Hochschulschrift:
Dissertation, Technische Universität Carolo-Wilhelmina zu Braunschweig, 2021
Anmerkungen:
Zusammenfassung in deutscher und englischer Sprache
Beschreibung:
C. difficile is a human pathogen of the gastrointestinal tract and the leading cause of severe nosocomial diarrhea and pseudomembranous colitis. During infection, this strictly anaerobic and spore-forming pathogen is exposed to varying osmolarities, oxygen concentrations and iron availability. In the present study, the adaptative responses to high salinity conditions and the regulatory network of the peroxide sensor PerR were characterized. Bioinformatic mining via structural comparisons of C. difficile transport systems with known osmolyte transporters identified two proteins: the annotated OpuC (CDIF630erm_01020/01021) and the unknown transporter system UtS" (CDIF630erm_03509/03510). The substrate spectrum of the OpuC-transporter was determined in a heterologous B. subtilis system and included the compatible solutes carnitine, glycinebetaine, choline, gamma-butyrobetaine, homobetaine, prolinebetaine, crotonobetaine, ectoine, and dimethylsulfoniopropionate (DMSP). Contrary the UtS showed no transport capacities. Investigations on osmotic stress tolerance showed significantly reduced growth of the wild type at a concentration of 350 mM NaCl, whereas growth of the opuCC mutant was almost abolished. Addition of carnitine, glycinebetaine, gamma-butyrobetaine, crotonobetaine, homobetaine, prolinebetaine, and DMSP restored the growth of the wild type but not that of the mutant. Choline did not appear to display any osmo-protective function. Interestingly, the commonly rod-shaped organism switched to a coccoid cell shape under salt stress and reverted to its natural morphology in the presence of carnitine. In addition, salt stress resulted in decreased toxin A concentrations as well as diminished levels of fermentation products. While toxin concentrations were only partially restored by carnitine, the metabolic fingerprint of the wild type was almost entirely recovered, but not the metabolic profile of the mutant. The H2O2-independent perR regulation was defined using a systems biology approach. Here, the perR mutant displayed a significant growth deficit in the stationary phase. Moreover, perR regulated pathways correlated highly with the Fur-dependent regulation. This observation, in combination with a potential Fur binding site upstream of the perR-operon and the upregulation of Fur in the absence of perR, suggest a co-regulation of Fur and perR.