Identification of the full scope of the CodY regulon in Clostridioides difficile

摘要

ABSTRACT Clostridioides (formerly Clostridium) difficile is a Gram-positive, sporulating, anaerobic bacterium that can cause severe disease, including antibiotic-associated diarrhea and pseudomembranous colitis, in humans. Pathogenic C. difficile produces at least two potent toxins, TcdA and TcdB, which cause major intestinal damage to the host. Formation of spores by C. difficile is critical for survival of the organism in the environment, initiation of infection (normally caused by spores), and recurrence of the disease due to re-germination of spores formed in the gut of infected humans or animals and their ability to cause a new round of infection. A global transcriptional regulator CodY appears to be the most important regulator of toxin-encoding genes, and codY mutants of C. difficile produce more toxins and are more virulent than wild-type strains in a mouse model of infection. CodY also controls expression of genes involved in sporulation, and codY mutants sporulate more efficiently. Moreover, CodY affects expression of multiple metabolic pathways that are likely to be important for growth and virulence. Although CodY is known to directly repress the tcdR gene, encoding a toxin-specific sigma factor, the mechanism of CodY’s effect on toxin gene expression has not been fully established. The direct targets of CodY that control C. difficile sporulation gene expression and spore formation remain completely unknown. To understand in full the mechanisms of CodY’s effect on C. difficile virulence, sporulation, and metabolism, it is essential to determine the full scope of the direct CodY targets via identification at single- nucleotide detail of the entire complement of CodY-binding sites. Therefore, we propose to determine the entire set of genes that are able to interact with CodY directly either in vivo or in vitro. Using a novel approach, in vitro DNA affinity purification coupled with massively parallel sequencing (IDAP-Seq), we are now able to visualize CodY-binding sites in vitro on a genome-wide basis at near single-nucleotide resolution and rank the sites according to their relative strengths. In addition, we will use ChIP-Seq experiments, also at near single- nucleotide resolution, to define the genome-wide set of sites that interact with CodY in vivo. Comparing the data generated by these approaches with the results of in vivo expression analysis (RNA-Seq) will allow us to (a) identify direct and indirect targets of CodY regulation; (b) determine whether the breadth of the CodY regulon is significantly greater than presently known; (c) uncover potential targets of CodY responsible for its effect on sporulation; (d) find the genes that are potentially subject to dual control by CodY and other regulators; and (e) establish whether premature transcription termination within coding sequences is involved in CodY-mediated regulation of toxin-encoding and other C. difficile genes. The precise information on the locations and relative strengths of CodY-binding sites for critical genes will provide the foundation for future research on the detailed mechanisms by which CodY regulates individual genes.