Transcriptional, proteomic and metabolic networks of the Fur regulated iron metabolism of Clostridium difficile

Clostridium difficile is known as a human pathogen causing diarrhea among patients in hospitals and health care units worldwide. C. difficile is a Gram-positive, strict anaerobic, spore-forming and toxin producing bacterium. In order to stop the growth of infectious microorganisms during infection the host restricts the excess to iron for the pathogens by producing iron- and siderophore-chelating proteins, by exporting iron from intracellular pathogen-containing compartments, and by limiting absorption of dietary iron. Consequently, infectious bacteria have to adjust their iron uptake and utilization strategies. Almost nothing was known about the regulation of iron uptake by C. difficile in the mammalian gut of the beginning of this thesis. Consequently, different possible iron sources were tested. A genomic knock out of the gene for the ferric uptake regulator Fur was generated using the ClosTron system. Fur displayed a clear growth deficiency compared to C. difficile wild type cells. Morphological experiments showed reduced flagella and pili formation in the fur mutant strain. Surprisingly, the toxin production was not affected by Fur. Subsequently, the wild type and fur mutant strain were subjected to a systems biology investigation using transcriptome, proteome and metabolome analyses to define the iron- and Fur regulon in C. difficile. Several iron-associated transporters as well as major energy metabolic pathways (e.g. L-leucine fermentation, proline metabolism, butyrate fermentation) are controlled in an iron dependent Fur regulated manner. Furthermore, flagella formation is positively regulated by iron and Fur. A consensus Fur-binding sequence was defined and potential Fur-boxes upstream of for example flagella associated genes and a ferrichrome transporter operon were found. Taken together, the regulator Fur is not only involved in the regulation of iron associated genes but also in processes of the central metabolism in C. difficile.