Cross-Resistance of Escherichia coli RNA Polymerases Conferring Rifampin Resistance to Different Antibiotics

Abstract

In this study we further defined the rifampin-binding sites in Escherichia coli RNA polymerase (RNAP) and determined the relationship between rifampin-binding sites and the binding sites of other antibiotics, including two rifamycin derivatives, rifabutin and rifapentine, and streptolydigin and sorangicin A, which are unrelated to rifampin, using a purified in vitro system. We found that there is almost a complete correlation between resistance to rifampin (Rif r ) and reduced rifampin binding to 12 RNAPs purified from different rpoB Rif r mutants and a complete cross-resistance among the different rifamycin derivatives. Most Rif r RNAPs were sensitive to streptolydigin, although some exhibited weak resistance to this antibiotic. However, 5 out of the 12 Rif r RNAPs were partially resistant to sorangicin A, and one was completely cross-resistant to sorangicin A, indicating that the binding site(s) for these two antibiotics overlaps. Both rifampin and sorangicin A inhibited the transition step between transcription initiation and elongation; however, longer abortive initiation products were produced in the presence of the latter, indicating that the binding site for sorangicin A is within the rifampin-binding site. Competition experiments of different antibiotics with 3 H-labeled rifampin for binding to wild-type RNAP further confirmed that the binding sites for rifampin, rifabutin, rifapentine, and sorangicin A are shared, whereas the binding sites for rifampin and streptolydigin are distinct. Because Rif r mutations are highly conserved in eubacteria, our results indicate that this set of Rif r mutant RNAPs can be used to screen for new antibiotics that will inhibit the growth of Rif r pathogenic bacteria.RNA polymerase (RNAP) is the sole enzyme responsible for transcribing RNA from DNA template in eubacteria (3). Because of its essential role in gene expression, RNAP has been a target for antibiotic studies since its discovery in the 1960s. Several antibiotics inhibiting the functions of RNAP have been discovered. Among them, rifampin, a derivative of rifamycin (35), is the most important in clinical use (22,33). Rifampin is part of the standard therapy of tuberculosis (8) which, after AIDS, is the leading cause of death by an infectious agent worldwide (23,32) and is also used in prophylaxis of meningitis and against staphylococcal infections (19,20).Rifampin binds to RNAP with high affinity (K eq ϭ 10 Ϫ9 M at 37°C) (39). The mode of action of rifampin has been studied in most detail using Escherichia coli RNAP as a model system, as the overall structure and function of RNAPs from different eubacteria have been conserved. Rifampin inhibits RNAP's function by blocking the transition from transcription initiation to transcription elongation (24). In the presence of rifampin, RNAP can only synthesize short RNA oligomers, and it was proposed that rifampin exerts a steric hindrance of RNAP translocation along the nascent RNA path. Indeed, cross-linking experiments have indicated that rifampin blocks the channel le...

The optimal deployment of synergistic antibiotics: a control-theoretic approach

“…Here M 3 and M 5 represent oligomers just a handful of nucleotides long that are the abortive mRNA-like transcripts produced when rifampicin-like drugs are bound to RNA polymerase [26]. However, this more complicated model does not provide any additional theoretical insight into the interaction of drugs targeting the same enzyme; (C 1) is already sufficient to have non-trivial, synergistic drug interactions as we now aim to show.…”

Novel Chemical Scaffolds for Inhibition of Rifamycin-Resistant RNA Polymerase Discovered from High-Throughput Screening

“…As precedence, Xu et al showed that E. coli H526Y RNAP had complete resistance to the RNAP inhibitor sorangicin A while mutants D516N and S531F, and other residues, showed only partial or no resistance. 29 Two of the four compounds in cluster 6 (186618 and 193157) showed activity against M. tuberculosis in culture (Table 1). Interestingly, compound 193157 was active in culture despite being inactive against WT MTB RNAP in vitro.…”

Identification of Rifampicin Resistance Mutations in <b><i>Escherichia coli</i></b>, Including an Unusual Deletion Mutation

“…Although RIF binds in the RNA tract, many of the residues in the RIF binding pocket do not directly contact the RNA. Numerous mutations in the RNA polymerase β-chain confer RIF resistance (RIF R ) in Escherichia coli and Mycobacterium tuberculosis [Xu et al, 2005;Makiela-Dzbenska et al, 2011;Zhou et al, 2013;Jin and Gross, 1989;Sandgren et al, 2009;Barrick et al, 2010]. These mutations are clustered in 4 regions of RNA polymerase β, termed RIF R -determining regions (RRDRs) N, I, II, and III [Molodtsov et al., 2016].…”