Project description:Gene expression level of Clostridioides difficile (C. difficile) strain R20291 comparing control C. difficile carring pMTL84151 as vector plasmid with C. difficile conjugated with a pMTL84151-03890 gene. Goal was to determine the effects of 03890 gene conjugation on C. difficile strain R20291 gene expression.
Project description:RNA-seq was conducted to uncover the regulatory roles of RsbW in the lifestyle of Clostridioides difficile strain R20291. RNA were extracted from planktonic cultures, which were grown in BHI-S (Sigma-Aldrich, USA) to early stationary phase (10h). The cells were re-suspended in LETS buffer and lysed using Fast-prep 24 instrument (MP Bioscience) and RNA was extracted using 1 ml Trizol (Ambion, USA). Samples were cleaned and sequenced by Novogene Company Limited, PE150 with approximately 10 million reads per sample. After QC, reads were aligned to R20291 (NC_013316.1) with Bowtie2 and readcounts were generated with Samtools and Bedtools. Differential gene expression analysis was conducted with DESeq2 with a p-value of 0.05, differential gene expression was determined with ± -2 logFC.
Project description:The intestines house a diverse microbiota that must compete for nutrients to survive, but the specific limiting nutrients that control pathogen colonization are not clearly defined. Clostridioides difficile colonization typically requires prior disruption of the microbiota, suggesting that outcompeting commensals for resources is key in establishing C. difficile infection (CDI). The immune protein calprotectin (CP) is released into the gut lumen during CDI to chelate zinc (Zn) and other essential nutrient metals. Yet, the impact of Zn limitation on C. difficile colonization is unknown. To define C. difficile responses to Zn limitation, we performed RNA sequencing on C. difficile exposed to CP. In media with CP, C. difficile upregulated genes involved in metal homeostasis and amino acid metabolism.
Project description:Clostridioides difficile is one of the most common nosocomial pathogens and a global public health threat. Upon colonization of the gastrointestinal tract, C. difficile is exposed to a rapidly changing polymicrobial environment and a dynamic metabolic milieu. Despite the link between the gut microbiota and susceptibility to C. difficile, the impact of synergistic interactions between the microbiota and pathogens on the outcome of infection is largely unknown. Here, we show that microbial cooperation between C. difficile and Enterococcus has a profound impact on the growth, metabolism, and pathogenesis of C. difficile.. Through a process of nutrient restriction and metabolite cross-feeding, E. faecalis shapes the metabolic environment in the gut to enhance C. difficile fitness and increase toxin production. These findings demonstrate that members of the microbiota, such as Enterococcus, have a previously unappreciated impact on C. difficile behavior and virulence.
Project description:We used RNA-seq to conduct a genome-wide transcriptomic analysis of the C. difficile strain R20291 carrying the newly introduced ϕCD38-2 prophage. A total of 39 bacterial genes were differentially expressed in the R20291 lysogen, 26 of them being downregulated. Several of the regulated genes encode transcriptional regulators and PTS subunits involved in glucose, fructose and glucitol/sorbitol uptake and metabolism. Also of note, the presence of ϕCD38-2 upregulated the expression of a group of regulatory genes located in phi-027, a resident prophage common to most ribotype 027 isolates. The most differentially expressed gene was the conserved phase-variable cell wall protein CwpV, which was upregulated by about 20-fold in the lysogen. This is the first study describing the global response of C. difficile to the presence of a newly introduced prophage.
Project description:Clostridioides difficile interactions with the gut mucosa are crucial for colonisation and establishment of infection, however key infection events during the establishment of disease are still poorly defined. To better understand the initial events that occur during C. difficile colonisation, we employed a dual RNA-sequencing approach to study the host and bacterial transcriptomic profiles during C. difficile infection in a dual-environment in vitro human gut model. Temporal changes in gene expression were analysed over 3-24h post infection and comparisons were made with uninfected controls.
Project description:We used RNA-seq to conduct a genome-wide transcriptomic analysis of the C. difficile strain R20291 carrying the newly introduced M-OM-^UCD38-2 prophage. A total of 39 bacterial genes were differentially expressed in the R20291 lysogen, 26 of them being downregulated. Several of the regulated genes encode transcriptional regulators and PTS subunits involved in glucose, fructose and glucitol/sorbitol uptake and metabolism. Also of note, the presence of M-OM-^UCD38-2 upregulated the expression of a group of regulatory genes located in phi-027, a resident prophage common to most ribotype 027 isolates. The most differentially expressed gene was the conserved phase-variable cell wall protein CwpV, which was upregulated by about 20-fold in the lysogen. This is the first study describing the global response of C. difficile to the presence of a newly introduced prophage. Two strains (WT and lysogen) were subjected for RNA-seq analysis with two biological replicates each.