Abstract
Tetracyclines are essential bacterial protein synthesis inhibitors under continual development to combat antibiotic resistance yet suffer from unwanted side effects. Therefore, next-generation drugs should better discriminate between prokaryotic and eukaryotic ribosomes to ensure host cells remain unaffected by treatment. Mitoribosomes - responsible for generating oxidative phosphorylation (OXPHOS) subunits - share evolutionary features with the bacterial machinery and may suffer from cross-reactivity. T cells depend upon OXPHOS upregulation to power clonal expansion and establish immunity. To this end, we compared important bacterial ribosome-targeting antibiotics for their ability to induce immortalized and primary T cell death. Tetracyclines tested were cytotoxic and tigecycline (third generation) was identified as the most potent. In human T cells in vitro, 5-10 mM tigecycline inhibited mitochondrial but not cytosolic translation; mitochondrial complex I, III, and IV function, and naïve and memory T cell expansion. To determine the molecular basis of these effects, we isolated mitochondrial ribosomes from Jurkat T cells for cryo-EM analysis. We discovered tigecycline not only obstructs A-site tRNA binding to the small subunit, as it does in bacteria, but also attaches to the peptidyl transferase center of the mitoribosomal large subunit. Intriguingly, a third binding site for tigecycline on the large subunit—absent in bacterial structures—aligned with helices analogous to those in bacterial ribosomes, albeit lacking methylation in humans. The data show tigecycline compromises T cell survival and activation by binding to the mitoribosome, providing a molecular mechanism to explain part of the anti-inflammatory effects of this drug class. The identification of species-specific binding sites guides antibiotic and OXPHOS inhibitor design.