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. 2019 Jun 17;58(25):8581-8584.
doi: 10.1002/anie.201903472. Epub 2019 May 16.

The Natural Product Elegaphenone Potentiates Antibiotic Effects against Pseudomonas aeruginosa

Weining Zhao  1 , Ashley R Cross  2   3   4 , Caillan Crowe-McAuliffe  5 , Angela Weigert-Munoz  1 , Erika E Csatary  6 , Amy E Solinski  6 , Joanna Krysiak  1 , Joanna B Goldberg  2   4   7 , Daniel N Wilson  5 , Eva Medina  8 , William M Wuest  4   6   7 , Stephan A Sieber  1
Affiliations

The Natural Product Elegaphenone Potentiates Antibiotic Effects against Pseudomonas aeruginosa

Weining Zhao et al. Angew Chem Int Ed Engl. .

Abstract

Natural products represent a rich source of antibiotics that address versatile cellular targets. The deconvolution of their targets via chemical proteomics is often challenged by the introduction of large photocrosslinkers. Here we applied elegaphenone, a largely uncharacterized natural product antibiotic bearing a native benzophenone core scaffold, for affinity-based protein profiling (AfBPP) in Gram-positive and Gram-negative bacteria. This study utilizes the alkynylated natural product scaffold as a probe to uncover intriguing biological interactions with the transcriptional regulator AlgP. Furthermore, proteome profiling of a Pseudomonas aeruginosa AlgP transposon mutant provided unique insights into the mode of action. Elegaphenone enhanced the elimination of intracellular P. aeruginosa in macrophages exposed to sub-inhibitory concentrations of the fluoroquinolone antibiotic norfloxacin.

Keywords: Pseudomonas aeruginosa; antibiotics; chemical proteomics; natural products; virulence.

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Figures

Figure 1:
Figure 1:
A) Structures of elegaphenone (EL) and derivatives. B) The amount of extracellular pyocyanin in P. aeruginosaPAO1 upon treatment with elegaphenone and derivatives in a dose-dependent manner. The data is based on two biological experiments with technical triplicates. C) MIC values of elegaphenone derivatives in different pathogenic bacterial strains. Lack of growth inhibition is highlighted in grey. E. coli BW25113 ΔbamBΔtolC is a hyperpermeable E. coli strain.[13]
Figure 2:
Figure 2:
A) Overall scheme of gel-free affinity-based protein profiling (AfBPP) employing isotope labeling. Avid: Avidin; CC: click chemistry; DM: stable isotope dimethyl labeling. B) and C) Volcano plots of gel-free AfBPP experiment in P. aeruginosa PAO1 treated with 50 μM ELP5 vs. a 10-fold excess of EL or DMSO, respectively (both soluble fraction). Blue dots depict targets that are enriched by ELP5 (criteria: log2-fold enrichment ≥ 2 and -log10(p-value) ≥ 2.5) and competed by EL (criteria: log2-fold enrichment ≥ 2 and -log10(p-value) ≥ 2.5). Green squares denote targets that are enriched by ELP5 but not competed by EL while black dots denote background proteins. For a full list of proteins please refer to supporting information.
Figure 3:
Figure 3:
A) Number of viable P. aeruginosa PAO1 cultured in the presence or absence of 50 μM EL or ELP5 for 5 h. Cultures were inoculated with 5 × 105 P. aeruginosa bacteria. B) Intracellular viable P. aeruginosa in human THP-1 macrophages. The number of viable intracellular P. aeruginosa was determined after lysing the infected THP-1 macrophages. Concentration of EL derivatives and norfloxacin was 50 μM and 1.5 μg/mL, respectively. Each bar represents the mean ± SD of the data from three independent experiments. **, p < 0.05; ***, p < 0.005; one-way ANOVA with Tukey’s multiple comparison test.
Scheme 1:
Scheme 1:
Synthesis of AfBPP Probe ELP. DCM: dichloromethane, RT: room temperature, THF: tetrahydrofuran, DMF: N,N-dimethylformamide, CSA: camphorsulfonic acid.
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