Deciphering the role of flavodoxins in the intestinal pathogen Clostridioides difficile

• Applicant: Dr. Susanne Sievers
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms
• Term: since 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 453440095

The strictly anaerobic bacterium and intestinal pathogen Clostridioides difficile causes one of the most problematic nosocomial infections nowadays. Patients suffering from a Clostridioides difficile infection (CDI) show symptoms ranging from mild diarrhea to life-threatening inflammation of the intestinal epithelium. Most challenging for the treatment of a CDI is the pathogen’s ability to form endospores, a highly resistant life-form that even survives antibiotic treatment, which also explains the high relapse rate accompanying the disease. Within the host, C. difficile is stressed with low levels of oxygen and different reactive oxygen species produced by the immune system. Despite its anaerobic lifestyle, the pathogen can survive in the host and even expedite an infection. Some C. difficile strains are characterized by an extraordinary high oxygen tolerance. However, detailed molecular knowledge on how the pathogen senses oxidative stress, how the signal is transduced and a stress response eventually regulated and initiated is missing. Cellular structures containing a flavin cofactor are often involved in the oxidative stress response of bacteria. We identified a much higher-than-average number of genes encoding for putative flavodoxins in C. difficile. Flavodoxins are small proteins of a specific fold and bound to the cofactor flavin mononucleotide (FMN). The FMN cofactor is capable of redox reactions involving the transfer of single electrons meaning that it can generate or neutralize radicals. None of the 8 putative flavodoxins of C. difficile has been associated with an exact function so far. Thus, their involvement in the bacterium’s metabolism or oxidative stress response is completely unknown. However, several facts turn them into well suited potential candidates for new antimicrobial therapies. Firstly, they do not occur in higher developed eukaryotes. Secondly, an essential cellular function has been assigned to flavodoxins in many other bacteria and finally, they were described to take up a vital role in virulence in several pathogens. This project aims at the functional characterization of the 8 putative flavodoxins of C. difficile to pinpoint those that are essential for the bacterium to survive and cause disease in the host. For the comprehensive characterization, the flavodoxins will be thoroughly studied in-silico, their gene expression profile will be recorded and in gene knock-down experiments their essentiality will be tested. A global proteomics approach comprising various physiological conditions and flavodoxin knock-down mutants will be applied to pinpoint interaction partners and flavodoxin interaction networks. Eventually, direct binding partners of flavodoxins will be pulled out in-vitro by using strep-tagged flavodoxins as bait. Findings of this enterprise will not only further enlighten the role of flavodoxins in bacteria in general, but will possibly support the identification of novel targets in CDI therapy.

• DFG Programme: Research Grants