T cell toxicity induced by tigecycline binding to the mitochondrial ribosome

Abstract

Tetracyclines are essential bacterial protein synthesis inhibitors under continual development to combat antibiotic resistance yet suffer from unwanted side effects. Mitoribosomes - responsible for generating oxidative phosphorylation (OXPHOS) subunits - share structural similarities with bacterial machinery and may suffer from cross-reactivity. Since lymphocytes rely upon OXPHOS upregulation to establish immunity, we set out to assess the impact of ribosome-targeting antibiotics on human T cells. We find tigecycline, a third-generation tetracycline, to be the most cytotoxic compound tested. In vitro, 5-10 μM tigecycline inhibits mitochondrial but not cytosolic translation, mitochondrial complex I, III and IV expression, and curtails the activation and expansion of unique T cell subsets. By cryo-EM, we find tigecycline to occupy three sites on T cell mitoribosomes. In addition to the conserved A-site found in bacteria, tigecycline also attaches to the peptidyl transferase center of the large subunit. Furthermore, a third, distinct binding site on the large subunit, aligns with helices analogous to those in bacteria, albeit lacking methylation in humans. The data provide a mechanism to explain part of the anti-inflammatory effects of these drugs and inform antibiotic design.

Fig. 1. Tigecycline compromises human T cell activation and proliferation by inhibiting mitochondrial translation.

A Representative dose-response curves of tetracycline antibiotic cytotoxicity towards PBMCs (left; n = 5) and Jurkat T cells (right; n = 3) measured 72 h after treatment; mean ± SEM is presented. Data are normalized to DMSO treatment without antibiotics. B Representative dose-response curves for additional bacterial protein synthesis inhibitors on PBMCs for 72 h. n = 6 biological replicates; mean ± SEM. Data are shown relative to DMSO treatment without antibiotics. C ³⁵S metabolic labeling assay of mitochondrial (left) and cytosolic (right) translation in Jurkat T cells after 18 h treatment with Q/D, tigecycline, or doxycycline. One representative result from n = 3 is shown. D Western blot OXPHOS profiling in Jurkat T cells after treatment with Q/D, tigecycline, and doxycycline and PBMCs after treatment with tigecycline for 6 days. n = 2 biological replicates, one shown. E Seahorse Mito Stress Test of PBMCs cultured with or without tigecycline for 6 days after stimulation with anti-CD3/CD28 antibodies + IL-2 (10 ng/ml). OCR is reported as picomoles (pmol) of O₂ per min and normalized according to protein amount/well. n = 4 biological replicates, mean ± SEM. Oligo, oligomycin; FCCP, carbonyl cyanide-p-tri-fluoromethoxyphenylhydrazone; Rot, rotenone; AA, antimycin A. F CD25 expression (MFI) on CD4⁺ and CD8⁺ T cells 18 h after anti-CD3/CD28 activation + IL-2 (10 ng/ml) in the presence or absence of tigecycline. n = 3 biological replicates, mean ± SEM. An RM one-way ANOVA with Tukey’s multiple comparisons test was used to analyze the data. G T cell proliferation assessed by flow cytometry. PBMC T cells were stimulated with anti-CD3/CD28 beads + IL-2 (10 ng/ml) in the presence or absence of tigecycline. Proliferation was assessed by CTV dilution in live cells after 6 days (n = 5 blood donor PBMC samples). PBMCs were labelled with CTV prior to stimulation. H Percentage of live T cells in division four or later after antibiotic treatment; data from (G), mean ± SEM. An RM one-way ANOVA with Tukey’s multiple comparisons test was applied to analyze the data. I Proliferation of FACS-isolated CD45RA⁺CD27⁺ naïve, CD45RA^-CD27⁺ central memory, and CD45RA^-CD27^- effector memory CD4⁺ T cells from healthy blood donors after 6 days of stimulation with anti-CD3/CD28 beads and IL-2 (10 ng/ml). FACS-isolated cells were seeded in the presence or absence of tigecycline (from 2.5 to 10 μM) and proliferation assessed by CTV dilution in live cells (n = 3 blood donor PBMC samples). Representative dot plots from non-treated and treated samples are shown. The FACS gating strategy is shown in Supplementary Fig. 2G. J Percentage of live CD4⁺ T naïve cells and central memory cells in each division; data from (I); mean ± SEM. Tn: naïve T cells; Tcm: central memory T cells; Tem: effector memory T cells. Source data for all panels are provided as a Source Data file.

Fig. 2. Overview of the interactions of tigecycline with the human mitoribosome.

A Chemical structure of tigecycline. B Overview of the three tigecycline (purple) binding sites in complex with the human mitoribosome comprising the mtSSU (yellow), mtLSU (blue), mRNA (red), and P-site tRNA (green). The mL45 (magenta) is depicted with its N-terminal extension occupying the exit tunnel. C mtSSU site: tigecycline overlaps with the A-tRNA binding site and is stabilized through multiple interactions. The residues from 12S rRNA which makes direct contact with tigecycline are shown. D mtLSU site-1: a novel tigecycline binding site located at helix 71 of the 16S rRNA, close to the acceptor stem of P-tRNA. E mtLSU site-2: tigecycline is positioned in the PTC where it stacks against the neighboring nucleotides and makes potential contact with mL45 N-terminus. Carved EM density is shown for tigecycline.

Fig. 3. The conserved binding site of tigecycline on the mtSSU.

A Tigecycline establishes canonical interactions with the 12S rRNA of the mtSSU accompanied by coordinated Mg²⁺ ions (lime) and waters (HOH) (red). Hydrogen bond interactions are indicated as dashed lines. B Structure of tigecycline (purple) (PDB: 5J91) (C) and sarecycline (yellow) (PDB: 6XQD), a tetracycline-derivative, in complex with the SSU of 70S ribosome from E.coli and T. thermophilis, respectively.

Fig. 4. Interaction of tigecycline with helix 71 of the mtLSU (mtLSU site-1) and its functional implications.

A Tigecycline stabilizes in the binding pocket through multiple interactions either directly with the neighbouring nucleotides or indirectly through Mg²⁺ ion coordination as compared to the non-treated mitoribosomes (PDB:7QI4) (B). C The superposition shows the large displacement of H71 upon the binding of tigecycline. D The movement of nucleotide to create a cavity for the binding of tigecycline. The direction of conformational changes of nucleotides occurring upon binding of the drug are shown by arrows. Nucleotides, C2603 and U2628, undergo a large conformational change (bold arrows), while A2604, C2605 and C2606 move downward (smaller arrows) as compared to the non-bound mitoribosome (PDB: 7QI4) resulting in a binding site for the antibiotic. In E. coli (PDB: 5J91), the flexible movement of this region is likely restricted by the presence of methylation of U1939. E The binding of tigecycline to the mtLSU site-1 might inhibit mitoribosome recycling. The RRF1-bound state (PDB: 7NSI) was superimposed on the tigecycline-bound mitoribosome (this study). The insertion represents a close view at mtLSU site-1 to show the clash of the drug (purple stick and transparent grey sphere) with the RRF1 protein (green).

Fig. 5. Interactions of tigecycline with the PTC (mtLSU site-2).

Tigecycline occupies the PTC, with its 9-t-butylglycylamido substituent stretching adjacent to the P-tRNA (green), and the A ring interacting with the mL45 N-terminal extension (pink). Upon tigecycline binding, A2725 nucleobase shifts and stacks against ring D in both the P-tRNA only and the A- (blue), P-tRNA mitoribosomal classes, as compared to the untreated (tigecycline) mitoribosome (PDB:7QI4). G2992, U2993, and G3063 in the PTC rearranges upon binding of the A-tRNA (as also observed for the untreated mitoribosomes).