The Novel Aminomethylcycline Omadacycline Has High Specificity for the Primary Tetracycline-Binding Site on the Bacterial Ribosome

Abstract

Omadacycline is an aminomethylcycline antibiotic with potent activity against many Gram-positive and Gram-negative pathogens, including strains carrying the major efflux and ribosome protection resistance determinants. This makes it a promising candidate for therapy of severe infectious diseases. Omadacycline inhibits bacterial protein biosynthesis and competes with tetracycline for binding to the ribosome. Its interactions with the 70S ribosome were, therefore, analyzed in great detail and compared with tigecycline and tetracycline. All three antibiotics are inhibited by mutations in the 16S rRNA that mediate resistance to tetracycline in Brachyspira hyodysenteriae, Helicobacter pylori, Mycoplasma hominis, and Propionibacterium acnes. Chemical probing with dimethyl sulfate and Fenton cleavage with iron(II)-complexes of the tetracycline derivatives revealed that each antibiotic interacts in an idiosyncratic manner with the ribosome. X-ray crystallography had previously revealed one primary binding site for tetracycline on the ribosome and up to five secondary sites. All tetracyclines analyzed here interact with the primary site and tetracycline also with two secondary sites. In addition, each derivative displays a unique set of non-specific interactions with the 16S rRNA.

Keywords: antibiotic resistance; antibiotics; chemical probing; omadacycline; ribosome structure; tetracycline; tetracycline resistance; tigecycline.

Figure 1

Tetracycline, tigecycline and omadacycline, the tetracycline binding sites and proximity of tetracycline rRNA resistance mutations to the primary binding site. (A) The chemical structures of tetracycline, tigecycline, and omadacycline drawn schematically with their common backbone ring structures (rings A–D) colored distinctly. Carbon atom assignments for the 4-ring backbone are indicated on tetracycline; (B) The primary and secondary tetracycline binding sites as observed in X-ray crystallography studies [2,3,4] are shown on the structure of the 30S ribosomal subunit (rRNA, light grey surface; ribosomal-proteins, dark grey surface). The primary (1°) and secondary (2°) TET binding sites observed by Brodersen et al. are colored pink, the primary (1°) site described by Jenner et al. is green but largely obscured underneath TET, and the TET binding sites 1–6 observed by Pioletti et al. are colored blue and labeled distinctly. The head, spur, platform (Pt) and body 30S subunit landmarks are labeled. (C) The primary tetracycline binding site according to Jenner et al. [4] is illustrated showing the two rRNA bases, G1058 and G966, whose mutation results in tetracycline resistance.

Figure 2

TET, TGC, and OMC affect DMS modification of bases in the16S rRNA. Empty E. coli 70S ribosomes (0.5–0.6 µM) were incubated with varying amounts of TET, TGC or OMC and methylated with DMS. Modification of nucleotides (A) C1054 and (B) A892 was detected by primer extension and analyzed by electrophoresis on denaturing 6% polyacrylamide gels, sections of which are shown in the panels (l) left of the plots (r) showing their respective quantification. The dideoxy sequencing lanes are indicated with A and C; the unmodified RNA with R; the unmodified rRNA in the presence of the antibiotics TET, TGC or OMC with T, G, and O respectively; the DMS-modified RNA in the absence of antibiotics with D; and the DMS modified RNA in the presence of antibiotic is indicated with wedges under the TET, TGC, OMC headers where the wedge represents the presence of antibiotics at 300, 30, 3, and 0.3 µM. The extent of DMS modification of the rRNA in the presence of increasing amounts of antibiotic was quantitated in a phosphorimager and is shown below the gel sections with a comparison to the control DMS-modified RNA in the absence of antibiotics (lanes designated as “D”). Quantification was adjusted for loading differences by normalization with regions unaffected by TET, TGC or OMC.

Figure 3

Fe²⁺-complexed with TET, TGC or OMC affects cleavage of bases in the 16S rRNA. Empty E. coli 70S ribosomes (2 µM) were incubated with increasing amounts of Fe²⁺-complexed TET, TGC or OMC (1–125 µM) and incubated with sodium ascorbate and hydrogen peroxide. Sites of cleavage were detected by primer extension and analyzed by electrophoresis on denaturing 6% polyacrylamide gels, sections of which are shown above the plots of their respective quantification. Dose-dependent changes in cleavage intensity were found at nucleotides (A) U965, (B) C1195, (C) A1197, (D) G894, and (E) G1053/C1054. The dideoxy sequencing lanes are indicated with A and C; the unmodified RNA with R; Fe²⁺ incubated rRNA in the absence of sodium ascorbate and hydrogen peroxide with H; Fenton-cleaved rRNA in the absence of antibiotics with F; unmodified rRNA in the presence of 125 µM antibiotic; TET, TGC, OMC with T, G, and O, respectively; Fenton-cleaved rRNA in the presence of the respective antibiotic under the TET, TGC, and OMC headers where the wedge represents the presence of 125, 25, 5, and 1 µM of the respective antibiotic. The extent of rRNA cleavage in the presence of increasing amounts of antibiotic was quantified in a phosphorimager and is shown below the gel sections with a comparison to the control Fenton-cleaved rRNA in the absence of antibiotic (shown in lanes designated “F”). Quantification was adjusted for loading differences by normalization to regions unaffected by TET, TGC or OMC. Note an identical gel slice is shown in panels B and C as the specified nucleotides are close in primary sequence. (F) Sites of increased (green: U965, C1195, and A1197) and decreased (red C1053, C1054) Fenton cleavage in the presence of the respective antibiotic within the primary tetracycline binding site [4] are shown. The 16S rRNA helices, h31 and h34, are colored purple and blue, respectively while the Mg²⁺ coordinated by tetracycline rings B and C is colored orange and the Mg²⁺ coordinated near tetracycline ring A is colored green. RNA residues are numbered according to the E. coli sequence.

Figure 4

Summary of the interaction sites of TET, TGC, and OMC with the 16S rRNA. The secondary structure of the E. coli 16S rRNA is shown schematically [44]. Located within the stippled boxes and shown in more detail in the enlarged sections are bases that (i) display altered reactivity towards DMS probing in the presence of TET, TGC or OMC (white diamond: TET only; black diamond: all three) [15,25]; (ii) lead to weak resistance against TET, TGC, and OMC when mutated (TET^R) [15,20,21,37]; show either (iii) Fe²⁺-mediated specific cleavage (white 4-pointed star, black star) [15], or (iv) protection from Fe²⁺-mediated cleavage in the presence of TET, TGC, and OMC (white 5-pointed star). In addition, the secondary structure contains (i) sites with altered reactivity towards DMS in the presence of tRNA (grey rectangle) [34] or the S7 protein (grey circle) [36]; (ii) direct photocrosslinks to TET (black arrow) [33,45]; (iii) RNA-RNA crosslinks affected by TET (black dumbbell) [35] or (iv) sites with Fe²⁺-mediated non-specific cleavage in the presence of TET (white triangle), TGC (grey triangle) or OMC (black triangle).