Targeting Bacterial Gyrase With Cystobactamid, Fluoroquinolone and Aminocoumarin Antibiotics Induces Distinct Molecular Signatures in Pseudomonas aeruginosa

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.