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, 116 (44), 22275-22281

Structure of Pseudomonas aeruginosa Ribosomes From an Aminoglycoside-Resistant Clinical Isolate


Structure of Pseudomonas aeruginosa Ribosomes From an Aminoglycoside-Resistant Clinical Isolate

Yehuda Halfon et al. Proc Natl Acad Sci U S A.


Resistance to antibiotics has become a major threat to modern medicine. The ribosome plays a fundamental role in cell vitality by the translation of the genetic code into proteins; hence, it is a major target for clinically useful antibiotics. We report here the cryo-electron microscopy structures of the ribosome of a pathogenic aminoglycoside (AG)-resistant Pseudomonas aeruginosa strain, as well as of a nonresistance strain isolated from a cystic fibrosis patient. The structural studies disclosed defective ribosome complex formation due to a conformational change of rRNA helix H69, an essential intersubunit bridge, and a secondary binding site of the AGs. In addition, a stable conformation of nucleotides A1486 and A1487, pointing into helix h44, is created compared to a non-AG-bound ribosome. We suggest that altering the conformations of ribosomal protein uL6 and rRNA helix H69, which interact with initiation-factor IF2, interferes with proper protein synthesis initiation.

Keywords: aminoglycoside; antibiotic; cystic fibrosis; resistance; ribosome.

Conflict of interest statement

The authors declare no competing interest.


Fig. 1.
Fig. 1.
Effect of rplF mutation on growth phenotype and gentamicin resistance. (A) Complementation assay of rplf mutation. The wild-type copy of rplF gene was expressed from a plasmid (pCrplF) in native and mutant strains and the generation time was assessed. The empty plasmid (pCE) was used as control. Error bars represent the SD of 3 independent replicates. Differences between conditions were computed by using the student test (P < 0.0001). (B) MIC for gentamicin antibiotic for the native and mutant strains containing the pCE empty plasmid, the pCrplF plasmid containing the wild-type copy of the rplF gene, and the pCL6M plasmid containing the mutated copy of the rplF gene. (C) Inhibition of translation as results of increasing concentrations of gentamicin antibiotic resulting from the translation of the firefly luciferase in vitro translation system. Note that at low gentamicin concentrations (1 × 10−4 to 3.6 × 10−3 μg⋅mL−1), the in vitro translational activity is comparable between wild-type (PAO1 and native) and mutant ribosomes. ns, not significant differences. The values represent the mean ± SE (n = 3) of the luminescence. Differences between the IC50 values were computed by using 1-way ANOVA followed by Tukey’s post hoc test (P < 0.01). (D) Molecular assessment of the ribosome functionality. Sedimentation profiles (10–50% sucrose gradient) of wild-type (PAO1 and native) and rplF mutant total ribosome fraction are shown.
Fig. 2.
Fig. 2.
(A) The cryo-EM structure of the PA ribosome where uL6, H69, H68, h44, h24, and paromomycin are shown as space-filling objects and are colored blue, yellow, magenta, green, orange, and red, respectively. The PA ribosome is shown in 2 views 90° apart. (BF) Structure comparison between PAnat, E. coli, S. aureus, and T. thermophilus. (B) Large subunit rRNA helix H45. (C) Large subunit rRNA helix H16. (D) Large subunit rRNA helix H63. (E) Small subunit rRNA helix h7. (F) Small subunit rRNA helix h33. PA, E. coli (PDB ID code 5NWY), S. aureus (PDB ID code 5TV7), and T. thermophilus (PDB ID code 5IMQ) are shown in orange, gray, blue, and green, respectively. (G) The location of the rRNA helices shown in BF is marked on the 2D map of the PA ribosome.
Fig. 3.
Fig. 3.
The specific differences found between the PA native and mutant ribosomes. (A) A comparison of the uL6 protein of PAnat (coral) and PAuL6m (cyan). The red arrow marks the structural alteration of the loop due to the GYKA deletion. (B) Differences at the structure of the main AG binding site in h44 of the 16S rRNA. The red arrows indicate the flipping of the nucleotides (PA and E. coli [in brackets] numbering are used). PAnat and PAuL6m 16S rRNA are shown in tan and blue, respectively. (C) Conformational changes of intersubunit bridge B2a: gentamycin (colored in red) is superimposed on its main binding site at h44 and its secondary binding site at H69, from PDB ID code 4V53 (45). H69 (cyan) and h44 (blue) of PAuL6m were superimposed on PAnat helices H69 (wheat) and h44 (orange) to show the conformational changes that occur in H69 that contribute to the phenotypic characteristics of the mutant. It may also harm the binding to the second AG binding site at H69. (D) Conformational changes at intersubunit bridge B2b: H68 (cyan) and h24 (blue) of PAuL6m were superimposed on PAnat helices H68 (wheat) and h24 (orange) to show the flexible region of h24 that was not modeled and contribute to the phenotypic characteristics of the mutant.
Fig. 4.
Fig. 4.
(A) IF2 bridges between uL6 and H69 IF2 (using PDB ID code 3JCJ) superimposed on PAnat (coral) and PAuL6m (cyan) suggests how IF2 is interacting with both uL6 and H69. IF2 domains G, II, III, IV, and h8 are shown in red, orange, blue, purple, and green, respectively. (B) Zoom into uL6 from PAnat (coral), PAUL6m (cyan), and E. coli (gray) interacting with the IF2 domain G (red). The shorter uL6 loop of PAuL6m (cyan) may hamper this interaction. (C) Zoom into H69 superimposed with P-site tRNA (PDB ID code 5LMV) and with the IF2 domain IV (purple). The shift of H69 in PAuL6m mutant toward IF2 domain IV, compared to PAnat, may lead to a clash between IF2 and H69 that may either interfere with IF2 proper binding and may hamper this function of H69. (D) Sequence alignment of uL6 protein of PA, E. coli (EC), T. thermophilus (TT), B. subtilis (BS), S. aureus (SA), and D. radiodurans (DR). The mutation site is marked with a red box.

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