Thiostrepton hijacks pyoverdine receptors to inhibit growth ofPseudomonas aeruginosa

Abstract

17Pseudomonas aeruginosa is a biofilm-forming opportunistic pathogen and intrinsically resistant to many 18 antibiotics. In a high-throughput screen for molecules that modulate biofilm formation, we discovered 19 that the thiopeptide antibiotic, thiostrepton (TS) -considered inactive against Gram-negative 20 bacteria -stimulated P. aeruginosa biofilm formation in a dose-dependent manner. This phenotype is 21 characteristic of exposure to antimicrobial compounds at sub-inhibitory concentrations, suggesting that 22 TS was active against P. aeruginosa. Supporting this observation, TS inhibited growth of a panel of 96 23 multidrug-resistant (MDR) P. aeruginosa clinical isolates at low micromolar concentrations. TS also had 24 activity against Acinetobacter baumannii clinical isolates. Expression of Tsr -a 23S rRNA-modifying 25 methyltransferase -in trans conferred TS resistance, confirming that the drug acted via its canonical 26 mode of action, inhibition of ribosome function. Deletion of oligopeptide permease systems used by 27 other peptide antibiotics for uptake failed confer TS resistance. TS susceptibility was inversely 28 proportional to iron availability, suggesting that TS exploits uptake pathways whose expression is 29 increased under iron starvation. Consistent with this finding, TS activity against P. aeruginosa and A.30 baumannii was potentiated by FDA-approved iron chelators deferiprone and deferasirox. Screening of P. 31 aeruginosa mutants for TS resistance revealed that it exploits pyoverdine receptors FpvA and FpvB to 32 cross the outer membrane. Our data show that the biofilm stimulation phenotype can reveal cryptic 33 sub-inhibitory antibiotic activity, and that TS has activity against select multidrug resistant Gram-34 negative pathogens under iron-limited growth conditions, similar to those encountered at sites of 35 infection.