6 research outputs found
Metabolic Profiling to Determine Bactericidal or Bacteriostatic Effects of New Natural Products using Isothermal Microcalorimetry.
Due to the global threat of rising antimicrobial resistance, novel antibiotics are urgently needed. We investigate natural products from Myxobacteria as an innovative source of such new compounds. One bottleneck in the process is typically the elucidation of their mode-of-action. We recently established isothermal microcalorimetry as part of a routine profiling pipeline. This technology allows for investigating the effect of antibiotic exposure on the total bacterial metabolic response, including processes that are decoupled from biomass formation. Importantly, bacteriostatic and bactericidal effects are easily distinguishable without any user intervention during the measurements. However, isothermal microcalorimetry is a rather new approach and applying this method to different bacterial species usually requires pre-evaluation of suitable measurement conditions. There are some reference thermograms available of certain bacteria, greatly facilitating interpretation of results. As the pool of reference data is steadily growing, we expect the methodology to have increasing impact in the future and expect it to allow for in-depth fingerprint analyses enabling the differentiation of antibiotic classes
Biomimetic Enterobactin Analogue Mediates Iron-Uptake and Cargo Transport into E. coli and P. aeruginosa
The design, synthesis
and biological evaluation of the artificial enterobactin analogue EntKL
and several fluorophore-conjugates thereof are described. EntKL
provides an attachment point for cargos such as fluorophores
or antimicrobial payloads. Corresponding conjugates are recognized by outer
membrane siderophore receptors of Gram-negative pathogens and retain the
natural hydrolyzability of the tris-lactone backbone, known to be key
for uptake into the cytosol. Initial density-functional theory (DFT)
calculations of the free energies of solvation (ΔG(sol)) and relaxed Fe-O force constants of the
corresponding [Fe-EntKL]3- complexes indicated a similar iron binding
constant compared to natural enterobactin (Ent). The synthesis of EntKL
was achieved via an iterative assembly based on a 3-hydroxylysine building block over 14 steps with an overall yield of
3%. A series of growth recovery assays under iron-limiting conditions with Escherichia coli
and Pseudomonas aeruginosa mutant strains that are defective in natural
siderophore synthesis revealed a potent concentration-dependent growth
promoting effect of EntKL similar to natural Ent.
Additionally, four cargo-conjugates differing in molecular size were able to
restore growth of E. coli indicating an uptake into the cytosol. P. aeruginosa
displayed a stronger uptake promiscuity as six different cargo-conjugates
were found to restore growth under iron-limiting conditions. Imaging studies utilizing
BODIPYFL-conjugates, demonstrated the ability of EntKL
to overcome the Gram-negative outer membrane permeability barrier and thus deliver
molecular cargos via the bacterial iron transport machinery of E. coli
and P. aeruginosa
Biomimetic enterobactin analogue mediates iron-uptake and cargo transport into E. coli and P. aeruginosa
The design, synthesis and biological evaluation of the artificial enterobactin analogue Ent(KL) and several fluorophore-conjugates thereof are described. Ent(KL) provides an attachment point for cargos such as fluorophores or antimicrobial payloads. Corresponding conjugates are recognized by outer membrane siderophore receptors of Gram-negative pathogens and retain the natural hydrolyzability of the tris-lactone backbone. Initial density-functional theory (DFT) calculations of the free energies of solvation (Delta G(sol)) and relaxed Fe-O force constants of the corresponding [Fe-Ent(KL)](3-) complexes indicated a similar iron binding constant compared to natural enterobactin (Ent). The synthesis of Ent(KL) was achieved via an iterative assembly based on a 3-hydroxylysine building block over 14 steps with an overall yield of 3%. A series of growth recovery assays under iron-limiting conditions with Escherichia coli and Pseudomonas aeruginosa mutant strains that are defective in natural siderophore synthesis revealed a potent concentration-dependent growth promoting effect of Ent(KL) similar to natural Ent. Additionally, four cargo-conjugates differing in molecular size were able to restore growth of E. coli indicating an uptake into the cytosol. P. aeruginosa displayed a stronger uptake promiscuity as six different cargo-conjugates were found to restore growth under iron-limiting conditions. Imaging studies utilizing BODIPYFL-conjugates, demonstrated the ability of Ent(KL) to overcome the Gram-negative outer membrane permeability barrier and thus deliver molecular cargos via the bacterial iron transport machinery of E. coli and P. aeruginosa
Darobactins Exhibiting Superior Antibiotic Activity by Cryo‐EM Structure Guided Biosynthetic Engineering
Over recent decades, the pipeline of antibiotics acting against Gram-negative bacteria is running dry, as most discovered candidate antibiotics suffer from insufficient potency, pharmacokinetic properties, or toxicity. The darobactins, a promising new small peptide class of drug candidates, bind to novel antibiotic target BamA, an outer membrane protein. Previously, we reported that biosynthetic engineering in a heterologous host generated novel darobactins with enhanced antibacterial activity. Here we utilize an optimized purification method and present cryo-EM structures of the Bam complex with darobactin 9 (D9), which served as a blueprint for the biotechnological generation of twenty new darobactins including halogenated analogs. The newly engineered darobactin 22 binds more tightly to BamA and outperforms the favorable activity profile of D9 against clinically relevant pathogens such as carbapenem-resistant Acinetobacter baumannii up to 32-fold, without observing toxic effects
Activity and cryo-EM structure guided biosynthetic pathway engineering yields non-natural Darobactin antibiotics with superior activity against Gram-negative pathogens
Over recent decades, the pipeline of antibiotics acting against Gram-negative bacteria is running dry, as most discovered candidate antibiotics suffer from insufficient potency, pharmacokinetic properties, or toxicity. The darobactins, a promising new small peptide class of drug candidates, bind to novel antibiotic target BamA, an outer membrane protein. Previously, we reported that biosynthetic engineering in a heterologous host generated non-natural darobactins with significantly enhanced antibacterial activity. Here we utilise an optimised purification procedure and present cryo-EM structures of the Bam-complex with darobactin 9 (D9), which served as a blueprint to further the biotechnological generation of twenty novel darobactins including halogenated analogues. The newly engineered darobactin 22 binds more tightly to BamA and outperforms the favorable activity profile of D9 against clinically relevant pathogens such as carbapenem-resistant Acinetobacter baumannii up to 32-fold, while toxic effects were not observed in human cells and zebrafish embryos up to 500 µg/mL
Darobactins Exhibiting Superior Antibiotic Activity by Cryo-EM Structure Guided Biosynthetic Engineering
Over recent decades, the pipeline of antibiotics acting against Gram-negative bacteria is running dry, as most discovered candidate antibiotics suffer from insufficient potency, pharmacokinetic properties, or toxicity. The darobactins, a promising new small peptide class of drug candidates, bind to novel antibiotic target BamA, an outer membrane protein. Previously, we reported that biosynthetic engineering in a heterologous host generated novel darobactins with enhanced antibacterial activity. Here we utilize an optimized purification method and present cryo-EM structures of the Bam complex with darobactin 9 (D9), which served as a blueprint for the biotechnological generation of twenty new darobactins including halogenated analogs. The newly engineered darobactin 22 binds more tightly to BamA and outperforms the favorable activity profile of D9 against clinically relevant pathogens such as carbapenem-resistant Acinetobacter baumannii up to 32-fold, without observing toxic effects