IMIB - Institute for Molecular Infection Biology

Identification and functional characterization of novel RNA-binding proteins in Clostridium difficile

Clostridium difficile (C. difficile) is a Gram-positive, obligate anaerobic and spore-forming bacterium. It is the leading cause of antibiotic-associated diarrhea. A disruption of the host microbiota due to the administration of antibiotics can lead to germination and outgrow of antibiotic-resistant C. difficile spores, followed by colonization of the gastrointestinal tract. The symptoms of a C. difficile infection (CDI) can range from mild diarrhea to tissue necrosis and are mediated by two large clostridial toxins, toxin A (TcdA) and toxin B (TcdB). Despite the important role of C. difficile in health care as one of the major nosocomial enteropathogens, the knowledge about mechanisms that control its virulence is still low.

In recent years, small noncoding RNAs (sRNAs) and RNA-binding proteins (RBPs) have been intensively studied in different bacterial species as important factors of post-transcriptional regulation. The investigation of these mechanisms contributed to gain further understanding of the regulation of different physiological, metabolic and virulence-associated processes. Initial studies of Soutourina et al. identified a large number and great diversity of potential regulatory sRNAs in C. difficile, suggesting the importance of RNA-based mechanisms for the regulation of gene expression in this organism [1].

The objective of my project is the further identification and characterization of such sRNAs along with their cognate binding proteins. First, RNA-protein complexes are identified by gradient profiling by sequencing (Grad-seq). RNA pull down approaches and Co-Immunoprecipitation are used to validade these complexes. This eventually leads to the identification of novel RBPs and unknown regulatory pathways, which will be further characterized with regard to their role in physiology, metabolism and virulence.

[1] A. Soutourina et al., “Genome-wide identification of regulatory RNAs in the human pathogen Clostridium difficile,” PLoS Genet., 2013.