Biophysical and Structural Characterization of the Metronidazole Resistance Protein NimB from Clostridioides difficile

Abstract

Clostridioides difficile nitroimidazole reductase B (CdNimB) is a proposed heme-dependent flavin enzyme associated with metronidazole resistance. This thesis characterized recombinant CdNimB to examine its folding, cofactor association, solution-state behaviour, and agreement with predicted structural models.

CdNimB was expressed in Escherichia coli, purified after His-SUMO tag removal, and verified by intact protein LC-MS as the expected mature protein. Circular dichroism showed that apo and heme-bound CdNimB were folded in solution. Differential scanning fluorimetry revealed strong hemin-dependent stabilization, increasing the apparent melting temperature from 64.7°C to 87.7°C, while UV-visible spectroscopy and cofactor extraction LC-MS supported heme association. In contrast, FAD supplementation produced limited additional evidence of stable association, leaving the flavin component unresolved.

SAXS supported an oligomeric, likely dimeric, solution-state architecture. Apo CdNimB was more consistent with AlphaFold-derived and homologue-informed models, whereas heme-bound CdNimB deviated more strongly, suggesting that heme binding may stabilize a distinct solution-state ensemble. Heme-bound CdNimB crystals diffracted to 2.75 Å, although molecular replacement was unsuccessful. Preliminary anaerobic metronidazole survival assays suggested possible CdNimB-associated protection.

Overall, this work supports a model in which CdNimB is a folded, heme-associated, likely dimeric protein whose behaviour changes upon heme binding. These findings provide a purified-protein framework for future studies defining the roles of heme, FAD, His55, and metronidazole binding in CdNimB-dependent resistance.