Project description:Clostridium difficile (C. difficile) strains belonging to PCR ribotype 027, PFGE type NAP1, REA type B1 and toxinotype III, termed NAP1/027, have been implicated in the increased frequency of outbreaks of Clostridium difficile-associated diarrhoea (CDAD) in North America and Europe. The NAP1/027 strains appears to be more virulent with an increased mortality and frequency of relapse. Current European C. difficile microarrays are designed to the first sequenced and annotated C. difficile complete genome - strain 630 (ribotype 12). A high density oligonucleotide microarray was designed to C. difficile 630 (CD630) sequence and extra probes corresponding to two PCR ribotypes O27 strains C. difficile R20291 and QCD-32g58 were also included. Comparative genomic hybridisation was used to identify markers of ribotype 027 strains and markers to identify CD630. Strains hybridised to the array included the most prevalent ribotypes found in the UK and Europe (106 and 001) as well as the emerging hypervirulent ribotype 078.
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:The strict anaerobe Clostridium difficile is an important nosocomial enteropathogen that has become the most common cause of antibiotic-associated diarrhea. The oxygen-resistant C. difficile spores play a central role in the infectious cycle, contributing to transmission, infection and recurrence. The spore surface layers, coat and exosporium, enable resistance of the spores to extreme physical and chemical stresses. Despite the critical importance of the spore in C. difficile infection, little is known about the mechanisms that orchestrate the assembly of its external layers. In this study, we identified and characterized a new C. difficile spore protein, named CotL, which is required for the assembly of the spore surface layers. The cotL gene was expressed in the mother cell compartment under the dual control of σE and σK. The CotL protein was localized in the C. difficile spore coat and cotL mutant spores had a major morphologic defect at the level of the coat/exosporium layers, as observed by transmission electron microscopy. Accordingly, spores of the cotL mutant contained a reduced amount of several proteins of the coat and exosporium, including CotB, CotE and CdeC. Additionally, cotL inactivation resulted in a defect in localization of late spore coat proteins in sporulating cells. Finally, spores of the cotL mutant were more sensitive to lysozyme and were impaired in germination. These defects are most likely associated with the structurally altered coat. Collectively, these results strongly suggest that CotL is a morphogenetic protein essential for the assembly of the spore coat in C. difficile.
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: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: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.
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