Clinically present mycobacterium tuberculosis RNA polymerase subunit RpoB K446 mutation confers broad-spectrum antibiotics resistance via pupylation.

Mycobacterium tuberculosis (Mtb) infection remains a serious global public health threat due to its inherent and acquired resistance to clinical drugs. Meanwhile, the genomic sequences of many clinical isolates are informative to find novel drugs. Most drug-resistant mutations are clustered in the antibiotics-binding region of the targets, reducing affinity with antibiotics and diminishing the efficacy of antibiotics. However, the roles of mutations outside the drug-binding regions remain elusive. RNA polymerase encoded by rpoB is the primary target of the first-front antibiotics of rifampicin. Here, we found that many clinical isolates with RpoB K446 residue mutation, which does not directly interact with rifampicin, are susceptible to rifampicin. Further genetic experiments demonstrated that defective prokaryotic ubiquitin-like protein modification (pupylation) of the RpoBK446R mutant underlies the phenotypes, and chemically inhibition of the pup-proteasome system (PPS) pathway via repurposed drug bithionol or bortezomib can accelerate the killing of M. bovis BCG and Mtb by four clinical antibiotics with different mechanisms of action, namely RNA polymerase-targeting rifampicin (RIF), ribosome-targeting streptomycin (SM), gyrase-targeting moxifloxacin (MOX) and ATPase-targeting bedaquiline (BDQ). The data showed pupylation-proteasome pathway is target for broad-spectrum antibiotics or potentiators, and repurposed drugs bithionol or bortezomib can be further explored to be included in tuberculosis chemotherapy regimen to shorten the duration of treatment.