Clostridium difficile, a Gram-positive, anaerobic, spore-former is the major cause of antibiotic associated diarrhoea and is also associated with more severe, sometimes life threatening disease. To date little research has been carried out on iron uptake mechanisms and their regulation in C. difficile. In common with other pathogens, iron is likely to be an essential growth factor necessary for the survival of the organism. Conversely, an over-abundance of iron might be toxic, therefore uptake is likely to be strictly regulated. Analysis of available genome sequences reveals the presence of several potential iron uptake systems and regulators. This study investigated for the first time the role of the ferrous iron uptake systems FeoBl, FeoB2, FeoB3 and the ferric uptake regulator Fur, iron dependent global gene regulator in C. difficile. The ClosTron mutagenesis system has been used to generate knockout mutants in the three annotated feoB homologues and in the single fur homologue in C. difficile 630. The fur mutants exhibited clear growth phenotype and were hypersensitive to hydrogen peroxide. Cytotoxicity assays revealed decreased levels of toxins TcdA and B in the fur mutants. The fur mutants demonstrated different phenotypes in sporulation and in swarming, were more susceptible to metronidazole and showed increased cellular iron content. RNA-Seq analysis investigated the fur regu lation in C. difficile and over 1600 genes were differentially expressed in the fur mutant Comparisons of the growth characteristics and cellular metal content suggest FeoBl but not FeoB2 and FeoB3 is involved in ferrous iron uptake. Levels of toxins TcdA and B were markedly reduced in the feoB1 mutant grown under iron limitation. In contrast, feoBl mutant was virulent in the hamster model of virulence. These findings highlight the role of Fur and FeoB systems in regulating key and diverse aspects of physiology and virulence of C. difficile.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:595313 |
| Date | January 2013 |
| Creators | Fit, Magdalena Katarzyna |
| Publisher | University of Nottingham |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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