Constrained RNA polymerase mutational pathways distinguish corallopyronin A resistance from rifampicin resistance in staphylococci

Corallopyronin A (CorA) is a natural RNA polymerase (RNAP) inhibitor active against Gram-positive pathogens, including Staphylococcus aureus. Although spontaneous resistance to CorA occurs less frequently than to rifampicin, the genetic basis and evolutionary constraints of CorA resistance remain incompletely defined. Spontaneous mutation frequencies to CorA and rifampicin were determined by fluctuation analysis in three S. aureus strains and two coagulase-negative staphylococci (CNS). Resistant mutants were phenotypically characterized and subjected to whole-genome sequencing. Double-resistant mutants were generated under rifampicin selection. Fitness costs were assessed by direct competition assays. Mutation frequencies to CorA were consistently lower than to rifampicin across all tested strains (Rif/CorA ratios 2.3-2.7). Whole-genome sequencing of 41 independently derived CorA-resistant mutants revealed resistance-associated substitutions confined to the RNAP subunits RpoB and RpoC. The mutational landscape was highly restricted, with recurrent hotspots at RpoC K334 and RpoB S1127. In coagulase-negative staphylococci (CNS), resistance remained RNAP-centred but displayed broader allelic diversity, including substitutions previously described in S. aureus but identified here for the first time in CNS species, as well as the previously undescribed RpoC D810Y substitution. Ten mutants with resistance to CorA and rifampicin retained the CorA-associated mutation and acquired additional rifampicin resistance substitutions at non-overlapping RNAP residues. CorA resistance imposed a moderate fitness cost (12-15%), while additional rifampicin resistance resulted in mutation-dependent increases in the fitness burden. CorA resistance in staphylococci arises at lower spontaneous frequencies than rifampicin resistance and is mediated by a constrained set of RNAP mutations. The restricted mutational landscape and associated fitness costs indicate that, under the in vitro conditions examined, CorA resistance arises through more constrained evolutionary pathways than rifampicin resistance. These findings provide a foundation for further evaluation of CorA as an RNA polymerase-targeting antimicrobial.