Uptake Mechanisms and Regulatory Responses to MECAM- and DOTAM-Based Artificial Siderophores and Their Antibiotic Conjugates in <i>Pseudomonas aeruginosa</i>

Abstract

The development of new antibiotics against Gram-negative bacteria has to deal with the low permeability of the outer membrane. This obstacle can be overcome by utilizing siderophore-dependent iron uptake pathways as entrance routes for antibiotic uptake. Iron-chelating siderophores are actively imported by bacteria, and their conjugation to antibiotics allows smuggling the latter into bacterial cells. Synthetic siderophore mimetics based on MECAM (1,3,5-<i>N</i>,<i>N</i>',<i>N</i>″-tris-(2,3-dihydroxybenzoyl)-triaminomethylbenzene) and DOTAM (1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane) cores, both chelating iron via catechol groups, have been recently applied as versatile carriers of functional cargo. In the present study, we show that MECAM and the MECAM-ampicillin conjugate <b>3</b> transport iron into <i>Pseudomonas aeruginosa</i> cells via the catechol-type outer membrane transporters PfeA and PirA and DOTAM solely via PirA. Differential proteomics and quantitative real-time polymerase chain reaction (qRT-PCR) showed that MECAM import induced the expression of <i>pfeA</i>, whereas <b>3</b> led to an increase in the expression of <i>pfeA</i> and <i>ampc</i>, a gene conferring ampicillin resistance. The presence of DOTAM did not induce the expression of <i>pirA</i> but upregulated the expression of two zinc transporters (<i>cntO</i> and <i>PA0781</i>), pointing out that bacteria become zinc starved in the presence of this compound. Iron uptake experiments with radioactive ⁵⁵Fe demonstrated that import of this nutrient by MECAM and DOTAM was as efficient as with the natural siderophore enterobactin. The study provides a functional validation for DOTAM- and MECAM-based artificial siderophore mimetics as vehicles for the delivery of cargo into Gram-negative bacteria.