Basis of narrow-spectrum activity of fidaxomicin on Clostridioides difficile

Fidaxomicin (Fdx) is widely used to treat Clostridioides difficile ( Cdiff ) infections, but the molecular basis of its narrow-spectrum activity in the human gut microbiome remains unknown. Cdiff infections are a leading cause of nosocomial deaths 1 . Fidaxomicin, which inhibits RNA polymerase, targets Cdiff with minimal effects on gut commensals, reducing recurrence of Cdiff infection 2 , 3 . Here we present the cryo-electron microscopy structure of Cdiff RNA polymerase in complex with fidaxomicin and identify a crucial fidaxomicin-binding determinant of Cdiff RNA polymerase that is absent in most gut microbiota such as Proteobacteria and Bacteroidetes. By combining structural, biochemical, genetic and bioinformatic analyses, we establish that a single residue in Cdiff RNA polymerase is a sensitizing element for fidaxomicin narrow-spectrum activity. Our results provide a blueprint for targeted drug design against an important human pathogen. Structural analysis of Clostridioides difficile RNA polymerase in complex with fidaxomicin combined with biochemical, genetic and bioinformatic analyses identifies a key residue that determines fidaxomicin sensitivity.