MiR-30d Inhibition Protects IPEC-J2 Cells Against Clostridium perfringens Beta2 Toxin-Induced Inflammatory Injury.
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ABSTRACT:
SUBMITTER: Xie K
PROVIDER: S-EPMC9253665 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundThe n6-methyladenosine (m6A) modification is present widely in mRNAs and long non-coding RNAs (lncRNAs), and is related to the occurrence and development of certain diseases. However, the role of m6A methylation in Clostridium perfringens type C infectious diarrhea remains unclear.MethodsHere, we treated intestinal porcine jejunum epithelial cells (IPEC-J2 cells) with Clostridium perfringens beta2 (CPB2) toxin to construct an in vitro model of Clostridium perfringens type C (C. perfringens type C) infectious diarrhea, and then used methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) to identify the methylation profiles of mRNAs and lncRNAs in IPEC-J2 cells.ResultsWe identified 6,413 peaks, representing 5,825 m6A-modified mRNAs and 433 modified lncRNAs, of which 4,356 m6A modified mRNAs and 221 m6A modified lncRNAs were significantly differential expressed between the control group and CPB2 group. The motif GGACU was enriched significantly in both the control group and the CPB2 group. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation analysis showed that the differentially methylated modified mRNAs were mainly enriched in Hippo signaling pathway and Wnt signaling pathway. In addition, the target genes of the differentially m6A modified lncRNAs were related to defense response to virus and immune response. For example, ENSSSCG00000042575, ENSSSCG00000048701 and ENSSSCG00000048785 might regulate the defense response to virus, immune and inflammatory response to resist the harmful effects of viruses on cells.ConclusionIn summary, this study established the m6A transcription profile of mRNAs and lncRNAs in IPEC-J2 cells treated by CPB2 toxin. Further analysis showed that m6A-modified RNAs were related to defense against viruses and immune response after CPB2 toxin treatment of the cells. Threem6A-modified lncRNAs, ENSSSCG00000042575, ENSSSCG00000048785 and ENSSSCG00000048701, were most likely to play a key role in CPB2 toxin-treated IPEC-J2 cells. The results provide a theoretical basis for further research on the role of m6A modification in piglet diarrhea.
| S-EPMC8646102 | biostudies-literature
Project description:Clostridium perfringens is a potent producer of a variety of toxins. Well studied from these are five toxins (alpha, Beta (CPB), epsilon, iota and CPE) that are produced by seven toxinotype strains (A-G) of C. perfringens. Besides these toxins, C. perfringens produces also another toxin that causes necrotizing enterocolitis in piglets. This toxin termed consensus Beta2 toxin (cCPB2) has a molecular mass of 27,620 Da and shows only little homology to CPB and no one to the other toxins of C. perfringens. Its primary action on cells remained unknown to date. cCPB2 was heterogeneously expressed as fusion protein with GST in Escherichia coli and purified to homogeneity. Although cCPB2 does not exhibit the typical structure of beta-stranded pore-forming proteins and contains no indication for the presence of amphipathic alpha-helices we could demonstrate that cCPB2 is a pore-forming component with an extremely high activity in lipid bilayers. The channels have a single-channel conductance of about 700 pS in 1 M KCl and are highly cation-selective as judged from selectivity measurements in the presence of salt gradients. The high cation selectivity is caused by the presence of net negative charges in or near the channel that allowed an estimate of the channel size being about 1.4 nm wide. Our measurements suggest that the primary effect of cCPB2 is the formation of cation-selective channels followed by necrotic enteritis in humans and animals. We searched in databases for homologs of cCPB2 and constructed a cladogram representing the phylogenetic relationship to the next relatives of cCPB2.
| S-EPMC8827211 | biostudies-literature
Project description:Beta2-toxin, encoded by cpb2, is implicated in the pathogenesis of Clostridium perfringens enteritis. However, cpb2 genes from nonporcine C. perfringens isolates were not always expressed, at least in vitro. Nucleotide sequencing identified atypical cpb2 genes with 70.2 to 70.7% DNA identity to previously identified (consensus) cpb2. Atypical beta2-toxin displayed 62.3% identity and 80.4% similarity to consensus beta2-toxin. No porcine type C isolates (n = 16) and only 3.3% of porcine type A isolates (n = 60) carried atypical cpb2 genes. However, 88.5% of nonporcine isolates carried atypical cpb2 (n = 78), but beta2-toxin was not expressed. Almost half of the nonporcine consensus cpb2 genes (44.4%) carried a frameshift mutation (n = 9), resulting in an absence of beta2-toxin expression. These findings strengthen the role of beta2-toxin in the pathogenesis of enteritis in neonatal pigs. However, the identification of apparently nonexpressed, atypical cpb2 genes raises the question of whether this protein plays the same role in enteritis in other animal species.
| S-EPMC538998 | biostudies-literature
Project description:Clostridium perfringens (C. perfringens) causes intestinal injury through overgrowth and the secretion of multiple toxins, leading to diarrhea and necrotic enteritis in animals, including pigs, chickens, and sheep. This study aimed to investigate the protective effects of Lactobacillus plantarum (L. plantarum) Lac16 on C. perfringens infection-associated injury in intestinal porcine epithelial cell line (IPEC-J2). The results showed that L. plantarum Lac16 significantly inhibited the growth of C. perfringens, which was accompanied by a decrease in pH levels. In addition, L. plantarum Lac16 significantly elevated the mRNA expression levels of host defense peptides (HDPs) in IPEC-J2 cells, decreased the adhesion of C. perfringens to IPEC-J2 cells, and attenuated C. perfringens-induced cellular cytotoxicity and intestinal barrier damage. Furthermore, L. plantarum Lac16 significantly suppressed C. perfringens-induced gene expressions of proinflammatory cytokines and pattern recognition receptors (PRRs) in IPEC-J2 cells. Moreover, L. plantarum Lac16 preincubation effectively inhibited the phosphorylation of p65 caused by C. perfringens infection. Collectively, probiotic L. plantarum Lac16 exerts protective effects against C. perfringens infection-associated injury in IPEC-J2 cells.
| S-EPMC8620398 | biostudies-literature
Project description:Introduction. Clostridium perfringens (C. perfringens) beta2 toxin (CPB2) is an important virulent factor of necrotic enteritis in both animals and humans. However, studies of its pathogenic roles and functional mechanisms have been hampered due to the difficulty of purification and lack of specific antibodies against this toxin. Methods. A recombinant His-tagged C. perfringens beta2 (rCPB2) toxin and monoclonal antibodies (McAbs) against CPB2 were generated and characterized by assays of cytotoxicity, immunoblotting, ELISA, neutralization, and immunofluorescence. Results. A His-tagged rCPB2 with integrity and cytotoxicity of native CPB2 was purified from E. coli expressing system, which exhibited a moderate cytotoxicity on NCM460 human intestinal epithelial cells. The rCPB2 could induce apoptotic cell death rather than necrotic death in part through a pathway involved in caspase-3 signaling. Mechanistically, rCPB2 was able to first bind to cell membrane and dynamically translocate into cytoplasm for its cytotoxic activity. Three McAbs 1E23, 2G7 and 2H7 were characterized to be able to immunologically react with CPB2 and neutralize rCPB2 cytotoxicity on NCM460 cells. Conclusion. These results indicated the rCPB2 and antibodies generated in this study are useful tools for studies of biological functions and pathogenic mechanisms of CPB2 in future, which warrants for further investigations.
| S-EPMC5031884 | biostudies-literature
Project description:The molecular mechanisms involved in host pathogen interactions at mucosal surfaces needs to be better understood in order to develop immune-mediated methods of protection against pathogens. Cholera toxin (CT) has the rare ability for a protein of inducing robust mucosal immunity in the gut and is therefore an excellent model with which to determine mechanisms of adjuvanticity and immunogenicity at intestinal mucosal surfaces. Jejunal epithelial cells are one of the first sites of antigen encounter. Therefore a porcine intestinal epithelial cell line, IPEC-J2, was cultured in 6-well transwell plates in the presence or absence of 50 ng/ml cholera toxin for up to 8 hours and the cell layer was harvested for gene expression analysis using the Affymetrix porcine genome array and real-time PCR analysis. Affymetrix analysis identified, and real-time PCR analysis of 15 genes confirmed, an increase in gene expression for 59 genes and a decrease in gene expression for 14 genes under CT treatment. An 8 hour time course of expression revealed that by 2-4 hours after CT treatment, all 10 upregulated genes were differentially expressed and by 4-6 hours after CT treatment 3 of the 5 downregulated genes were differentially expressed. These data suggest that the potent mucosal adjuvanticity and immunogenicity of CT derives from rapid alterations in gene expression at the site of first antigen encounter with the immune system. Characterization of early immune gene expression may elucidate potential biological mechanisms for mucosal immune induction leading to the development of effective vaccines against enteric pathogens. Keywords: Treatment comparison, cholera toxin vs untreated at 8h time point
2008-04-01 | GSE10314 | GEO
Project description:In both humans and animals, Clostridium perfringens is an important cause of histotoxic infections and diseases originating in the intestines, such as enteritis and enterotoxemia. The virulence of this Gram-positive, anaerobic bacterium is heavily dependent upon its prolific toxin-producing ability. Many of the ?16 toxins produced by C. perfringens are encoded by large plasmids that range in size from ?45 kb to ?140 kb. These plasmid-encoded toxins are often closely associated with mobile elements. A C. perfringens strain can carry up to three different toxin plasmids, with a single plasmid carrying up to three distinct toxin genes. Molecular Koch's postulate analyses have established the importance of several plasmid-encoded toxins when C. perfringens disease strains cause enteritis or enterotoxemias. Many toxin plasmids are closely related, suggesting a common evolutionary origin. In particular, most toxin plasmids and some antibiotic resistance plasmids of C. perfringens share an ?35-kb region containing a Tn916-related conjugation locus named tcp (transfer of clostridial plasmids). This tcp locus can mediate highly efficient conjugative transfer of these toxin or resistance plasmids. For example, conjugative transfer of a toxin plasmid from an infecting strain to C. perfringens normal intestinal flora strains may help to amplify and prolong an infection. Therefore, the presence of toxin genes on conjugative plasmids, particularly in association with insertion sequences that may mobilize these toxin genes, likely provides C. perfringens with considerable virulence plasticity and adaptability when it causes diseases originating in the gastrointestinal tract.
| S-EPMC3668675 | biostudies-literature
Project description:The incidence of a new, yet unassigned toxin type of Clostridium perfringens containing the genes for the alpha-toxin and the recently described beta2-toxin in horses with intestinal disorders is reported. The study included 18 horses suffering from typical typhlocolitis, 7 horses with atypical typhlocolitis, 16 horses with other intestinal disorders, and 58 horses without intestinal disease. In total, 20 samples of ingesta of the small and large intestines, five biopsy specimens of the intestinal wall, and 74 fecal samples were analyzed bacteriologically. C. perfringens isolates were typed for the presence of the alpha-, beta-, beta2-, and epsilon-toxin and enterotoxin genes by PCR, including a newly developed PCR for the detection of the beta2-toxin gene cpb2. beta2-Toxigenic C. perfringens was detected in samples from 13 of 25 (52%) horses with typical or atypical typhlocolitis, with a particularly high incidence in specimens of ingesta and biopsy specimens (75%), whereas only 6 of 16 specimens from horses with other intestinal diseases yielded beta2-toxigenic C. perfringens. No beta2-toxigenic C. perfringens was found in the samples from the 58 control horses, of which only one fecal sample contained C. perfringens type A. Among the samples from the 15 horses with fatal cases of typical and atypical typhlocolitis 9 (60%) were positive for beta2-toxigenic C. perfringens, whereas samples from only 4 of the 10 (40%) animals with nonfatal cases of infection were positive. We found an interesting correlation between the antibiotic-treated horses which were positive for beta2-toxigenic C. perfringens and lethal progression of the disease. No C. perfringens strains isolated in this study contained genes for the beta- and epsilon-toxins and enterotoxin. The high incidence of beta2-toxigenic C. perfringens in samples of ingesta, biopsy specimens of the intestinal wall, and feces from horses suffering or dying from typhlocolitis together with the absence of this organism in healthy horses provides strong evidence that beta2-toxigenic C. perfringens play an important role in the pathogenesis of typhlocolitis.
| S-EPMC84307 | biostudies-literature
Project description:Clostridium perfringens alpha-toxin is a key mediator of gas gangrene, which is a life-threatening infection that manifests as fever, pain, edema, myonecrosis, and gas production. Alpha-toxin possesses phospholipase C and sphingomyelinase activities. The toxin is composed of an N-terminal domain (1-250 aa, N-domain), which is the catalytic site, and a C-terminal domain (251-370 aa, C-domain), which is the membrane-binding site. Immunization of mice with the C-domain of alpha-toxin prevents the gas gangrene caused by C. perfringens, whereas immunization with the N-domain has no effect. The central loop domain (55-93 aa), especially H….SW(84)Y(85)….G, plays an important role in the interaction with ganglioside GM1a. The toxin binds to lipid rafts in the presence of a GM1a/TrkA complex, and metabolites from phosphatidylcholine to diacylglycerol through the enzymatic activity of alpha-toxin itself. These membrane dynamics leads to the activation of endogenous PLC?-1 via TrkA. In addition, treatment with alpha-toxin leads to the formation of diacylglycerol at membrane rafts in ganglioside-deficient DonQ cells; this in turn triggers endocytosis and cell death. This article summarizes the current the membrane-binding mechanism of alpha-toxin in detail.
| S-EPMC4690130 | biostudies-literature
Project description:The virulent properties of the common human and livestock pathogen Clostridium perfringens are attributable to a formidable battery of toxins. Among these are a number of large and highly modular carbohydrate-active enzymes, including the mu-toxin and sialidases, whose catalytic properties are consistent with degradation of the mucosal layer of the human gut, glycosaminoglycans, and other cellular glycans found throughout the body. The conservation of noncatalytic ancillary modules among these enzymes suggests they make significant contributions to the overall functionality of the toxins. Here, we describe the structural basis of an ultra-tight interaction (K(a) = 1.44 x 10(11) M(-1)) between the X82 and dockerin modules, which are found throughout numerous C. perfringens carbohydrate-active enzymes. Extensive hydrogen-bonding and van der Waals contacts between the X82 and dockerin modules give rise to the observed high affinity. The mu-toxin dockerin module in this complex is positioned approximately 180 degrees relative to the orientation of the dockerin modules on the cohesin module surface within cellulolytic complexes. These observations represent a unique property of these clostridial toxins whereby they can associate into large, noncovalent multitoxin complexes that allow potentiation of the activities of the individual toxins by combining complementary toxin specificities.
| S-EPMC2527888 | biostudies-literature