Abstract
ABSTRACTThe 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 subinhibitory concentrations could be distinguished clearly by RNA sequencing as well as metabolomics. We observed a strong (2.8- to 212-fold) induction of autolysis-triggering pyocins in all gyrase inhibitors, which correlated with extracellular DNA (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.IMPORTANCE Novel antibiotics are urgently needed to tackle the growing worldwide problem of antimicrobial resistance. Bacterial pathogens possess few privileged targets for a successful therapy: the majority of existing antibiotics as well as current candidates in development target the complex bacterial machinery for cell wall synthesis, protein synthesis, or DNA replication. An important mechanistic question addressed by this study is whether inhibiting such a complex target at different sites with different compounds has similar or differentiated cellular consequences. Using transcriptomics and metabolomics, we demonstrate that three different classes of gyrase inhibitors can be distinguished by their molecular signatures in P. aeruginosa. We describe the cellular effects of a promising, recently identified gyrase inhibitor class, the cystobactamids, in comparison to those of the established gyrase A-binding fluoroquinolones and the gyrase B-binding aminocoumarins. The study results have implications for mode-of-action discovery approaches based on target-specific reference compounds, as they highlight the intraclass variability of cellular compound effects.
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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