Characterization of the RNA-binding protein KhpB - mechanisms of RNA binding and the regulation of metabolism and virulence in Clostridioides difficile

• Applicant: Professorin Dr. Franziska Faber
• Subject Area: Medical Microbiology and Mycology, Hygiene, Molecular Infection Biology
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 503186380

Bacteria have evolved multi-layered strategies to respond to changes in their environment including post-transcriptional networks that allow them to fine-tune their gene expression almost instantaneous. RNA-binding proteins are an integral part of these post-transcriptional networks, regulating the stability and translational efficiency of targeted mRNAs either directly, or indirectly through facilitating sRNA-mRNA base pairing. The best characterized bacterial RNA chaperones are CsrA, Hfq and ProQ. Whereas they have been extensively characterized in gram-negative model organisms, such as S. enterica and E. coli, they are often absent in gram-positive bacteria or their functions appear to be different, respectively. We have recently published the first characterization of a novel RNA-binding protein in the gram-positive, obligate anaerobic, human pathogen Clostridioides difficile, that we called KhpB, and demonstrated pervasive mRNA and sRNA binding activity as well as a function in stabilizing some of its sRNA ligands. In addition, we identified a role for KhpB in regulating the production of the central virulence factor clostridial toxin A. This function in toxin regulation appears to be connected to the regulation of central metabolic pathways by KhpB but the underlying mechanisms of this regulation remain unknown. KhpB homologs are widely conserved in bacteria and harbor a modular domain architecture containing two putative RNA-binding domains, a KH-II and a R3H domain, and a Jag domain of unknown function. In this proposal, we will focus on the RNA-binding domains of KhpB, identifying the RNA-binding sites of KhpB on a genome-wide scale and at single-nucleotide resolution using CLIP-seq to characterize its RNA binding modalities. We will perform these analyses using defined mutants in RNA-binding sites of its KH-II and R3H domains, respectively, to dissect the contribution of each domain to RNA binding. With this atlas of KhpB ligands we will also analyze the functional consequences of KhpB binding for its RNA targets in vivo. Further, we will integrate our data on the KhpB targetome with targeted metabolite analysis of a khpB mutant to reveal how KhpB controls central metabolic pathways and toxin production. Our analyses will reveal a novel, post-transcriptional layer in the regulation of C. difficile virulence which will expand our understanding of how this intestinal pathogen fine-tunes its metabolism, and consequently its virulence, in response to the nutritional environment. More generally, it will present a major step forward in understanding the mechanism of RNA regulation by this emerging class of global RNA binding proteins.

• DFG Programme: Research Grants