Novel antibiotic mode of action by repression of promoter isomerisation

Rising levels of antibiotic resistance dictate that new antibiotics with novel modes of action must be found. Here, we investigated the mode of action of a novel antibiotic that is a member of a family of synthetic DNA minor groove binding (MGB) molecules. MGB-BP-3 has successfully completed a Phase II clinical trial in humans as an orally administered drug for the treatment of chronic Clostridioides (Clostridium) difficile infections, where it outperformed the existing benchmark (vancomycin). MGB-BP-3 is active against a variety of Gram-positive pathogens including Staphylococcus aureus, which was used as the model for this study. The transcriptomic response of S. aureus to MGB-BP-3 identified downregulated promoters. DNase I and permanganate footprinting demonstrated binding to essential SigA promoters and the inhibition of promoter isomerisation by RNA polymerase holoenzyme. Promoters controlling DNA replication and peptidoglycan biosynthesis are amongst those affected by MGB-BP-3. Thus, MGB-BP-3 binds to and inhibits multiple essential promoters on the S. aureus chromosome, suggesting that evolution of resistance by drug target mutation should be unlikely. In confirmation, laboratory-directed evolution against sub-inhibitory concentrations of MGB-BP-3 resulted in no resistance whereas resistance to the single target RNA-polymerase inhibitor rifampicin arose rapidly.

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Faculty Opinions recommendation of Structural organization of bacterial RNA polymerase holoenzyme and the RNA polymerase-promoter open complex.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1004911.55358 ◽

2002 ◽

Author(s):

Robert Dutnall

Keyword(s):

Rna Polymerase ◽

Structural Organization ◽

Bacterial Rna ◽

Rna Polymerase Holoenzyme ◽

Rna Polymerase Promoter

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Utilization of variably spaced promoter-like elements by the bacterial RNA polymerase holoenzyme during early elongation

Molecular Microbiology ◽

10.1111/j.1365-2958.2009.07021.x ◽

2010 ◽

Vol 75 (3) ◽

pp. 607-622 ◽

Cited By ~ 8

Author(s):

Pukhrambam Grihanjali Devi ◽

Elizabeth A. Campbell ◽

Seth A. Darst ◽

Bryce E. Nickels

Keyword(s):

Rna Polymerase ◽

Bacterial Rna ◽

Rna Polymerase Holoenzyme

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Effect of disulfide and sulfhydryl reagents on abortive and productive elongation catalyzed by Escherichia coli RNA polymerase.

Acta Biochimica Polonica ◽

10.18388/abp.1994_4686 ◽

1994 ◽

Vol 41 (4) ◽

pp. 415-419

Author(s):

M Radłowski ◽

D Job

Keyword(s):

Escherichia Coli ◽

Rna Polymerase ◽

Fold Increase ◽

Sulfhydryl Reagents ◽

Nitrobenzoic Acid ◽

Steady State Kinetic ◽

Transcription Complexes ◽

The Stability ◽

Rna Polymerase Holoenzyme ◽

Productive Elongation

The effect of disulfide and sulfhydryl reagents on the rate of abortive and productive elongation has been studied using Escherichia coli RNA polymerase holoenzyme and poly[d(A-T)] as template. In the presence of UTP as a single substrate and UpA as a primer, the enzyme catalyzed efficiently the synthesis of the trinucleotide product UpApU. Incubation of RNA polymerase with 1 mM 2-mercaptoethanol resulted in a 5-fold increase of the rate of UpApU synthesis. In contrast, incubation of the enzyme with 1 mM 5,5'-dithio-bis(2-nitrobenzoic) acid resulted in a 6-fold decrease of the rate of abortive elongation. Determination of the steady state kinetic constants associated with UpApU synthesis disclosed that the disulfide and sulfhydryl reagents mainly affected the rate of UpApU release from the ternary transcription complexes and therefore influenced the stability of such complexes.

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A Mutation of RNA Polymerase β′ Subunit (RpoC) Converts Heterogeneously Vancomycin-Intermediate Staphylococcus aureus (hVISA) into “Slow VISA”

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00135-15 ◽

2015 ◽

Vol 59 (7) ◽

pp. 4215-4225 ◽

Cited By ~ 13

Author(s):

Miki Matsuo ◽

Tomomi Hishinuma ◽

Yuki Katayama ◽

Keiichi Hiramatsu

Keyword(s):

Staphylococcus Aureus ◽

Cell Wall ◽

Rna Polymerase ◽

Stringent Response ◽

Whole Genome ◽

Multicopy Plasmid ◽

Β Subunit ◽

Whole Genome Analysis ◽

Content Type ◽

Peptidoglycan Biosynthesis

ABSTRACTVarious mutations in therpoBgene, which encodes the RNA polymerase β subunit, are associated with increased vancomycin (VAN) resistance in vancomycin-intermediateStaphylococcus aureus(VISA) and heterogeneously VISA (hVISA) strains. We reported thatrpoBmutations are also linked to the expression of the recently found “slow VISA” (sVISA) phenotype (M. Saito, Y. Katayama, T. Hishinuma, A. Iwamoto, Y. Aiba, K Kuwahara-Arai, L. Cui, M. Matsuo, N. Aritaka, and K. Hiramatsu, Antimicrob Agents Chemother 58:5024–5035, 2014,http://dx.doi.org/10.1128/AAC.02470-13). Because RpoC and RpoB are components of RNA polymerase, we examined the effect of therpoC(P440L) mutation on the expression of the sVISA phenotype in the Mu3fdh2*V6-5 strain (V6-5), which was derived from a previously reported hVISA strain with the VISA phenotype. V6-5 had an extremely prolonged doubling time (DT) (72 min) and high vancomycin MIC (16 mg/liter). However, the phenotype of V6-5 was unstable, and the strain frequently reverted to hVISA with concomitant loss of low growth rate, cell wall thickness, and reduced autolysis. Whole-genome sequencing of phenotypic revertant strain V6-5-L1 and comparison with V6-5 revealed a second mutation, F562L, inrpoC. Introduction of the wild-type (WT)rpoCgene using a multicopy plasmid resolved the sVISA phenotype of V6-5, indicating that therpoC(P440L) mutant expressed the sVISA phenotype in hVISA. To investigate the mechanisms of resistance in the sVISA strain, we independently isolated an additional 10 revertants to hVISA and VISA. In subsequent whole-genome analysis, we identified compensatory mutations in the genes of three distinct functional categories: therpoCgene itself as regulatory mutations, peptidoglycan biosynthesis genes, andrelQ, which is involved in the stringent response. It appears that therpoC(P440L) mutation causes the sVISA phenotype by augmenting cell wall peptidoglycan synthesis and through the control of the stringent response.

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