Replacement of S14 Protein in Ribosomes of Zinc-Starved Mycobacteria Reduces Spectinamide Sensitivity

Zinc is an essential micronutrient for mycobacteria, and its depletion induces multiple adaptive changes in cellular physiology, the most remarkable of which are remodeling and hibernation of ribosomes. Ribosome remodeling, induced upon relatively moderate depletion of zinc, involves replacement of multiple ribosomal proteins containing the zinc-binding CXXC motif (called C+ r proteins) by their motif-free C− paralogs. Severe zinc depletion induces binding of mycobacterial protein Y (Mpy) to the 70S C− ribosome, thereby stabilizing the ribosome in an inactive state that is also resistant to kanamycin and streptomycin. Because the Mpy binding region on the ribosome is proximal to the binding pocket of spectinamides (Spa), the preclinical drug candidates for tuberculosis, we addressed the impact of remodeling and hibernation of ribosomes on Spa sensitivity. We report here that while Mpy binding has no significant effect on Spa sensitivity to the ribosome, replacement of S14(C+) with its C− counterpart reduces the binding affinity of the drug by ∼2-fold, causing increased Spa tolerance in Mycobacterium smegmatis and Mycobacterium tuberculosis cells harboring the C− ribosome. The altered interaction between Spa and ribosomes likely results from new contact points for D67 and R83 residues of S14(C−) with U1138 and C1184 of 16S rRNA helix 34, respectively. Given that M. tuberculosis induces ribosome remodeling during progression from the acute to chronic phase of lung infection, our findings highlight new considerations in the development of Spa as effective drugs against tuberculosis.