The ribosomal S10 protein is a general target for decreased tigecycline susceptibility

Abstract

Tigecycline is a translational inhibitor with efficacy against a wide range of pathogens. Using experimental evolution, we adapted Acinetobacter baumannii, Enterococcus faecium, Escherichia coli, and Staphylococcus aureus to growth in elevated tigecycline concentrations. At the end of adaptation, 35 out of 47 replicate populations had clones with a mutation in rpsJ, the gene that encodes the ribosomal S10 protein. To validate the role of mutations in rpsJ in conferring tigecycline resistance, we showed that mutation of rpsJ alone in Enterococcus faecalis was sufficient to increase the tigecycline MIC to the clinical breakpoint of 0.5 μg/ml. Importantly, we also report the first identification of rpsJ mutations associated with decreased tigecycline susceptibility in A. baumannii, E. coli, and S. aureus. The identified S10 mutations across both Gram-positive and -negative species cluster in the vertex of an extended loop that is located near the tigecycline-binding pocket within the 16S rRNA. These data indicate that S10 is a general target of tigecycline adaptation and a relevant marker for detecting reduced susceptibility in both Gram-positive and -negative pathogens.

FIG 1

S10 mutations identified in strains that underwent TGC exposure. The symbol “Δ” indicates a deleted residue. Eight colonies were sampled from each population. For some populations, multiple types of rpsJ mutations were identified between different clones, and therefore the number of observations among the different mutations is sometimes greater than the total number of populations.

FIG 2

TGC selects for mutations on a loop of S10 that is in close proximity to the 16S rRNA and composes the TGC binding pocket. (A) Image of the crystal structure of TGC bound to the Thermus thermophilus ribosome (PDB 4G5T) (21). Labels on amino acids indicate positions on the loop of S10 where mutations were identified in Gram-positive species (red) or both Gram-positive and -negative species (blue). For clarity, the amino acid numbering corresponds to the E. coli S10 sequence, and only rRNA structure (gray) proximal to the loop is shown. The inset shows the entire structure of S10 with colored spheres at the α-carbon of residues where mutations where identified in Gram-positive (red) or both Gram-positive and -negative (blue) organisms. (B) Alignment of the T. thermophilus S10 sequence and the susceptible S10 sequences for species where mutations in S10 have been identified in response to TGC adaptation. The sequence logo shows the S10 consensus sequence. The black box outlines the residues that compose the loop structure (positions 53 to 61). The blue and red boxes outline the residues where mutations were identified in Gram-positive species (red) or both Gram-positive and -negative species (blue).

FIG 3

The S10^{R53Q-Δ54-57ATHK} mutation in E. faecalis confers improved growth in the presence of TGC. The growth of S613 and S613(S10^{R53Q-Δ54-57ATHK}) were measured for 12 h at 0 μg/ml TGC (A), 0.031 μg/ml TGC (B), 0.063 μg/ml TGC (C), 0.125 μg/ml TGC (D), and 0.25 μg/ml TGC (E). The averages for three replicates run in parallel were plotted; error bars show standard deviations.