Global transcriptional control by glucose and carbon regulator CcpA in Clostridium difficile

Laboratoire Pathogenèse des Bactéries Anaérobies, Département de Microbiologie Institut Pasteur, Université Paris Diderot, Sorbonne Paris Cité, Cellule Pasteur, 25 rue du Docteur Roux, Paris 75015, France, Institute for Information Transmission Problems, Russian Academy of Sciences, Moscow 127994, Russia and Sanford-Burnham Medical Research Institute, La Jolla, CA 92037, USA.
Nucleic Acids Research (Impact Factor: 9.11). 09/2012; 40(21). DOI: 10.1093/nar/gks864
Source: PubMed

ABSTRACT The catabolite control protein CcpA is a pleiotropic regulator that mediates the global transcriptional response to rapidly catabolizable carbohydrates, like glucose in Gram-positive bacteria. By whole transcriptome analyses, we characterized glucose-dependent and CcpA-dependent gene regulation in Clostridium difficile. About 18% of all C. difficile genes are regulated by glucose, for which 50% depend on CcpA for regulation. The CcpA regulon comprises genes involved in sugar uptake, fermentation and amino acids metabolism, confirming the role of CcpA as a link between carbon and nitrogen pathways. Using combination of chromatin immunoprecipitation and genome sequence analysis, we detected 55 CcpA binding sites corresponding to ∼140 genes directly controlled by CcpA. We defined the C. difficile CcpA consensus binding site (cre(CD) motif), that is, 'RRGAAAANGTTTTCWW'. Binding of purified CcpA protein to 19 target cre(CD) sites was demonstrated by electrophoretic mobility shift assay. CcpA also directly represses key factors in early steps of sporulation (Spo0A and SigF). Furthermore, the C. difficile toxin genes (tcdA and tcdB) and their regulators (tcdR and tcdC) are direct CcpA targets. Finally, CcpA controls a complex and extended regulatory network through the modulation of a large set of regulators.

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    • "The LacI family contains a number of global regulators for central carbohydrate metabolic pathways, in particular the previously known regulators CcpA in Firmicutes and FruR in Enterobacteria. Here, we report a comparative genomics reconstruction of orthologous FruR regulons in γ-Proteobacteria, while the reconstructions of CcpA regulons in different lineages of Firmicutes has been reported previously (Ravcheev et al., 2011, 2013b; Antunes et al., 2012; Leyn et al., 2013) and is available in "
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    ABSTRACT: DNA-binding transcription factors (TFs) are essential components of transcriptional regulatory networks in Bacteria. LacI-family TFs (LacI-TFs) are broadly distributed among certain lineages of bacteria. The majority of characterized LacI-TFs sense sugar effectors and regulate carbohydrate utilization genes. The comparative genomics approaches enable in silico identification of TF-binding sites and regulon reconstruction. To study function and evolution of LacI-TFs, we performed genomics-based reconstruction and comparative analysis of their regulons. For over 1,300 LacI-TFs from over 270 bacterial genomes, we predicted their cognate DNA-binding motifs and identified target genes. Using the genome context and metabolic subsystem analyses of reconstructed regulons we tentatively assigned functional roles and predicted candidate effectors for 78% and 67% of the analyzed LacI-TFs, respectively. Nearly 90% of the studied LacI-TFs are local regulators of sugar utilization pathways, whereas the remaining 125 global regulators control large and diverse sets of metabolic genes. The global LacI-TFs include the previously known regulators CcpA in Firmicutes, FruR in Enterobacteria, and PurR in Gammaproteobacteria, and the three novel regulators, GluR, GapR, and PckR, that are predicted to control the central carbohydrate metabolism in three lineages of Alphaproteobacteria. Phylogenetic analysis of regulators combined with the reconstructed regulons provides a model of evolutionary diversification of LacI-TFs. The obtained genomic collection of in silico reconstructed regulons in Bacteria is available in the RegPrecise database ( It provides a framework for future structural and functional classification of the LacI protein family and identification of molecular determinants of the DNA and ligand specificity. The inferred regulons can be also used for functional gene annotation and reconstruction of sugar catabolic networks in diverse bacteria.
    Frontiers in Microbiology 06/2014; 5:294. DOI:10.3389/fmicb.2014.00294 · 3.94 Impact Factor
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    • "In both organisms , carbon catabolite repression is thought to be responsible since glucose inhibition of toxin production is not observed in ccpA mutants that no longer produce the carbon catabolite repressor protein CcpA (Antunes et al., 2011; Mendez et al., 2012). Further support for the role of CcpA in mediating glucose-dependent inhibition of toxin production in C. difficile comes from the finding that the tcdA, tcdB and toxin regulator genes tcdR and tcdC are direct targets of the CcpA protein in vitro (Antunes et al., 2012). The observation that C. difficile ccpA mutants produce a reduced amount of toxin in comparison to the wild-type strain (Antunes et al., 2011) suggests that toxin regulation is complex and that unidentified regulators must also be involved in the CcpA-regulation cascade. "
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    ABSTRACT: The genus Clostridium comprises a large, heterogeneous group of obligate anaerobic, Gram-positive spore forming bacilli. Members of this genus are ubiquitous in the environment and although most species are considered saprophytic, several are pathogenic to both humans and animals. These bacteria cause a variety of diseases including neuroparalysis, gas gangrene, necrotic enteritis, food poisoning, toxic shock syndrome and pseudomembraneous colitis, which in most cases arise as a consequence of the production of potent exotoxins. Treatment options are often limited, underscoring the need for new treatment strategies and novel therapeutics. Understanding the fundamental mechanisms and signals that control toxin production in the pathogenic clostridia may lead to the identification of novel therapeutic targets that can be exploited in the development of new antimicrobial agents.
    Molecular Microbiology 01/2014; 91(2):221-31. DOI:10.1111/mmi.12469 · 5.03 Impact Factor
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    • "The native promoters that mediate expression of C. difficile tcdA and tcdB genes are subject to multiple levels of transcriptional regulation that increase toxin production under conditions of nutrient limitation while suppressing yields during exponential growth (Antunes et al., 2012; Dineen et al., 2007). To maximize expression of recombinant toxoids in standard culture media, we sought a promoter from which gene expression was relatively insensitive to regulation by nutrients or phase of growth. "
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    ABSTRACT: The Clostridium difficile toxins A and B are primarily responsible for symptoms of C. difficile associated disease and are prime targets for vaccine development. We describe a plasmid-based system for the production of genetically modified toxins in a non-sporulating strain of C. difficile that lacks the toxin genes tcdA and tcdB. TcdA and TcdB mutations targeting established glucosyltransferase cytotoxicity determinants were introduced into recombinant plasmids and episomally expressed toxin mutants purified from C. difficile transformants. TcdA and TcdB mutants lacking glucosyltransferase and autoproteolytic processing activities were ~10,000-fold less toxic to cultured human IMR-90 cells than corresponding recombinant or native toxins. However, both mutants retained residual cytotoxicity that could be prevented by preincubating the antigens with specific antibodies or by formalin treatment. Such non-toxic formalin-treated mutant antigens were immunogenic and protective in a hamster model of infection. The remaining toxicity of untreated TcdA and TcdB mutant antigens was associated with cellular swelling, a phenotype consistent with pore-induced membrane leakage. TcdB substitution mutations previously shown to block vesicular pore formation and toxin translocation substantially reduced residual toxicity. We discuss the implications of these results for the development of a C. difficile toxoid vaccine.
    Microbiology 04/2013; 159(Pt 7). DOI:10.1099/mic.0.066712-0 · 2.84 Impact Factor
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