Project description:Clostridioides difficile (formerly Clostridium difficile) colonizes the gastrointestinal tract following disruption of the microbiota and can initiate a spectrum of clinical manifestations ranging from asymptomatic to life-threatening colitis. Following antibiotic treatment, luminal oxygen concentrations increase, exposing gut microbes to potentially toxic reactive oxygen species (ROS). Though typically regarded as a strict anaerobe, C. difficile can grow at low oxygen concentrations. How the bacterium adapts to a microaerobic environment and whether those responses to oxygen are conserved amongst strains is not entirely understood. Here, two C. difficile strains (630 and CD196) were cultured in 1.5% oxygen and the transcriptional response was evaluated via RNA-sequencing. During growth in a microaerobic environment, several genes predicted to protect against oxidative stress were upregulated, including ruberythrins and rubredoxins. Genes involved in metal homeostasis were positively correlated with increasing oxygen levels and were also amongst the most differentially transcribed. These included ferrous iron transporters (feo), a zinc transporter (zupT), and predicted siderophore transporters. To directly compare the transcriptional landscape between C. difficile strains, a ‘consensus-genome’ was generated. On the basis of the identified conserved genes, basal transcriptional differences as well as variations in the response to oxygen were evaluated. While several responses were similar between the strains, there were significant differences in the abundance of transcripts for amino acid and carbohydrate metabolism. Furthermore, homologous metal homeostasis genes were similarly transcribed, but the intracellular metal concentrations significantly varied both in an oxygen-dependent and independent manner. Overall, these results indicate that C. difficile adapts to grow in a low oxygen environment through transcriptional changes, though the specific strategy employed varies between strains.
Project description:Clostridium difficile is a gram-positive, spore-forming enteric anaerobe which can infect humans and a wide variety of animal species. Recently, the incidence and severity of human C. difficile infection has markedly increased. In this study, we evaluated the genomic content of 73 C. difficile strains isolated from humans, horses, cattle, and pigs by comparative genomic hybridization with microarrays containing coding sequences from C. difficile strains 630 and QCD-32g58. The sequenced genome of C. difficile strain 630 was used as a reference to define a candidate core genome of C. difficile and to explore correlations between host origins and genetic diversity. Approximately 16% of the genes in strain 630 were highly conserved among all strains, representing the core complement of functional genes defining C. difficile. Absent or divergent genes in the tested strains were distributed across the entire C. difficile 630 genome and across all the predicted functional categories. Interestingly, certain genes were conserved among strains from a specific host species, but divergent in isolates with other host origins. This information provides insight into the genomic changes which might contribute to host adaptation. Due to a high degree of divergence among C. difficile strains, a core gene list from this study offers the first step toward the construction of diagnostic arrays for C. difficile.
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:Clostridium difficile is a gram-positive, spore-forming enteric anaerobe which can infect humans and a wide variety of animal species. Recently, the incidence and severity of human C. difficile infection has markedly increased. In this study, we evaluated the genomic content of 73 C. difficile strains isolated from humans, horses, cattle, and pigs by comparative genomic hybridization with microarrays containing coding sequences from C. difficile strains 630 and QCD-32g58. The sequenced genome of C. difficile strain 630 was used as a reference to define a candidate core genome of C. difficile and to explore correlations between host origins and genetic diversity. Approximately 16% of the genes in strain 630 were highly conserved among all strains, representing the core complement of functional genes defining C. difficile. Absent or divergent genes in the tested strains were distributed across the entire C. difficile 630 genome and across all the predicted functional categories. Interestingly, certain genes were conserved among strains from a specific host species, but divergent in isolates with other host origins. This information provides insight into the genomic changes which might contribute to host adaptation. Due to a high degree of divergence among C. difficile strains, a core gene list from this study offers the first step toward the construction of diagnostic arrays for C. difficile.investigated by determining changes in transcript profiles when aerobic steady-state cultures were depleted of air.
Project description:We compared transcriptomes of wild-type and ∆vanS strains of Clostridioides difficile 630 growing in the presence or absence of peptidoglycan-targeting antibiotics, vancomycin or ramoplanin. VanS is a histidine kinase of a two-component system that regulates expression of the vancomycin-induced vanG operon.
Project description:The Gram-positive bacterium Clostridium difficile, a leading cause of antibiotic-associated pseudomembranous colitis, has received increasing attention due to a rising incidence of clinical C. difficile infections (CDI). Despite progress understanding bacterial factors that promote CDI-associated morbidity and mortality, many fundamental aspects of C. difficile biology remain to be explored. Compared to other Gram-positive pathogens, little is known about the bacterium’s transcriptome architecture and in particular mechanisms of post-transcriptional control. To close this knowledge gap, we have applied a suite of transcriptome-focused techniques, including transcription start site mapping (dRNA-seq), transcription termination mapping, and Hfq RIP-seq, resulting in a single-nucleotide resolution RNA map of C. difficile strain 630.
Project description:Investigation of whole genome gene expression level changes in a Clostridium difficile fur (ferric uptake regulator) mutant, compared to the wild type strain 630 erm. The fur mutant analyzed in this study is further described in Ho and Ellermeier (2015) J. Bacteriology A microarray study using total RNA recovered from three separate wild type cultures of Clostridium difficile 630 erm strain and three separate cultures of a fur mutant strain (ltrA::ermR) were grown in Tryptone-Yeast Extract medium containing 0.25 mM ferric chloride . Each chip measures the expression level of 3,786 of the 3,787 open reading frames of the C. difficile 630 genome with 18 probes (60 oligomers each) for each gene.
Project description:We illustrate how metabolically distinct species of Clostridia can protect against or worsen Clostridioides difficile infection, modulating the pathogen's colonization, growth, and virulence to impact host survival. Gnotobiotic mice colonized with the amino acid fermenter Paraclostridium bifermentans survived infection while mice colonized with the butyrate-producer, Clostridium sardiniense, more rapidly succumbed. Systematic in vivo analyses revealed how each commensal altered the gut nutrient environment, modulating the pathogen's metabolism, regulatory networks, and toxin production. Oral administration of P. bifermentans rescued conventional mice from lethal C. difficile infection via mechanisms identified in specifically colonized mice. Our findings lay the foundation for mechanistically informed therapies to counter C. difficile disease using systems biologic approaches to define host-commensal-pathogen interactions in vivo.