Mutations in rpoB that confer rifampicin resistance can alter levels of peptidoglycan precursors and affect β-lactam susceptibility

Abstract

Bacteria can adapt to stressful conditions through mutations affecting the RNA polymerase core subunits that lead to beneficial changes in transcription. In response to selection with rifampicin (RIF), mutations arise in the RIF resistance determining region (RRDR) of rpoB that reduce antibiotic binding. These changes can also alter transcription and thereby have pleiotropic effects on bacterial fitness. Here, we studied the evolution of resistance in Bacillus subtilis to the synergistic combination of RIF and the {beta}-lactam cefuroxime (CEF). Two independent evolution experiments led to the recovery of a single rpoB allele (S487L) that was able to confer resistance to RIF and CEF through a single mutation. Two other common RRDR mutations made the cells 32x more sensitive to CEF (H482Y) or led to only modest CEF resistance (Q469R). The diverse effects of these three mutations on CEF resistance are correlated with differences in the expression of peptidoglycan (PG) synthesis genes and in the levels of two metabolites crucial in regulating PG synthesis, glucosamine-6-phosphate (GlcN-6-P) and UDP-N-acetylglucosamine (UDP-GlcNAc). We conclude that RRDR mutations can have widely varying effects on pathways important for cell wall biosynthesis, and this may restrict the spectrum of mutations that arise during combination therapy. ImportanceRifampicin (RIF) is one of the most valued drugs in the treatment of tuberculosis. TB treatment relies on a combination therapy, and for multidrug resistant strains may include {beta}-lactams. Mutations in rpoB present a common route for emergence of resistance to RIF. In this study, using B. subtilis as a model, we evaluate the emergence of resistance for the synergistic combination of RIF and the {beta}-lactam cefuroxime (CEF). One clinically-relevant rpoB mutation conferred resistance to both RIF and CEF, whereas two others increased CEF sensitivity. We were able to link these phenotypes to accumulation of specific PG precursors. Mainly, UDP-GlcNAc through its GlmR mediated influence on GlmS activity has a strong impact on CEF resistance. Since these mutations are clinically relevant, these effects on CEF sensitivity may help refine the use of {beta}-lactams in TB therapy.