The development of novel drugs against Gramnegative bacteria represents an urgent medical need. To overcome their outer cell membrane, we synthesized conjugates of antibiotics and artificial siderophores based on the MECAM core, which are imported by bacterial iron uptake systems. Structures, spin states, and iron binding properties were predicted in silico using density functional theory. The capability of MECAM to function as an effective artificial siderophore in Escherichia coli was proven in microbiological growth recovery and bioanalytical assays. Following a linker optimization focused on transport efficiency, five β-lactam and one daptomycin conjugates were prepared. The most potent conjugate 27 showed growth inhibition of Gram-positive and Gram-negative multidrug-resistant pathogens at nanomolar concentrations. The uptake pathway of MECAMs was deciphered by knockout mutants and highlighted the relevance of FepA, CirA, and Fiu. Resistance against 27 was mediated by a mutation in the gene encoding ExbB, which is involved in siderophore transport.
The diagnosis of bacterial infections at deep body sites benefits from noninvasive imaging of molecular probes that can be traced by positron emission tomography (PET). We specifically labeled bacteria by targeting their iron transport system with artificial siderophores. The cyclen-based probes contain different binding sites for iron and the PET nuclide gallium-68.A panel of 11 siderophores with different iron coordination numbers and geometries was synthesized in up to eight steps, and candidates with the best siderophore potential were selected by a growth recovery assay. The probes [ 68 Ga]7 and [ 68 Ga]15 were found to be suited for PET imaging based on their radiochemical yield, radiochemical purity, and complex stability in vitro and in vivo. Both showed significant uptake in mice infected with E.coli and were able to discern infection from LPS-triggered, sterile-inflammation. The study qualifies cyclen-based artificial siderophores as readily accessible scaffolds for the in vivo imaging of bacteria. 7 and 8 from cyclen: (i) bromoacetyl bromide, K2CO3,
The sensitive detection of bacterial infections is a prerequisite for their successful treatment. The use of a chemiluminescent readout was so far hampered by an insufficient probe enrichment at the pathogens. We coupled siderophore moieties, that harness the unique iron transport system of bacteria, with enzyme-activatable dioxetanes and obtained seven trifunctional probes with high signal-to-background ratios (S/B = 426-859). Conjugates with efficient iron transport capability into bacteria were identified through a growth recovery assay. All ESKAPE pathogens were labelled brightly by desferrioxamine conjugates, while catechols were weaker due to self-quenching. Bacteria could also be detected inside lung epithelial cells. The best probe 8 detected 9.1 × 10 3 CFU mL À 1 of S. aureus and 5.0 × 10 4 CFU mL À 1 of P. aeruginosa, while the analogous fluorescent probe 10 was 205-305fold less sensitive. This qualifies siderophore dioxetane probes for the selective and sensitive detection of bacteria.
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.
The growing antibiotic resistance, foremost in Gram-negative bacteria, requires novel therapeutic approaches. We aimed to enhance the potency of well-established antibiotics targeting the RNA polymerase (RNAP) by utilizing the microbial...
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