Details

Autor(en) / Beteiligte

• Deshpande, Aditi

Titel

Iron Metabolism Intersects Metronidazole Resistance and Virulence in Clostridioides difficile

Ort / Verlag

ProQuest Dissertations & Theses

Erscheinungsjahr

2020

Link zum Volltext

Quelle

ProQuest Dissertations & Theses A&I

Beschreibungen/Notizen

• At the turn of the 20th century, Clostridioides difficile infections (CDI) escalated both in incidence and severity. CDI is the leading cause of hospital-acquired antibiotic-associated diarrhea (AAD). Traditionally, metronidazole (MTZ) and vancomycin (VAN) were used to treat these infections with >80% clinical success. This has now declined with the emergence of epidemic strains and MTZ has become inferior to VAN. This situation appears to have arisen from the evolution of MTZ resistance, but the underlying genetic basis is unknown. Chapter II of this dissertation addressed this knowledge gap by showing how C. difficile evolves MTZ resistance, under laboratory conditions. MTZ is a prodrug that undergoes one electron reduction to form reactive species, in reactions catalyzed by oxidoreductase enzymes. Our key discovery was the construction of a mismatch repair non-toxigenic mutator strain, which abrogated genetic barriers for de novo resistance development. This study revealed a new epistatic mechanism of resistance in which loss of the ferrous iron transporter FeoB1 synergized with CRISPR-dCas driven gene knockdowns of iron-dependent proteins like pyruvate-ferredoxin/flavodoxin oxidoreductase (PFOR), xanthine dehydrogenase (XDH) and iron-sulfur cluster regulator (IscR). Some findings were substantiated in the clinical strains.Because the in vitro evolution of MTZ resistance was deterministic, following feoB1 deletion, in Chapter III we appraised the clinical relevance of this mechanism. This was guided by the view that FeoB1 is crucial for iron acquisition and if C. difficile adopted this path for MTZ resistance in the clinic, such strains would be severely unfit. Hence, we deleted feoB1 in a toxigenic strain, whose virulence potential was assessed in the modified colitis model of CDI. This identified that loss of feoB1 attenuated colonization and virulence by inadequate production of the toxins TcdA and TcdB. Thus, we provide the first report that FeoB1 is relevant for C. difficile pathogenesis and certainly explains why FeoB1 is not disrupted in MTZ-resistant strains. This dissertation has successfully advanced the current knowledge of C. difficile pathophysiology, presenting new insights of its evolutionary responses to drug selection pressure and information that could be harnessed for therapeutic interventions.