Thiostrepton Hijacks Pyoverdine Receptors To Inhibit Growth of Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa is a biofilm-forming opportunistic pathogen and is intrinsically resistant to many antibiotics. In a high-throughput screen for molecules that modulate biofilm formation, we discovered that the thiopeptide antibiotic thiostrepton (TS), which is considered to be inactive against Gram-negative bacteria, stimulated P. aeruginosa biofilm formation in a dose-dependent manner. This phenotype is characteristic of exposure to antimicrobial compounds at subinhibitory concentrations, suggesting that TS was active against P. aeruginosa. Supporting this observation, TS inhibited the growth of a panel of 96 multidrug-resistant (MDR) P. aeruginosa clinical isolates at low-micromolar concentrations. TS also had activity against Acinetobacter baumannii clinical isolates. The expression of Tsr, a 23S rRNA-modifying methyltransferase from TS producer Streptomyces azureus, in trans conferred TS resistance, confirming that the drug acted via its canonical mode of action, inhibition of ribosome function. The deletion of oligopeptide permease systems used by other peptide antibiotics for uptake failed to confer TS resistance. TS susceptibility was inversely proportional to iron availability, suggesting that TS exploits uptake pathways whose expression is increased under iron starvation. Consistent with this finding, TS activity against P. aeruginosa and A. baumannii was potentiated by the FDA-approved iron chelators deferiprone and deferasirox and by heat-inactivated serum. Screening of P. aeruginosa mutants for TS resistance revealed that it exploits pyoverdine receptors FpvA and FpvB to cross the outer membrane. We show that the biofilm stimulation phenotype can reveal cryptic subinhibitory antibiotic activity, and that TS has activity against select multidrug-resistant Gram-negative pathogens under iron-limited growth conditions, similar to those encountered at sites of infection.

Forging new antibiotic combinations under iron-limiting conditions

“…CLI antagonized DSX at the highest concentration; however, growth was still below 20% of control (Fig. 4C), which we previously established as equivalent to the MIC in the growth medium used for this work (22). When the data were plotted against TS concentration (Fig.…”

“…The spectrophotometric assay was repeated for all compounds listed in Table 1 except for clioquinol (CLI). CLI was identified in our previous screen as a P. aeruginosa growth inhibitor (22) but precipitated at concentrations above 8 µg/mL. Ciclopirox olamine (CO) and tropolone (TRO) showed shifts in their absorption spectra (Fig.…”

“…We previously showed that iron chelation potentiated the effects of TS, as DSX alone had no anti- Pseudomonas activity (22). CLI, TRO, DOXY, and CO can inhibit P. aeruginosa growth, suggesting that the innate activity of the compounds could be partly responsible for synergy with TS.…”

“…Double (TS + compound) and triple combinations (TS + DSX + compound) were tested against same panels of P. aeruginosa and A. baumannii clinical isolates we previously assayed for susceptibility to TS + DSX (Fig. 5) (22). GN was omitted due to its weak synergy with TS against PA14 and antagonism with iron chelators ( Fig.…”

“…Our group recently discovered that the thiopeptide antibiotic thiostrepton (TS) hijacks pyoverdine receptors under iron-limited conditions to cross the outer membranes of the World Health Organization’s top two critical priority pathogens, P. aeruginosa and Acinetobacter baumannii (22). TS activity was potentiated in heat-inactivated mouse and human serum, and by FDA-approved iron chelators, deferiprone (DFP) and deferasirox (DSX).…”