Project description:Toxin A and B from Clostridium difficile are the primary virulence factors in Clostridium difficile disease. The changes in gene transcription of human colon epithelial cells were investigated in vitro in order to better understand the many effects of both toxins.
Project description:Transcriptional analysis of Clostridium difficile R20291 in biofilm formation, planktonic state and grown on blood agar RNA sequencing was performed on Clostridium difficile R20291 in three different conditions: Biofilm formation, plantonic state and grown on blood agar plates. Each condtion has 3 replicates.
Project description:Analysis of Clostridium difficile (Cd) from the cecal contents of germ-free mice or Bacteroides thetaiotaomicron (Bt)-monocolonized mice on a standard, polysaccharide rich diet or polysaccharide deficient diet 5 days after infection. Results identify genes that are involved in the Cd response to diet, in vivo colonization and in interactions with Bt. In vitro transcriptional profiles of Clostridium difficile obtained from cecal contents of germ-free or Bt-monocolonized mice on a standard, polysaccharide rich or polysaccharide deficient diet. 4 samples/group. 2 control genomic DNA samples for Clostridium difficile and 2 reference genomic DNA samples for Bacteroides thetaiotaomicron Please note that 4 control samples (genomic DNA) were used to determine whether the genomic DNA correctly bound to the probes and thus, were not included in data processing (i.e no processed/normalized data).
Project description:Clostridium difficile is an anaerobic spore-forming rod-shaped gram-positive bacterium that can infect both humans and animals. Most studies on the pathogenesis of C. difficile have focused on its toxins and their effect on the host cells. Recently, we utilized microarrays to identify conserved and divergent genes associated with virulence in C. difficile isolates from humans and animals. Our data provided the first clue toward a complex mechanism underlying host adaptation and pathogenesis. Microarray technology offers an efficient high-throughput tool to study the transcriptional profiles of pathogens and infected host cells. Transcriptomes of C. difficile after exposure to environmental and antibiotic stresses and those of human epithelial colorectal Caco-2 cells upon TcdA treatment have been analyzed. To our knowledge, there are still no reports on the transcriptomic study of host-pathogen interactions for C. difficile infection (CDI). In vitro analyses of interplay between host and pathogen are essential to unravel the mechanisms of infection and to investigate the host response to infection. We therefore employed microarrays to study both bacterial and human cellular transcriptome kinetics during CDI to Caco-2 cells. Here we present a large-scale analysis of transcriptional profiles to reveal molecular determinants playing a role in C. difficile pathogenesis and the host response. We found that there were 254 and 224 differentially-expressed genes after CDI in C. difficile and Caco-2 cells, respectively. These genes are clustered according to their functional categories and their potential roles in pathogenesis and host response are discussed. Our results will not only increase our understanding on the host-pathogen interaction, but may also provide targets for drug development. Clostridium difficile: Control vs Infection (time course) mRNA with genomic DNA of tested and reference strains Caco-2 cells: Control vs Infected with Clostridium difficile Time-course experiments of Caco-2 cells infected with C. difficile for 30, 60 and 120 min
Project description:Clostridium difficile is an important nosocomial pathogen and the leading cause of hospital-acquired diarrhea. Antibiotic use is the primary risk factor for the development of C. difficile-associated disease because it disrupts normal protective gut flora and enables C. difficile to colonize the colon. C. difficile damages host tissue by secreting toxins and disseminates by forming spores. The toxin-encoding genes, tcdA and tcdB are part of a pathogenicity locus, which also encodes the gene tcdR that codes for the toxin genes positive regulator. TcdR is an alternate sigma factor that initiates transcription of tcdA and tcdB at their promoters. Alternative sigma factors are known to regulate virulence and virulence associated genes in many pathogenic bacteria. We created a tcdR mutant in the epidemic-type C. difficile R20291 strain in an attempt to identify the global role of tcdR. A site-directed mutation in tcdR affected both toxin production and sporulation in C. difficile R20291. Spores derived from the tcdR mutant were found to be mildly temperature sensitive. Moreover, nearly two fold more taurocholate was needed to germinate spores from the tcdR mutant than the spores prepared from the wild-type parent strain. Comparison of the tcdR mutant transcriptome with the parent strain revealed many differentially expressed late sporulation genes in the tcdR mutant. These data suggests that gene regulatory networks of toxin production and sporulation in Clostridium difficile are linked with each other.
Project description:The incidence of Clostridium difficile infection has been steadily rising over the past decade. Its increased rate is associated with the specific NAP1/BI/027 strains which are “hypervirulent” and have led to several large outbreaks since their emergence. However, the relation between their outbreaks and virulence regulation mechanisms remains unclear. It has been reported that the major virulence factor TcdA and TcdB in C. difficile could be repressed by cysteine. Here, we investigated functional and virulence-associated regulation of C. difficile R20291 in response to cysteine stress by using a time-resolved genome-wide transcriptional analysis. Dramatic changes of gene expression in C. difficile were revealed in functional categories related to transport, metabolism, and regulators under cysteine stress during different phases of growth.
Project description:Toxin A and B from Clostridium difficile are the primary virulence factors in Clostridium difficile disease. The changes in gene transcription of human colon epithelial cells were investigated in vitro in order to better understand the many effects of both toxins. HCT-8 cells were treated with 100 ng/ml of either Toxin A or B (TcdA or TcdB). RNA was isolated 2, 6, and 24 hours after addition of toxin from untreated and toxin-treated cells.