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

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-N,N′,N″-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 3 transport iron into Pseudomonas aeruginosa 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 pfeA, whereas 3 led to an increase in the expression of pfeA and ampc, a gene conferring ampicillin resistance. The presence of DOTAM did not induce the expression of pirA but upregulated the expression of two zinc transporters (cntO and PA0781), 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.

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

“…Previously, we have employed 55 Fe uptake assays to demonstrate that MECAM or DOTAM have the ability to shuttle iron into bacteria through the TBDTs PfeA and PirA, or through PirA only, respectively. 46 Unfortunately, small precipitations of iron loaded conjugates we prohibited the execution of 55 Fe uptake assays with DOTAM and MECAM peptide conjugates with a sufficiently strong 55 Fe signal-to-noise ratio (data not shown). Consequently we exploited the property of ferri-siderophores to interact with the two component system PfeS/PfeR and induce pfeA transcription.…”

“…showed a one to six (log2)fold induction of pfeA transcription in the PAO1 wildtype and ΔpvdFΔpchA mutant. 46 The free peptides 5-10 had no effect. For all tested MECAM and DOTAM conjugates, an induction of pfeA transcription was observed.…”

“…For compounds that do not exert antimicrobial activities, a growth reduction after TBDT knock-out proves that the missing siderophore transporter was crucial for the internalization of ferric chelates and thus permitted bacterial growth. 54,46 The free peptides 5-7 exerted no effect (Figure 5). The growth of DOTAMbased conjugates was hardly impaired by missing PfeA, but a strong growth inhibition was observed for all mutants lacking PirA, indicating that all these compounds enter the cells through this TBDT.…”

“…We have recently shown that unconjugated MECAM induced the transcription and expression of pfeA by interacting with PfeS. 46 This induction was accompanied by a transcription repression of the genes involved in acquisition of iron by the siderophore pyochelin (PCH). 46 The PYO and PCH-deficient ∆pvdF∆pchA strain was grown over eight hours in iron-restricted CAA medium in the absence or presence of the peptides, free DOTAM and MECAM cores, and a selection of conjugates (11, 12, 13, 17, 22 and 28, each at 10 µM).…”

“…46 This induction was accompanied by a transcription repression of the genes involved in acquisition of iron by the siderophore pyochelin (PCH). 46 The PYO and PCH-deficient ∆pvdF∆pchA strain was grown over eight hours in iron-restricted CAA medium in the absence or presence of the peptides, free DOTAM and MECAM cores, and a selection of conjugates (11, 12, 13, 17, 22 and 28, each at 10 µM). The expression of the genes encoding pfeA (ferri-ENT TBDT), 36 pirA (ferri-ENT and ferri-catecholamines TBDT), 49,50 fpvA (ferri-PYO TBDT), 24 and fptA (ferri-PCH TBDT) 51,35,52 was analyzed by differential quantitative real-time PCR (qRT-PCR) (Figure 4).…”