Project description:Multiple Sclerosis (MS) is a complex disease of the CNS believed to require one or more environmental triggers and is characterized by episodic formation of inflammatory demyelinating lesions in the brain and spinal cord. Gut dysbiosis is a common feature in MS and here, using enhanced and quantitative PCR detection, we show that people with MS are more likely to harbor and have higher abundance of epsilon toxin (ETX)-producing strains of Clostridium perfringens within their gut microbiome compared to healthy controls (HC). MS patient-derived isolates produce functional ETX and have a genetic architecture typical of highly conjugative plasmids. In the active immunization model of experimental autoimmune encephalomyelitis (EAE), where pertussis toxin (PTX) is used to overcome CNS immune privilege, we find that ETX can substitute for PTX in disease induction. In contrast to PTX-induced EAE, where inflammatory demyelination is largely restricted to the spinal cord, ETX-induced EAE results in multifocal demyelination in the corpus callosum, thalamus, cerebellum, brainstem, and spinal cord, more akin to the lesion distribution observed in MS. Transcriptional profiles from CNS endothelial cells reveal ETX-induced genes that are known to play a role in overcoming CNS immune privilege. Together, these findings support ETX-producing strains of C. perfringens as biologically plausible pathogens in MS to trigger inflammatory demyelination in the context of circulating myelin autoreactive lymphocytes.
Project description:Analysis of proteins from botulinum neurotoxin-producing Clostridium butyricum strains grown upon exposure to air. This strain usually lives in anaerobic environments, but we found that it is able to keep growing and producing toxin in vitro upon exposure to air. This project analyzes the proteins differentially expressed both in anaerobic and aerobic conditions.
Project description:Enterotoxin-producing C. perfringens type A is a common cause of food poisonings. The cpe encoding the enterotoxin can be chromosomal (genotype IS1470) or plasmid-borne (genotypes IS1470-like-cpe or IS1151-cpe). The chromosomal cpe-carrying C. perfringens are a more common cause of food poisonings than plasmid-borne cpe-genotypes. The chromosomal cpe-carrying C. perfringens type A strains are generally more resistant to most food-processing conditions than plasmid-borne cpe-carrying strains. On the other hand, the plasmid-borne cpe-positive genotypes are more commonly found in human feces than chromosomal cpe-positive genotypes, and humans seem to be a reservoir for plasmid-borne cpe-carrying strains. Thus, it is possible that the epidemiology of C. perfringes type A food poisonings caused by plasmid-borne and chromosomal cpe-carrying strains is different. A DNA microarray was designed for analysis of genetic relatedness between the different cpe-positive and cpe-negative genotypes of C. perfringens strains isolated from human, animal, environmental and food samples. The DNA microarray contained two probes for all protein-coding sequences in the three genome-sequenced strains (C. perfringens type A strains 13, ATCC13124, and SM101). The chromosomal and plasmid-borne C. perfringens genotypes were grouped into two distinct clusters, one consisting of the chromosomal cpe-genotypes and the other consisting of plasmid-borne cpe-genotypes. Analysis of the variable gene pool complemented with the growth studies demonstrate different carbohydrate and amine metabolism in the chromosomal and plasmid-borne cpe-carrying strains, suggesting different epidemiology of the cpe-positive C. perfringens strain groups.
Project description:Necrotic enteritis (NE) in broiler chickens, caused by the overgrowth of toxin-producing strains of Clostridium (C.) perfringens, results in the development of necrotic lesions, compromised intestinal health, and significant economic losses in poultry production. This study aims to analyze the blood proteome of broiler chickens affected by NE, providing insights into the host's response to the infection. Using MS/MS-based proteomics, blood plasma samples from broilers with necrotic lesions of different severity were analyzed and compared to healthy controls. A total of 412 proteins were identified, with 63 showing significant differences and (for some of those) correlating with disease severity. Gene Set Enrichment Analysis (GSEA) revealed that proteins affected by NE were predominantly associated with the immune and signaling processes and extracellular matrix (ECM) structures. Notably, regulated proteins were significantly involved in bioprocesses related to complement activation, acute phase reaction, proteolysis and humoral immune response. The findings suggest that the changes in plasma proteins in response to NE are driven by the host's intensified efforts to counteract the infection, demonstrating a.o. a notable reduction in peptides from ECM-related proteins in the blood of NE-affected birds. Overall, proteomics results underscored the attempts of the host to manage tissue damage and inflammation, indicating a coordinated effort to mitigate the pathogenic impact of C. perfringens. This study provides a deeper understanding of the host-pathogen interactions and potential targets for therapeutic intervention.
Project description:Purpose: RNA-Seq has become a powerful tool for investigating transcriptional profiles in gene expression analysis, which would help to reveal the molecular mechanism of Clostridium perfringens type C infecting the piglets. In this study, we analyzed the transcriptome profiles of the spleen of piglets caused by Clostridium perfringens type Cens type C. Methods: 30 normal 7-day-old piglets (Y x L), without infecting Clostridium perfringens type C, Escherichia coli and Salmonella, were selected as experimental subjects. 25 piglets were randomly selected as the experimental group, which were disposed once a day for 5 days. Each piglet was dosed with 1 ml of bouillon culture-medium inoculated Clostridium perfringens type C at 37℃ for 16h, which approximate to 1 x109 CFU per ml. Then, 5 piglets were randomly selected as the control group (SC), which were taken the equal volume medium for 5 days.Based on total diarrhea scores, 25 piglets were ranked from high to low. The top and last five piglet were considered as sensitive group (SS) and resistant group (SR), respectively. Finally, spleen were collected and sequenced for lncRNA and mRNA. Results: RNA libraries constructed from spleen of piglets caused by Clostridium perfringens type C were sequenced. A total of 1,450,292,484 clean reads were generated. Among them, 2056 novel lncRNA transcripts corresponding to 1561 lncRNA genes were identified, including 1811 intergenic lncRNAs and 245 anti-sense lncRNAs. The identified spleen lncRNAs shared some characteristics, such as fewer exons and shorter length, with the lncRNAs in other animal. Notably, in pairwise comparisons between the libraries of spleen tissue at the different group, a total of 247 lncRNA and 2170 mRNA were differentially expressed (P < 0.05). Function analyses indicated that these differentially expressed lncRNAs and mRNAs play roles in defensing Clostridium perfringens type C, which were enriched in immune-related biological processes, such as the antigen processing and presentation, TNF signaling pathway, NF-kappa B signaling pathway, B cell receptor signaling pathway and MAPK signaling pathway. Conclusions: This study provides the information of spleen-related lncRNAs in swine diarrhea with Clostridium perfringens type C. We also analyzed all lncRNA’s genomic feature and expression. Bioinformatic analysis indicates that some lncRNAs participated in important biological processes associated with defeasing Clostridium perfringens type C, such as antigen processing and presentation, the MHC protein complex and regulation of autophagy.
Project description:Purpose: The purpose of this study is to clarify the response of Clostridium perfringens ATCC 13124 to host polysaccharide. Methods: Clostridium perfringens ATCC 13124 cells were cultured anaerobically in a medium containing Minimal medium-like condition Poor + medium, medium in which hyaluronic acid or mucin was added to Poor + medium. Total RNA was extracted from bacterial cells by the Hot-Phenol method. Samples for RNA-seq were prepared according to the Illmina protocol available from the manufacturer. Array leads passed through quality filters were analyzed at the transcript isoform level using bowtie v 1.1.2. Results: Using the optimized data analysis workflow, we mapped about 50 million sequence leads per sample to the whole genome of Clostridium perfringens ATCC 13124. In addition, 2735 transcripts in C. perfringens ATCC 13124 were identified using a Bowtie aligner. Lead counts per genome were extracted from known gene annotations using the HTSeq program.
Project description:Purpose: RNA-Seq has become a powerful tool for investigating transcriptional profiles in gene expression analysis, which would help to reveal the molecular mechanism of Clostridium perfringens type C infecting the piglets. In this study, we analyzed miRNA profiles of the ileum of piglets caused by Clostridium perfringens type C. Methods: 30 normal 7-day-old piglets (Y x L), without infecting Clostridium perfringens type C, Escherichia coli and Salmonella, were selected as experimental subjects. 25 piglets were randomly selected as the experimental group, which were disposed once a day for 5 days. Each piglet was dosed with 1 ml of bouillon culture-medium inoculated Clostridium perfringens type C at 37℃ for 16h, which approximate to 1 x10e9 CFU per ml. Then, 5 piglets were randomly selected as the control group (IC), which were taken the equal volume medium for 5 days.Based on total diarrhea scores, 25 piglets were ranked from high to low. The top and last five piglet were considered as sensitive group (IS) and resistant group (IR), respectively. Finally, ileum were collected and sequenced for miRNA. Result: 53 differentially expressed miRNAs were found. KEGG pathway analysis for target genes revealed that these miRNAs were involved in ErbB signaling pathway, MAPK signaling pathway, Jak-STAT signaling pathway and Wnt signaling pathway. The expression correlation analysis between miRNAs and target genes revealed that the expression of miR-7134-5p had negative correlation with target NFATC4, miR-500 had negative correlation with target ELK1, HSPA2 and IL7R, and miR-92b-3p had negative correlation with target CLCF1 in ileum of IR vs IS group, suggesting that miR-7134-5p targeting to NFATC4, miR-500 targeting to ELK1, HSPA2 and IL7R, and miR-92b-3p targeting to CLCF1 were probably involved in piglet resisting C. perfringens type C. Conclusions: The results will provide value resources for better understanding of the genetic basis of C. perfringens type C resistance in piglet and lays a new foundation for identifying novel markers of C. perfringens type C resistance
Project description:Purpose: RNA-Seq has become a powerful tool for investigating transcriptional profiles in gene expression analysis, which would help to reveal the molecular mechanism of Clostridium perfringens type C infecting the piglets. In this study, we analyzed miRNA profiles of the spleen of piglets caused by Clostridium perfringens type C. Methods: 30 normal 7-day-old piglets (Y x L), without infecting Clostridium perfringens type C, Escherichia coli and Salmonella, were selected as experimental subjects. 25 piglets were randomly selected as the experimental group, which were disposed once a day for 5 days. Each piglet was dosed with 1 ml of bouillon culture-medium inoculated Clostridium perfringens type C at 37℃ for 16h, which approximate to 1 x109 CFU per ml. Then, 5 piglets were randomly selected as the control group (SC), which were taken the equal volume medium for 5 days.Based on total diarrhea scores, 25 piglets were ranked from high to low. The top and last five piglet were considered as sensitive group (SS) and resistant group (SR), respectively. Finally, spleen were collected and sequenced for miRNA. Result: 88 differentially expressed miRNAs were found. KEGG pathway analysis for target genes revealed that these miRNAs were involved in MAPK signaling pathway, mTOR signaling pathway, FoxO signaling pathway, p53 signaling pathway and ECM-receptor interaction. And 4 miRNAs (miR-133b, miR-532-3p, miR-339-5p and miR-331-3p) of closely related to piglets resistance to C. perfringens type C were obtained. The expression correlation analysis between these miRNAs and target genes revealed that the expression of miR-133b and miR-532-3p had negative correlation with their target NFATC4, miR-339-5p had negative correlation with target HTRA3, and miR-339-5p and miR-331-3p had negative correlation with target TNFAIP8L2 in spleen, suggesting that miR-133b and miR-532-3p targeting to NFATC4, miR-339-5p targeting to HTRA3, and miR-339-5p and miR-331-3p targeting to TNFAIP8L2 were probably involved in piglet resisting C. perfringens type C. Conclusions:The results will provide value resources for better understanding of the genetic basis of C. perfringens type C resistance in piglet and lays a new foundation for identifying novel markers of C. perfringens type C resistance.
Project description:Clostridium difficile is an emerging nosocomial pathogen of increasing importance and virulence but our ability to study the molecular mechanisms underlying the pathogenesis of C. difficile-associated disease has been limited because of a lack of tools for its genetic manipulation. We have now developed a reproducible method for the targeted insertional inactivation of chromosomal C. difficile genes. The approach relies on the observation that an Escherichia coli?Clostridium perfringens shuttle vector is unstable in C. difficile and can be used as a form of conditional lethal vector to deliver gene constructs to the chromosome. We have used this methodology to insertionally inactivate two putative response regulator genes, rgaR and rgbR, which encode proteins with similarity to the toxin gene regulator, VirR, from C. perfringens. Transcriptomic analysis demonstrated that the C. difficile RgaR protein positively regulated four genes, including a putative agrBD operon. The RgaR protein was also purified and shown to bind specifically to sites that contained two consensus VirR boxes located just upstream of the putative promoters of these genes. The development of this methodology will significantly enhance our ability to use molecular approaches to develop a greater understanding of the ability of C. difficile to cause disease. Data is also available from http://bugs.sgul.ac.uk/E-BUGS-39