Abstract
The design of novel antibiotics relies on a profound understanding of their mechanism of action. While it has been shown that cellular effects of antibiotics cluster according to their molecular targets, we investigated whether compounds binding to different sites of the same target can be differentiated by their transcriptome or metabolome signatures. The effects of three fluoroquinolones, two aminocoumarins and two cystobactamids, all inhibiting bacterial gyrase, on Pseudomonas aeruginosa at sub-inhibitory concentrations could be clearly distinguished by RNA sequencing as well as metabolomics. We observed a strong (2.8-212-fold) induction of autolysis-triggering pyocins in all gyrase inhibitors, which correlated with eDNA release. Gyrase B-binding aminocoumarins induced the most pronounced changes, including a strong downregulation of phenazine and rhamnolipid virulence factors. Cystobactamids led to a downregulation of a glucose catabolism pathway. The study implies that clustering cellular mechanisms of action according to the primary target needs to take class-dependent variances into account.
Highlights
The design of novel antibiotics relies on a profound understanding of their mechanism of action
In order to identify and compare interclass versus intraclass effects of gyrase inhibitors on the bacterial transcriptome and metabolome, the P. aeruginosa model strain PA14 was exposed to the three fluoroquinolones ciprofloxacin, levofloxacin, and lomefloxacin, the two aminocoumarins novobiocin and coumermycin A1, and the two cystobactamids CN-DM-861 and AR351 (Fig. 1a)
We show that the level of pyocin induction depends very much on the inhibitor class, with the fluoroquinolones inducing the strongest effects, and that the release of extracellular DNA (eDNA) correlates directly with the endolysin expression
Summary
The design of novel antibiotics relies on a profound understanding of their mechanism of action. While it has been shown that cellular effects of antibiotics cluster according to their molecular targets, we investigated whether compounds binding to different sites of the same target can be differentiated by their transcriptome or metabolome signatures. We wanted to probe this assumption and questioned whether or not antibiotics that bind to the same target but belong to different structural classes induced distinguishable molecular signatures. For this purpose, representatives of three classes of DNA gyrase inhibitors, the fluoroquinolones, the aminocoumarins, and the recently discovered cystobactamids, were selected and compared with respect to their metabolome and transcriptome effects on the opportunistic human pathogen Pseudomonas aeruginosa. In contrast to cleavage-complex-stabilizing compounds or gyrase poisons, aminocoumarins do not cause DNA double-strand breaks [25]
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