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: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:The gut microbiome engenders colonization resistance against the diarrheal pathogen Clostridioides difficile but the molecular basis of this colonization resistance is incompletely understood. A prominent class of gut microbiome-produced metabolites important for colonization resistance against C. difficile is short chain fatty acids (SCFAs). In particular, one SCFA (butyrate) decreases the fitness of C. difficile in vitro and is correlated with C. difficile-inhospitable gut environments, both in mice and in humans. Here, we demonstrate that butyrate-dependent growth inhibition in C. difficile occurs under conditions where C. difficile also produces butyrate as a metabolic end product. Furthermore, we show that exogenous butyrate is internalized into C. difficile cells and is incorporated into intracellular CoA pools where it is metabolized in a reverse (energetically unfavorable) direction to crotonyl-CoA and (S)-3-hydroxybutyryl-CoA and/or 4-hydroxybutyryl-CoA. This internalization of butyrate and reverse metabolic flow of butyrogenic pathway(s) in C. difficile coincides with alterations in toxin release and sporulation. Together, this work highlights butyrate as a marker of a C. difficile inhospitable environment to which C. difficile responds by releasing its diarrheagenic toxins and producing environmentally-resistant spores necessary for transmission between hosts. These findings provide foundational data for understanding the molecular and genetic basis of how C. difficile growth is inhibited by butyrate and how butyrate alters C. difficile virulence in the face of a highly competitive and dynamic gut environment.
Project description:Clostridioides difficile can cause severe infections in the gastrointestinal tract and affects almost half a million people in the U.S every year. Upon establishment of infection, a strong immune response is induced. We sought to investigate the dynamics of the mucosal host response during C. difficile infection.
Project description:The ability of bacterial pathogens to establish recurrent and persistent infections is frequently associated with their ability to form biofilms. Clostridioides difficile infections have a high rate of recurrence and relapses and it is hypothesised that biofilms are involved in its pathogenicity and persistence. Biofilm formation by C. difficile is still poorly understood. It has been shown that specific molecules such as deoxycholate (DCA) or metronidazole induce biofilm formation, but the mechanisms involved remain elusive. In this study, we describe the role of the lipoprotein CD1687 in the DCA-induced biofilm formation of C. difficile. We showed that the expression of CD1687, which is part of an operon within the CD1685-CD1689 gene cluster, is controlled by multiple transcription starting sites induced for some of them in response to DCA. Only CD1687 is required for biofilm formation and the overexpression of CD1687 is sufficient to induce biofilm formation. Using RNAseq analysis, we showed that CD1687 affects the expression of transporters and metabolic pathways and we identified several potential binding partners by pull-down assay, including transport-associated extracellular proteins. We then demonstrated that CD1687 is surface exposed in C. difficile, and this localization is required for DCA-induced biofilm formation. Given this localization and that C. difficile forms eDNA-rich biofilms, we confirmed that CD1687 binds DNA in a non-specific manner. We thus hypothesize that CD1687 is a component of the downstream response to DCA leading to biofilm formation by promoting interaction between the cells and the biofilm matrix by binding eDNA.
Project description:Metabolomic and transcriptomic analysis of changes in the exponential and stationary phase of Clostridioides difficile after cultivation in casamino acids medium (reference) and supplemented with L-lactate and the connection to toxin production.
Project description:Clostridioides difficile is a Gram-positive opportunistic pathogen that results in 220,000 infections, 12,000 deaths, and upwards of $1 billion in medical costs in the US each year. C. difficile is highly resistant to a variety of antibiotics, but we have a poor understanding of how C. difficile senses and responds to antibiotic stress, and how such sensory systems affect clinical outcomes. There has been recent interest in using a daptomycin analog, Surotomycin, to treat C. difficile infections. We have identified a spontaneous C. difficile mutant that displays increased daptomycin resistance. We performed whole-genome sequencing that this mutant possessed a nonsense mutation, S605*, in draS which encodes a putative sensor histidine kinase of a two-component system (TCS). The draSS605* mutant has approximately a 4-8-fold increase in daptomycin MIC compared to WT. We found that expression of constitutively active DraRD54E in WT increases daptomycin resistance 8-16-fold and increases bacitracin resistance ~4-fold. We found that a selection of lipid II inhibiting compounds leads to increased activity of the luciferase-based reporter, PdraR-sLucopt including vancomycin, bacitracin, ramoplanin, and daptomycin. Using RNA-seq we identified the DraRS regulon. Interestingly, we found that DraRS can induce expression of the previously identified hex locus required for synthesis of a novel glycolipid produced in C. difficile. Our data suggest that induction of the hex locus by DraR explains some, but not all, of the DraR-induced daptomycin and bacitracin resistance.
Project description:Systems biology approach of Clostridioides difficile to analyze the temporal changes in the intracellular and extracellular metabolme, transcriptome and proteome along the growth curve in casamino acids medium and the connection to toxin production.