Novel molecular mechanisms of transcription targeting antibiotics

Abstract

Transcription, the first stage of gene expression, is performed by the multi-subunit RNA polymerase (RNAP). The indispensable nature of transcription and sequence divergence from eukaryotic counterparts make bacterial RNAP an excellent target for antibiotics. Yet very few clinical antibiotics target RNAP. The growing prevalence of antibiotic resistance amongst pathogenic bacteria demands the identification of novel antibacterial compounds acting through novel molecular mechanisms. This work consists of four distinct projects in which we investigated the molecular mechanisms of several previously uncharacterised transcription inhibitors. (i) Most clinical antibiotics are derived from the natural products of actinomycete bacteria. Consequently, we screened a library of actinomycetes compiled by our industrial collaborators DemurisTM for producers of novel inhibitors of bacterial transcription. From this screen we identified Antibiotic A39079S-1, produced by Streptomyces strain DEM40380, as an inhibitor of bacterial RNAP. The compound is an ansamycin type antibiotic with a previously uncharacterised mechanism of action. Here, we show the compound inhibits bacterial RNAP through a steric occlusion mechanism typical of rifamycins. (ii) Recently, the antibiotic ureidothiophene (Urd) was identified within a commercial screen of synthetic compounds in which inhibition of S. aureus RNAP was analysed. Here, we characterised the molecular mechanism of action by which Urd inhibits bacterial RNAP. We show the inhibitor targets regulatory sub-region 1.2 of the sigma subunit to prevent melting of the -10 promoter element. Consequently, Urd inhibits formation of the open promoter complex, a key step in transcription initiation. (iii) A prior screening program conducted by DemurisTM had identified the rifamycin type natural product kanglemycin A (KglA) as an inhibitor of rifampicin resistant RNAPs. Here, we show the unique ansa-bridge substituents of the compound act to form new binding contacts with RNAP. We also show II KglA inhibits transcription through a unique steric occlusion mechanism by preventing extension of the nascent transcript at an earlier stage than rifampicin. (iv) Finally, we investigated ADP-ribosylation as a mechanism of KglA inactivation by Mycobacterium smegmatis and Mycobacterium abscessus Rifampin ADP-ribosyltransferase (Arr) enzymes. We show KglA is not a substrate of the rifampicin inactivating Arr purified from Mycobacterium smegmatis, but remains a substrate of Arr purified from the extensively drug resistant pathogen Mycobacterium abscessus.