Trojan Horse siderophore conjugates induce P. aeruginosa suicide and qualify the TonB protein as a novel antibiotic target

Abstract

Rising infection rates with multidrug-resistant bacterial pathogens such as Pseudomonas combined with a shallow antibiotic pipeline urgently call for antibiotics with novel modes of action. Herein, we identify the inner membrane protein TonB, motor of active uptake in Gram negative bacteria, as a novel target in antimicrobial therapy. The interaction of the TonB box, the periplasmic N-terminal domain of ferri-siderophore transporters, with the inner membrane protein TonB is crucial for the internalization of essential bacterial metabolites. Overexpression of a TonB box-containing peptide fragment in P. aeruginosa resulted in a growth repression, even in the presence of ferric heme as an iron source. The coupling of three TonB box peptides to synthetic DOTAM and MECAM siderophores with covalent or cleavable linkers of varying length and attachment sites yielded a panel of 24 conjugates in up to 32 synthetic steps. The transporters mediating iron uptake through these conjugates were identified by molecular approaches and transporter knockout mutants to be PfeA and PirA. The conjugates 11, 13 and 17 repressed bacterial growth in P. aeruginosa strains with minimal inhibitory concentrations of 0.5, 4 and 0.1 μM, respectively. The study illustrates a variant of cellular suicide therapy where a transporter imports its own inhibitor; it also demonstrates that artificial siderophores are capable to import large cargo with molecular weights of up to 4 kDa, and suggests that TonB constitutes an attractive target for antimicrobial therapy.