• The Peptide Chain Release Factor Methyltransferase PrmC Influences the Pseudomonas aeruginosa PA14 Endo- and Exometabolome.

      Depke, Tobias; Häussler, Susanne; Brönstrup, Mark; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MDPI, 2020-10-18)
      Pseudomonas aeruginosa is one of the most important nosocomial pathogens and understanding its virulence is the key to effective control of P. aeruginosa infections. The regulatory network governing virulence factor production in P. aeruginosa is exceptionally complex. Previous studies have shown that the peptide chain release factor methyltransferase PrmC plays an important role in bacterial pathogenicity. Yet, the underlying molecular mechanism is incompletely understood. In this study, we used untargeted liquid and gas chromatography coupled to mass spectrometry to characterise the metabolome of a prmC defective P. aeruginosa PA14 strain in comparison with the corresponding strain complemented with prmC in trans. The comprehensive metabolomics data provided new insight into the influence of prmC on virulence and metabolism. prmC deficiency had broad effects on the endo- and exometabolome of P. aeruginosa PA14, with a marked decrease of the levels of aromatic compounds accompanied by reduced precursor supply from the shikimate pathway. Furthermore, a pronounced decrease of phenazine production was observed as well as lower abundance of alkylquinolones. Unexpectedly, the metabolomics data showed no prmC-dependent effect on rhamnolipid production and an increase in pyochelin levels. A putative virulence biomarker identified in a previous study was significantly less abundant in the prmC deficient strain.
    • Targeting Bacterial Gyrase with Cystobactamid, Fluoroquinolone, and Aminocoumarin Antibiotics Induces Distinct Molecular Signatures in Pseudomonas aeruginosa.

      Franke, Raimo; Overwin, Heike; Häussler, Susanne; Brönstrup, Mark; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (ASM, 2021-07-13)
      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 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.