection of the atypical β2-toxin (CPB2) of Clostridium perfringens. Polyclonal (PAbs) and monoclonal (MAbs) antibodies were previously obtained employing recombinant CPB2 produced in the baculovirus system as antigen. In the current study, PAbs were used as capture molecules, while purified MAbs conjugated to horseradish peroxidase (MAbs-HRP) were used for the detection of atypical CPB2 toxin. MAbs 5C11E6 and 2G3G6 showed high reactivity, sensitivity and specificity when tested on 232 C. perfringens cell culture isolates. In addition, a reactivity variation among different strains producing atypical CPB2 toxin was observed using the conformation-dependent MAb 23E6E6, suggesting the hypothesis of high instability and/or the existence of different three-dimensional structures of this toxin. Results obtained by sELISA and Western blotting performed on experimentally CPB2-contaminated feces revealed a time-dependent proteolytic degradation as previously observed with the consensus allelic form of CPB2. Finally, the sELISA and an end-point PCR, specific for the atypical cpb2 gene, were used to test field samples (feces, rectal swabs and intestinal contents) from different dead animal species with suspected or confirmed clostridiosis. The comparison of sELISA data with those obtained with end-point PCR suggests this method as a promising tool for the detection of atypical CPB2 toxin. Full article

N6-methyladenosine (m6A) modification can accommodate mRNA processing, stability, and translation in mammals, and fat mass and obesity associated protein (FTO) is a vital demethylase in the m6A modification pathway. Clostridium perfringens type C (C. perfringens type C) causes diarrhea in piglets and has a serious impact on the pig industry. However, our understanding of the effect of m6A in the process of C. perfringens type C infectious piglet diarrhea (CPTCIPD) is limited. Here, an in vitro model of CPTCIPD was constructed by treating the intestinal porcine epithelial cell line-J2 (IPEC-J2) with Clostridium perfringens beta2 (CPB2) toxin, and the role of FTO was analyzed using quantitative real-time polymerase chain reaction, Western blotting, and flow cytometry. The results revealed that the overall RNA m6A contents at the tissue and cell levels were significantly up-regulated after C. perfringens infection (p < 0.05). FTO expression was significantly reduced in CPB2-treated IPEC-J2 cells. Functionally, FTO knockdown in the treated cells inhibited their proliferation and promoted apoptosis and the inflammation phenotype, whereas FTO overexpression had the opposite effects. Inhibiting FTO prolonged the half-life and up-regulated the expression of Caspase 3, leading to apoptosis. Therefore, this work explored the regulation of FTO in IPEC-J2 cells after CPB2 treatment and enhanced our understanding of the effect of the m6A modification in CPTCIPD. Full article

Several food-poisoning outbreaks have been attributed to Clostridium perfringens (C. perfringens) worldwide. Despite that, this crisis was discussed in a few studies, and additional studies are urgently needed in this field. Therefore, we sought to highlight the prevalence, antimicrobial resistance, toxin profiles, and toxinotypes of C. perfringens isolates. In this study, 50 C. perfringens isolates obtained from 450 different animal origin samples (beef, chicken meat, and raw milk) were identified by phenotypic and genotypic methods. The antimicrobial susceptibility results were surprising, as most of the isolates (74%) showed multidrug-resistant (MDR) patterns. The phenotypic resistance to tetracycline, lincomycin, enrofloxacin, cefoxitin/ampicillin, and erythromycin was confirmed by the PCR detections of tet, lnu, qnr, bla, and erm(B) genes, respectively. In contrast to the toxinotypes C and E, toxinotype A prevailed (54%) among our isolates. Additionally, we found that the genes for C. perfringens enterotoxin (cpe) and C. perfringens beta2 toxin (cpb2) were distributed among the tested isolates with high prevalence rates (70 and 64%, respectively). Our findings confirmed that the C. perfringens foodborne crisis has been worsened by the evolution of MDR strains, which became the prominent phenotypes. Furthermore, we were not able to obtain a fixed association between the toxinotypes and antimicrobial resistance patterns. Full article

In this study, we determined the prevalence and toxin types of antibiotic-resistant Clostridium perfringens in chicken, pigeons, camels, and humans. We investigated the inhibitory effects of AgNPs on biofilm formation ability of the isolates and the genetic relatedness of the isolates from various sources determined using RAPD-PCR. Fifty isolates were identified using PCR, and all the isolates were of type A. The cpe and cpb2 genes were detected in 12% and 56% of the isolates, respectively. The effect of AgNPs on biofilm production of six representative isolates indicated that at the highest concentration of AgNPs (100 µg/mL), the inhibition percentages were 80.8–82.8%. The RAPD-PCR patterns of the 50 C. perfringens isolates from various sources revealed 33 profiles and four clusters, and the discriminatory power of RAPD-PCR was high. Multidrug-resistant C. perfringens isolates are predominant in the study area. The inhibition of biofilm formation by C. perfringens isolates was dose-dependent, and RAPD-PCR is a promising method for studying the genetic relatedness between the isolates from various sources. This is the first report of AgNPs’ anti-biofilm activity against C. perfringens from chickens, pigeons, camels, and humans, to the best of our knowledge. Full article

We analyze the application filed for the marketing and cultivation of genetically engineered Bt cowpea (event AAT 709A) approved in Nigeria in 2019. Cowpea (Vigna ungiguiculata) is extensively grown throughout sub-Saharan Africa and consumed by around two hundred million people. The transgenic plants produce an insecticidal, recombinant Bt toxin meant to protect the plants against the larvae of Maruca vitrata, which feed on the plants and are also known as pod borer. Our analysis of the application reveals issues of concern regarding the safety of the Bt toxins produced in the plants. These concerns include stability of gene expression, impact on soil organisms, effects on non-target species and food safety. In addition, we show deficiencies in the risk assessment of potential gene flow and uncontrolled spread of the transgenes and cultivated varieties as well as the maintenance of seed collections. As far as information is publicly available, we analyze the application by referring to established standards of GMO risk assessment. We take the provisions of the Cartagena Protocol on Biosafety (CPB) into account, of which both Nigeria and the EU are parties. We also refer to the EU standards for GMO risk assessment, which are complementary to the provisions of the CPB. Full article

Beta toxin (CPB) is a small hemolysin beta pore-forming toxin (β-PFT) produced by Clostridium perfringens type C. It plays a central role in the pathogenesis of necro-hemorrhagic enteritis in young animals and humans via targeting intestinal endothelial cells. We recently identified the membrane protein CD31 (PECAM-1) as the receptor for CPB on mouse endothelial cells. We now assess the role of CD31 in CPB cytotoxicity against human endothelial and monocytic cells using a CRISPR/Cas9 gene knockout and an antibody blocking approach. CD31 knockout human endothelial and monocytic cells were resistant to CPB and CPB oligomers only formed in CD31-expressing cells. CD31 knockout endothelial and monocytic cells could be selectively enriched out of a polyclonal cell population by exposing them to CPB. Moreover, antibody mediated blocking of the extracellular Ig6 domain of CD31 abolished CPB cytotoxicity and oligomer formation in endothelial and monocytic cells. In conclusion, this study confirms the role of CD31 as a receptor of CPB on human endothelial and monocytic cells. Specific interaction with the CD31 molecule can thus explain the cell type specificity of CPB observed in vitro and corresponds to in vivo observations in naturally diseased animals. Full article

The insect integument (exoskeleton) is an effective physiochemical barrier that limits disease-causing agents to a few portals of entry, including the gastrointestinal and reproductive tracts. The bacterial biopesticide Bacillus thuringiensis (Bt) enters the insect host via the mouth and must thwart gut-based defences to make its way into the body cavity (haemocoel) and establish infection. We sought to uncover the main antibacterial defences of the midgut and the pathophysiological features of Bt in a notable insect pest, the Colorado potato beetle Leptinotarsa decemlineata (CPB). Exposing the beetles to both Bt spores and their Cry3A toxins (crystalline δ-endotoxins) via oral inoculation led to higher mortality levels when compared to either spores or Cry3A toxins alone. Within 12 h post-exposure, Cry3A toxins caused a 1.5-fold increase in the levels of reactive oxygen species (ROS) and malondialdehyde (lipid peroxidation) within the midgut – key indicators of tissue damage. When Cry3A toxins are combined with spores, gross redox imbalance and ‘oxidation stress’ is apparent in beetle larvae. The insect detoxification system is activated when Bt spores and Cry3A toxins are administered alone or in combination to mitigate toxicosis, in addition to elevated mRNA levels of candidate defence genes (pattern-recognition receptor, stress-regulation, serine proteases, and prosaposin-like protein). The presence of bacterial spores and/or Cry3A toxins coincides with subtle changes in microbial community composition of the midgut, such as decreased Pseudomonas abundance at 48 h post inoculation. Both Bt spores and Cry3A toxins have negative impacts on larval health, and when combined, likely cause metabolic derangement, due to multiple tissue targets being compromised. Full article