Resistance mutations generate divergent antibiotic susceptibility profiles against translation inhibitors

Abstract

Mutations conferring resistance to translation inhibitors often alter the structure of rRNA. Reduced susceptibility to distinct structural antibiotic classes may, therefore, emerge when a common ribosomal binding site is perturbed, which significantly reduces the clinical utility of these agents. The translation inhibitors negamycin and tetracycline interfere with tRNA binding to the aminoacyl-tRNA site on the small 30S ribosomal subunit. However, two negamycin resistance mutations display unexpected differential antibiotic susceptibility profiles. Mutant U1060A in 16S Escherichia coli rRNA is resistant to both antibiotics, whereas mutant U1052G is simultaneously resistant to negamycin and hypersusceptible to tetracycline. Using a combination of microbiological, biochemical, single-molecule fluorescence transfer experiments, and X-ray crystallography, we define the specific structural defects in the U1052G mutant 70S E. coli ribosome that explain its divergent negamycin and tetracycline susceptibility profiles. Unexpectedly, the U1052G mutant ribosome possesses a second tetracycline binding site that correlates with its hypersusceptibility. The creation of a previously unidentified antibiotic binding site raises the prospect of identifying similar phenomena in antibiotic-resistant pathogens in the future.

The third-generation tetracycline, KBP-7072, exploits and reveals a new potential of the primary tetracycline binding pocket

“…In this respect, it should be noted that the 4-ring backbone of KBP-7072 is a structural feature that is common to both the parental Tetracycline (TET) molecule and with the first third generation tetracycline developed, Tigecycline (TIG) (Figure 1). Accordingly, this 4-ring moiety of KBP-7072 makes similar interactions with the binding pocket as described in previous work on Tetracycline or Tigecycline (8,9,12). Briefly, as illustrated in Figure 3A-B and summarized schematically in Supplemental Figure 1, these shared interactions include (i) the coordination of a Mg2+ ion between the polar groups of rings B and C, (ii) the coordination of a second Mg2+ ion by the phosphate backbone of G966 in helix 31 and ring A, (iii) hydrogen bonds to residues G1198 and C1195, and (iv) a stacking interaction between C1054 and ring D. In addition to these common interactions, the strong density observed in both the initial Fo-Fc difference map (Figure 2B) and final 2Fo-Fc electron density map (Figure 2C) allowed us to confidently model and describe interactions for the unique 3-methyl-3-azabicyclo[3.1.0]hexane sidechain moiety that differentiates KBP-7072 from other Tetracycline derivatives.…”

“…Although KBP-7072 was soaked into the 30S subunit crystal using relatively high concentrations (17-44 µM) KBP-7072 was observed to be bound at only the primary tetracycline binding site. This suggests that KBP-7072, like Tigecycline, shows higher binding specificity to the 30S subunit than the parent compound, Tetracycline, which has been observed at several secondary binding sites (10)(11)(12)25). As this initial unbiased Fo-Fc difference Fourier map unambiguously established the presence of KBP-7072 in the ribosomal complex, the KBP-7072 structure was added to the 30S model and subsequent refinement at the resolution of 3.1 Å led to a final structure with crystallographic Rwork/Rfree values of 19.3%/23.4% ( Table 1).…”

“…It should be noted that a bent but distinct conformation has also been observed in the structure of Tigecycline bound to the 70S ribosome (Figure 4; purple molecule). In these structures, however, the secondary amine in the tert-butylglycylamide sidechain of Tigecycline is not positioned to allow significant direct interactions with the 16S rRNA phosphate backbone and is not discussed in previous Tigecycline-70S structures (9,12). This difference in the ability of KBP-7072 and Tigecycline to form direct interactions with the 16S rRNA phosphate backbone could stem from the fact that the tert-butylglycylamide side chain of tigecycline harbors a peptide bond (Figure 1), that should adopt a planar conformation due to its partial double bond character (in conjugation with the heteroaromatic D ring of the drug) and restrict its rotational freedom.…”

“…Additional biochemical and biophysical studies aimed at comparing Tigecycline and other 3rd generation Tetracycline derivatives, including Fluorocycline, Azatetracycline, Omadacycline and Pentacycline, indicate that the derivative's efficiency can be rationalized in terms of its mode of binding to the 30S ribosomal subunit (8,9). In this regard, structural studies, conducted so far only on tetracycline (10,11) and Tigecycline (8,9,12), have revealed that although Tigecycline interacts with the same pocket on the ribosome as Tetracycline, the sidechain extension at the C9 position of TIG establishes additional contacts, via the glycyl nitrogen and the secondary amine present in the 9-tert-butylglycylamide moiety, with the ribosomal nucleotide C1054. These additional interactions can rationalize the increased affinity observed for TIG in comparison to TET, as well as the ability of TIG to overcome resistance mechanisms mediated by the ribosomal protection protein Tet(M) (9).…”

Deletion of RsmE 16S rRNA methyltransferase leads to low level increase in aminoglycoside resistance inMycobacterium smegmatis

“…However, deletion of rsmE had no significant effect on growth and survival of E. coli (Basturea 2006). Apart from its key roles in ribosomal function and fidelity, U1498 also likely to affect the response to antibiotics, as U1498 lies in the vicinity of binding site of various aminoglycosides as seen in the crystal structures of ribosome-antibiotic complexes (Borovinskaya et al, 2007(Borovinskaya et al, , 2008Demirci et al, 2013;Cocozaki et al, 2016). A mutation of U1498C is among three mutations that confer different levels of hygromycin resistance in M. smegmatis in vitro (Pfister et al, 2003).…”