Bifunctional Nitrone-Conjugated Secondary Metabolite Targeting the Ribosome

Abstract

Many microorganisms possess the capacity for producing multiple antibiotic secondary metabolites. In a few notable cases, combinations of secondary metabolites produced by the same organism are used in important combination therapies for treatment of drug-resistant bacterial infections. However, examples of conjoined roles of bioactive metabolites produced by the same organism remain uncommon. During our genetic functional analysis of oxidase-encoding genes in the everninomicin producer Micromonospora carbonacea var. aurantiaca, we discovered previously uncharacterized antibiotics everninomicin N and O, comprised of an everninomicin fragment conjugated to the macrolide rosamicin via a rare nitrone moiety. These metabolites were determined to be hydrolysis products of everninomicin P, a nitrone-linked conjugate likely the result of nonenzymatic condensation of the rosamicin aldehyde and the octasaccharide everninomicin F, possessing a hydroxylamino sugar moiety. Rosamicin binds the erythromycin macrolide binding site approximately 60 Å from the orthosomycin binding site of everninomicins. However, while individual ribosomal binding sites for each functional half of everninomicin P are too distant for bidentate binding, ligand displacement studies demonstrated that everninomicin P competes with rosamicin for ribosomal binding. Chemical protection studies and structural analysis of everninomicin P revealed that everninomicin P occupies both the macrolide- and orthosomycin-binding sites on the 70S ribosome. Moreover, resistance mutations within each binding site were overcome by the inhibition of the opposite functional antibiotic moiety binding site. These data together demonstrate a strategy for coupling orthogonal antibiotic pharmacophores, a surprising tolerance for substantial covalent modification of each antibiotic, and a potential beneficial strategy to combat antibiotic resistance.

Figure 1.

Analysis of genetic replacements of evdN1 and evdO1. (A) LC/MS analysis of wildtype M. carbonacea var. aurantiaca, gene replacements evdN1 (ΔevdN1::ac(3)IV), evdO1 (ΔevdO1::aac(3)IV), and genetic complementation of evdO1 gene replacement (ΔevdO1::aac(3)IV GC). The chromatogram shows summed ion intensities in negative mode (orange) and positive mode (black) for Evn D–G and new metabolites. Chromatograms are extracted ion currents for positive (M+H) and negative (M−H) ions for the indicated compounds. Masses of all everninomicins in this study are found in Supplementary Table 1. (B) Structures of wildtype metabolites Evn A–G and deletion metabolite Evn H.

Figure 2.

evdO1 reveals nitrone linked macrolide congeners. (A) Structure of Evn M from evdN1 gene replacement strain. (B) Formation of nitrone moiety from the hydroxylamine of Evn F and aldehyde of Rosa. (C) Antibiotics Evn N and Evn O, identified in extracts of genetic complementation mutants of ΔevdO1 are likely hydrolysis products of Evn P, subsequently isolated from the wildtype M. carbonacea var. aurantiaca.

Figure 3.

Mapping the binding site of Evn P on the bacterial ribosome. (A) Overview of the binding sites of macrolides, such as Rosa (orange), and orthosomycins, such as everninomicin A (Evn A, purple) on the large ribosomal 50S subunit (grey). For reference, the P-site tRNA (green) is shown and central protuberance (CP) and L1 stalk are indicated. (B) Filter binding assay monitoring bound radiolabeled Ery in the presence of increasing concentrations of Evn H (purple), Rosa (orange), cold erythromycin (green) and Evn P (salmon). The error bars show the standard deviation from the mean of three independent experiments. (C–E) 23S rRNA nucleotides protected from chemical modification by DMS in the presence of (C) Evn H, (D) Rosa and (E) Evn P. (F) Electron density (grey mesh) for Evn P bound within the exit tunnel of the ribosome, with model for Rosa (orange) fitted. (G) Comparison of binding position of Rosa/Evn P relative to Ery (green) with 23S rRNA nucleotides A2058 and A2059 (blue) shown for reference. (H) Electron density (grey mesh) for Evn P bound to orthosomycin binding site of the ribosome, with model for Evn P (salmon) fitted. (I) Comparison of binding position of Evn P relative Evn A (purple) with 23S rRNA helices H89 and H91 (blue) and ribosomal protein L16 (green) shown for reference.

Figure 4.

Toeprinting assay monitoring translation inhibition by Evn H, Rosa and Evn P using the ErmBL mRNA. Translation reactions were performed using the ermBL mRNA in the absence of antibiotics (−), the presence of thiostrepton (Ths), as well as increasing concentrations of Evn H, Rosa, and Evn P. Reverse transcription stops indicating ribosomes trapped during initiation with the AUG start codon in the P-site, as well as ribosomes stalled at Asp codon or Lys codon, are indicated with arrows. Sequencing lanes C, U, A and G are shown for reference with relevant ErmBL sequence. Because the ribosome protects 12 and 15 nucleotides from the A-site and P-site codons to the 3′ end of the mRNA where the reverse transciptase stops, the position of the toeprints (Asp and Lys arrows) are shifted compared to the sequencing lanes.

Figure 5.

Effect of Evn P on translation using wildtype, Evn, and macrolide resistant ribosomes. (A-D) in vitro translation using firefly luciferase as a reporter to monitor antibiotic inhibition on (A) wildtype E. coli 70S ribosomes, (B) E. coli A2059G macrolide-resistant ribosomes, (C) E. coli A2471C orthosomycin-resistant ribosomes, and (D) E. coli A2059G/A2471C macrolide and/or orthosomycin-resistant ribosomes. In (A-C) and (D), the error bars show the standard version from the mean for 2 and 3 independent experiments, respectively.