Structural Basis of T Cell Toxicity Induced by Tigecycline Binding to the Mitochondrial Ribosome

Structural Basis of T Cell Toxicity Induced by Tigecycline Binding to the Mitochondrial Ribosome | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Structural Basis of T Cell Toxicity Induced by Tigecycline Binding to the Mitochondrial Ribosome Joanna Rorbach, Qiuya Shao, Anas Khawaja, Minh Nguyen, Vivek Singh, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4671643/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 May, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version 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. Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy Biological sciences/Immunology/Lymphocytes antibiotics immunometabolism mitochondrial ribosomes tetracyclines T cells Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Highlights • Mitoribosome inhibition by tetracyclines curtails T cell function. • First cryo-EM analysis of human lymphoid mitoribosomes. • Tigecycline blocks A-site tRNA binding and binds to the PTC. • A human-specific, third binding site of tigecycline serves as a guide for antibiotic design. Introduction Antibiotic resistance is a major One Health problem that has led to an intensive search for new antibacterials, as well as the modification of existing entities to broaden their spectrum of activity. Many antibiotics on the WHO’s list of essential medicines (such as tetracycline and doxycycline) are bacterial protein synthesis inhibitors, which mediate their effects by binding to bacterial ribosomes and halting translation. Although these entities have been of clinical importance for many decades, serious complications are known to arise due to therapy. For example, tetracyclines (which bind both 30S and 50S subunits and are used to treat plague, brucellosis, and Lyme disease) have long been noted for nephro- and hepato-toxicity, together with anti-inflammatory effects that have shown benefits in patients with chronic inflammatory skin, autoimmune and neurodegenerative diseases 1 . Similarly, chloramphenicol - which binds the bacterial 50S ribosomal subunit and is used to treat meningitis, cholera, and typhoid fever - is known to induce bone marrow suppression 2 , 3 . As antibiotic resistance mechanisms and drugs evolve, so too will the need for efforts to dissect treatment effects on different foreign organisms, as well as host cells. Mitochondria possess their own genome that encodes core components of the oxidative phosphorylation (OXPHOS) machinery - translated by the organelle’s specialized ribosomes, mitoribosomes, in proximity to the inner mitochondrial membrane to efficiently generate ATP. Evolutionarily descended from an α-proteobacterial ancestor, mitoribosomes share several features with their bacterial counterparts, and it has been long recognized that bacterial protein synthesis inhibitors can interfere with mitochondrial translation and cellular aerobic capacity, although the molecular bases of such effects have only been described in a handful of cases. Unlike interactions between bacterial or eukaryotic cytosolic ribosomes and antibiotics, which have been well characterized at atomic resolution, only more recently have insights emerged concerning the mitoribosome, the function of which is increasingly implicated in a range of clinical conditions. For example, streptomycin was recently shown to bind the mitoribosomal small subunit (mtSSU) 4 , while dalfopristin/quinupristin (Q/D) binds the large subunit (mt-LSU), effectively suppressing glioblastoma stem cell growth 5 . The interactions between these antibiotics and mitoribosomes closely resemble that of bacterial ribosome-antibiotic interactions. In recent years, the multifaceted and central role mitochondria play in the immune system has become increasingly clear. Via dynamic rearrangements in specialized immune lineages, the organelle determines cellular fate via metabolic reprogramming and innate signalling. Naive lymphocytes fundamentally depend upon OXPHOS to power clonal expansion 6 – 9 , something which if inhibited can affect the effector-memory response 6 – 9 . On the other hand, such inhibition could explain the beneficial effects of mitochondria-targeting drugs in other clinical contexts 10 , 11 , such as autoimmunity 12 – 14 , although the concomitant disruption of the metagenome and increased risk of antibiotic resistance may limit their application to these conditions. To this end, we explored the effects of several important antibiotics targeting protein synthesis on human T cells. For the most potent inhibitor of mitochondrial translation and proliferation identified, tigecycline, we then sought to determine the mechanism of action at atomic resolution, guiding the design of tetracyclines with reduced off-target binding. Results Tetracyclines compromise lymphocyte survival in vitro We first compared the survival of immortalized Jurkat T cells and HeLa cells in the presence of chloramphenicol or doxycycline, two important bacterial ribosome-targeting antibiotics associated with white blood cell phenotypes 13 , 15 – 21 . We found chloramphenicol to reduce the survival of both cell lines after 3 and 5 days of treatment (Jurkat IC50 = 25.85 µM; HeLa IC50 = 41.71 µM, after 5 days). However, Jurkat T cells were uniquely sensitive to lower concentrations of doxycycline (IC50 = 6.93 µM after 5 days) (Fig. S1 A). Tetracyclines (such as doxycycline) are broad-spectrum protein synthesis inhibitor agents characterized by a four-ring core structure 22 . Their mechanism of action involves reversible binding to the 30S bacterial ribosomal subunit, thereby preventing the attachment of aminoacyl-tRNA to the mRNA-ribosome complex 1 . Given our results, we chose to investigate six additional tetracyclines, including newer derivates reserved for difficult to treat infections (medocycline, methacycline, minocycline, oxycycline, tetracycline, tigecycline). In both Jurkat T cells and peripheral blood mononuclear cells (PBMCs) isolated from healthy blood donor samples, we found the third-generation tigecycline to have the greatest negative effect on cell survival (IC50 = 2.94–3.08 µM for Jurkat T cells; 2.02–9.42 µM for PMBCs after 3 days of treatment (Fig. 1 A). Tigecycline is a glycylcycline with a N, N-dimethyglycylamido (DMG) moiety attached to position 9 of tetracycline ring D that confers enhanced activity against tetracycline-resistant bacteria. We then compared the effect of tigecycline to an additional set of widely used bacterial protein synthesis inhibitors (spanning different drug classes) described in the literature (dalfopristin/quinupristin (Q/D), azithromycin, tiamulin, linezolid, and clindamycin) (Fig. 1 B). In this comparison, tigecycline again showed the greatest toxicity to PBMCs, followed by Q/D, which was previously reported to bind the mitochondrial large subunit 5 . Given our prior observations with doxycycline, it was interesting to observe that HeLa cells were also relatively resistant to 10 µM tigecycline, although Q/D did have a potent effect on their survival (IC50 = 16.66 µM) (Fig. S1 B). Q/D also had a greater influence on Hek293 cell survival than did tigecycline and doxycycline (Fig. S1 B), together illustrating that different cell lines have differential susceptibility to the same compounds, likely due to differences in their metabolism. Tigecycline inhibits oxidative phosphorylation and primary T cell expansion To ascertain whether tigecycline affected mitoribosomal function, we profiled mitochondrial and cytosolic translation with/without antibiotic treatment in Jurkat T cells. Metabolic labelling using 35 S-methionine was performed after 18 h of treatment with tigecycline; doxycycline and Q/D were used as comparators. We found tigecycline and Q/D inhibited mitochondrial translation at 5 µM, while weaker inhibition was observed for doxycycline (Fig. 1 C). On the other hand, 10 µM of these compounds did not have profound effects on cytosolic translation, suggesting mitochondrial protein synthesis is more greatly affected than cytosolic in these rapidly dividing cells (Fig. 1 C). The defect in translation could be confirmed at the protein level. OXPHOS subunit profiling of Jurkat T cells and human PBMCs by western blot revealed the levels of subunits of complex I (NADH: ubiquinone oxidoreductase subunit B (NDUFB8)), complex III (ubiquinol-cytochrome c reductase core protein 2 (UQCRCII)) and complex IV (cytochrome c oxidase subunit 2 (COX2)), which contain mitochondrially-encoded molecules, to drop in response to 5 µM tigecycline (Fig. 1 D). In contrast, subunits of complex II (succinate dehydrogenase complex iron sulphur subunit B (SDHB)) and complex V (ATP Synthase F1 subunit alpha (ATP5A)) were much less affected, indicating treatment-induced nuclear-mitochondrial protein imbalance in the electron transport chain. Given the pivotal role OXPHOS plays in T cell clonal expansion and memory generation, we wanted to determine whether these deficits correlated with reduced OXPHOS capacity at the cellular level. To do this, we activated T cells within PBMCs via plate-bound anti-CD3/CD28 antibodies and analyzed oxygen consumption using the Seahorse MitoStress test 6 days after stimulation. 5 and 10 µM tigecycline reduced basal, ATP-coupled, maximal and spare oxygen consumption rates (Fig. 1 E, S1C). In the shorter term (18 h post-stimulation), the drug reduced expression of the T cell activation marker, CD25 ( IL2RA ), by stimulated CD4 + and CD8 + T cells, suggesting cells are affected prior to cell division (Fig. 1 F). To determine whether treatment inhibited T cell expansion, we assessed proliferation using flow cytometry. After 6 days of anti-CD3/CD28 stimulation in the presence of IL-2, both CD4 + and CD8 + T cells in healthy donor PBMCs showed dose-dependent reductions in proliferation after tigecycline treatment (Fig. 1 G-H). The effects on T cell proliferation by other antibiotics included in the study are presented in Fig. S1 D. Given our observations in cell lines (Fig. S1 B) and previous research indicating differential susceptibility to antibiotics by specialized T cell subsets 12 , 13 , we FACS-isolated naïve (CD45RA + CD27 + ) and memory (CD45RA - CD27 + ) CD4 + T cells and compared proliferation in the presence or absence of tigecycline (Fig. 1 I-J). As observed for total T cells, dose-dependent inhibition was observed for both subsets between 2.5–10 µM. Structure determination of tigecycline-mitoribosome complexes To explore the molecular basis of the off-target effects of tigecycline, we isolated 55S mitochondrial ribosomes (mitoribosomes) from Jurkat T cells, incubated them with tigecycline, and analyzed the resulting complexes using single-particle cryo-EM (see “Methods”). The initial reconstruction of the mitoribosome-tigecycline complex yielded an initial cryo-EM density map with an overall resolution of 2.4 Å (Fig. S2 and Table 1). The particles underwent 3D classification with a solvent mask to get rid of poorly aligned particles, which yielded three major classes of monosomes: (class 1) mitoribosomes with no tRNA (‘empty class’), (class 2) with tRNA in the P-site only, (class 3) with A- and P- tRNAs (Fig. S2 ). Further local refinements led to a resolution of 2.2 Å for the core region of the monosome (Fig. S2 and Table 1), detecting all known methylations of 12S and 16S rRNAs, along with 2 iron-sulphur (2Fe-S) clusters in the mtSSU, and 1 Fe-S in the mtLSU, previously identified in the mitoribosomes isolated from Hek293 cells 4 . Interestingly, other reported cofactors of the mitoribosome such as NAD, spermine, and spermidine were not found in any of the classes of tigecycline-bound monosomes. The absence of these cofactors might result from the preferential occupancy of these cofactors in specific cell types (T cells versus embryonic kidney cells). Third-generation tetracyclines are based on a common naphthacene-carboxamide core comprising four rings (A, B, C, and D). The optimization of tigecycline consists of structural modifications of ring D at positions C7 (dimethyl-amino group) and C9 ( tert- butylglycylamide) 23 (Fig. 2 A). Our analyses unambiguously identified three densities corresponding to tigecycline molecules (Fig. S3A-C); one on the mtSSU (‘mtSSU site’) and two on the mtLSU (mtLSU site-1 and site-2) (Fig. 2 B-E). This is in contrast to previous structural data from bacteria, which reported a single tigecycline binding site on the SSU 24 – 26 . The antibiotic's relative occupancy in the three sites was notably high, particularly in the mtSSU and mtLSU site-1, where the P-site-only class of mitoribosomes showed an occupancy exceeding 80% (Fig. S3D). Tigecycline blocks aminoacyl-tRNA binding to the mtSSU A site Tigecycline density on the mtSSU was detected in two mitoribosomal classes, ‘P-site tRNA’ and ‘empty monosome’, but not in the class containing A-site tRNA. Based on bacterial studies, it is established that tigecycline specifically targets the head region of the SSU and competes with the binding of A-site tRNA, in a similar manner observed for other tetracycline derivatives 25 . The interaction between tigecycline and the mtSSU is analogous to how tetracyclines bind to the small ribosomal subunits of various bacterial species (Fig. 3 A-C) 24 – 30 . In the mtSSU, tigecycline interacts with helix 34 of the 12S rRNA through its polar edge and through ring A, which establish potential hydrogen bonds with the sugar-phosphate backbone of several nucleotides (A1258, U1259, U1325, A1326, G1327, and G1328) or make indirect contact via coordinated Mg 2+ ions (G1328) (Fig. 2 C and S3A). Ring D stacks on top of nucleobase U1259, which further stacks on A1326, stabilizing the binding of tigecycline to the mtSSU A-site. Tigecycline coordinates a Mg 2+ ion (denoted as Mg-1) via its keto-enol system (C11 and C12) and a second one (denoted as Mg-2) via hydroxyl and amide groups of ring A (O3 and O21, respectively). This coordination of the two Mg 2+ ions is a conserved feature of the tetracycline-ribosome interaction (Fig. 3 A-C). We detected the density for an additional Mg 2+ ion (Mg-3) in close proximity to tigecycline, likely interacting with the oxygen atom from the 9-t - butylglycylamido moiety adopting an extended conformation. Furthermore, the sugar phosphate of U1259 is within hydrogen-bonding distance from nitrogen atoms of the C9 butylglycylamido substituent (Fig. 3 A). These extensive interactions stabilize the overall binding of tigecycline to the decoding centre of mtSSU. Overall, the interactions observed for the tigecycline-bound mtSSU are comparable to tetracycline binding with the bacterial SSU, since the functional core of the ribosome including the decoding centre is universally conserved 31 . A unique tigecycline binding site on helix 71 of the mtLSU In all our mitoribosome classes, we observed additional density in the region of mtLSU comprising helix 69 and 71 (denoted as mtLSU site-1). We identified tigecycline in this density and observed multiple stabilizing interactions of the antibiotic with the residues forming its binding pocket (Fig. 4 A and S3B). In this position, the polar edge of the tigecycline molecule is directed towards the 16S rRNA where it forms multiple interactions, while its C7 extension is near the acceptor stem of the P-tRNA (Fig. 4 A). To form a tigecycline binding site that consists primarily of a hydrophobic cavity between the base of U2628 and the nucleoside of A2604, helix 71 must undergo large conformational changes (Fig. 4 A and Fig S4B). The rearrangement of nucleotides U2626 and U2628 contributes to opening-up a space where the drug can bind. Nucleotide U2626 shifts outward and forms stacking interactions with A2598 and C2625. U2628 changes conformation such that its nucleobase becomes intercalated between ring D of tigecycline and the nucleobase of A2629. These conformational changes are accompanied by a downward shift of helix 71 (~ 4–5 Å) as compared to the untreated monosome (Fig. 4 B) 32 . Additionally, nucleotide C2603 flips inward together with A2600, with which it forms stacking interactions (Fig. 4 C). This rearrangement positions the nucleobase of A2604 such that it stacks against ring C of tigecycline. Overall, stacking interactions between rings C and D of tigecycline and residues U2628 and A2604 of the 16S rRNA help to stabilize the binding of the drug to this region (Fig. S3B). Moreover, these conformational changes result in the formation of a non-canonical U-U pair between nucleotides 2599 and 2606, which further contributes to stabilizing the drug-bound conformation of helix 71 (Fig. 4 A). As observed for the tigecycline molecule in the mtSSU, the drug molecule bound to mtLSU site-1 forms metal ion complexes with the phenol-ketone system of rings B and C, and via the oxygen atom of ring A’s amide group and the hydroxyl oxygen at position C3. Interestingly, similarly to the mtSSU, we located an additional Mg 2+ ion density, which coordinates with the hydroxyl of ring D and facilitates indirect interaction with the backbone phosphate of A2604 (Fig. 2 D and S3B). Through its polar edge, tigecycline forms potential hydrogen bonds with the phosphate groups of adjacent nucleotide G2627 of 16S rRNA and indirectly through Mg 2+ ion coordination. The 9-t-butylglycylamido moiety of tigecycline extends towards the PTC center of the mitoribosome, causing nucleotide A3089 to shift (Fig. 4 C). As of now, there is no documentation of antibiotics binding to a comparable location within the bacterial LSU. Interestingly, helix 71 from multiple bacterial species comprises conserved methylations of 23S rRNA surrounding the region where tigecycline binds to the 16S rRNA of the mitoribosome (Fig. S4A). In E. coli and T. thermophilus these correspond to positions m 5 U1939 and m 5 C1962, and in C. acnes , positions m 5 U2122 and m 5 C2145. There is additional methylation of cytosine m 5 C1942 in T. thermophilus (Fig. S4A). In contrast, these modifications are absent in the mitochondrial LSU’s rRNA. The methylations in helix 71 of 23S rRNA in bacteria are not expected to directly clash with tigecycline; nevertheless, they may confer rigidity of the region, preventing antibiotic binding. Especially, m 5 C1942 is likely to prevent the conformational change needed for U1940 to flip out and the drug to bind to the bacterial ribosome (Fig. 4 D). To understand the potential consequences of the binding of tigecycline to the mtLSU site-1 on mitochondrial translation, we compared our structure with previously reported structures of translating mitoribosome. As depicted in Fig. 4 E, the tigecycline binding site likely hinders the interaction of mitoribosome with the ribosomal recycling factor 1 (RRF1) 33 , 34 . This obstruction may impede mitoribosome recycling, leading to the accumulation of non-functional mitoribosomes prone to aggregation. Furthermore, the mtLSU site-1 is positioned between the P and A site tRNA, and binding of the drug to this site may interfere with the translocation of a tRNA from the A site to the P site during the elongation phase of translation. Indeed, the dimethylamine group at C4 of tigecycline might interfere with the phosphate backbone of C71, as well as the OH group of the ribose ring of C70 in the hybrid (A/P site) tRNA (Fig. S4B). Tigecycline binds to the peptidyl transferase center of the mtLSU The third density corresponding to a tigecycline molecule was identified at the catalytic peptidyl transferase center (PTC). There have been previous reports of tetracycline-derivative such as sarecycline, anthracycline-like tetracenomycin X (TcmX), as well as the macrolide erythromycin, binding to the PTC and mediating translation arrest in bacteria 28 , 29 , 35 – 37 . Though the occupancy of tigecycline at the PTC in our study is the lowest, as compared to the other two sites in the mtLSU and mtSSU (Fig. S3D), it represents a unique occurrence for tigecycline (Fig. 2 E). In this position, the ring A of tigecycline faces the lumen of the nascent polypeptide exit tunnel while the 9-t-butylglycylamido extension is in proximity of the terminal adenosine A76 of the P-site tRNA, potentially hindering the progression of the elongating nascent chain along the tunnel (Fig. 5 ). As observed for the mtSSU and mtLSU site-1, tigecycline mediates canonical metal Mg 2+ ion complexes through its polar groups at rings B and C, and via the oxygen atoms of the carboxamide group and C3. Further stabilization of tigecycline at the PTC occurs via nucleobase C3073 which stacks on ring D and forms a non-canonical base-pair with C2502 (Fig. 2 E and S3C). In the PTC of C. acnes 70S, similar non-canonical C1965-C2768 base pairing is observed, contributing to sarecycline binding, whereas corresponding U-U (U1782-U2586) base-pairing in the E. coli 23S rRNA is proposed to be crucial for TcmX accommodation (Fig. S6) 28 , 35 . In the absence of a nascent chain, the exit tunnel in mitoribosomes is occupied by the mito-specific N-terminal extension of mL45 38 . A2725 interacts with either the mL45 N-terminal extension or the nascent chain in the exit tunnel, which may contribute to the stabilization of the mL45 or the nascent chain 39 . However, in the tigecycline-bound mtLSU, the A2725 nucleobase is rotated 90° and stacks onto ring D, further securing the binding of tigecycline to the PTC (Fig. 5 ). The binding sites for tigecycline and sarecycline at the PTC overlap, but surprisingly sarecycline is rotated 180° and shifted laterally relative to the tigecycline, resulting in its polar edge arranged in the opposite direction (Fig. S6). These differences can be either species-specific or antibiotic-specific, requiring further investigations. Ring A of tigecycline is placed away from the N-terminal extension of mL45 in the exit tunnel, and it coordinates with the Mg 2+ ion through the oxygen atom of the carboxamide group and hydroxyl group at C3 (Fig. 5 and S3C). Additionally, the oxygen atom at C1 of ring A forms a hydrogen bond with C2502, which forms a cis Watson-Crick/Watson-Crick base-pair with C3073 and stacks against the ring D (Fig. 5 ). The overall positioning of tigecycline allows 9-t-butylglycylamido moiety to extend towards the acceptor arm of the P-tRNA. 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 32 ). G2992, U2993, and G3063 in the PTC rearranges upon binding of the A-tRNA (as also observed for the untreated mitoribosomes). Discussion In this study, we investigated the off-target effects of bacterial ribosome-binding antibiotics on OXPHOS-dependent lymphocytes. Our screening pinpointed tigecycline as an antibiotic exhibiting toxicity towards human T cell lines and primary cells, although other members of this class (such as doxycycline) showed similar phenotypes at the cellular level. While our experiments were not designed to assess antibiotic toxicity in a comparative manner between bacteria and eukaryotic cells, the observation of three binding sites for tigecycline on the mitochondrial ribosome, as opposed to one on the bacterial ribosome, implies that this antibiotic is a potent inhibitor of mitochondrial translation. Extensive studies in bacterial systems have shown that high concentrations of antibiotics may result in secondary binding sites in in vitro experiments, which may not be physiologically relevant 40 . To avoid this issue, we employed a notably lower concentration of tigecycline (30 µM) than that previously used to investigate its interactions with ribosomes isolated from various bacterial species (concentrations ranging from ~ 60 to 300 µM) 24 – 26 . Nevertheless, unlike in previous studies on bacterial ribosomes, where the tigecycline molecule was located only on the SSU, we found two additional binding sites on the mtLSU (Fig. 2 ), with relatively high occupancy for identified new sites (Fig. S3D). The binding to the PTC (mtLSU site-2) overlapped with the recently identified binding site for tetracenomycin X (TcmX) and sarecycline, both with a very similar architecture to tigecycline (Fig. S5). Increasing evidence indicates that many ribosome-targeting antibiotics act in a context-dependent manner, influenced by the nature of the nascent protein. This was recently demonstrated for TcmX, where it was shown to sequester the 3′ adenosine of peptidyl-tRNALys in the nascent polypeptide exit tunnel of the ribosome upon translation of a QK motif 37 . Analysis of our tigecycline-PTC site did not detect any changes in the conformation of P-tRNA in either of the mitoribosomal classes comprising P-tRNA only or A- and P-tRNA. This could be due to the absence of the nascent polypeptide in our structures. Unfortunately, current mitoribosome purification methods often do not retain the nascent polypeptide, instead, the exit tunnel in mitoribosomes is occupied by the mito-specific N-terminal extension of mL45, which enters the tunnel once mitoribosomes are detached from the inner mitochondrial membrane. Therefore, the context specificity of tigecycline binding to the PTC needs to be addressed in the future using other methods, for example, mitoribosome profiling 41 . One of the most intriguing observations arising from our structural studies was the identification of the novel tigecycline binding site on the mtLSU (mtLSU site-1). This specific region has not previously been recognized as a prominent site for antibiotic binding on bacterial ribosomes, raising the question of its mitochondrial specificity. To create sufficient space for the drug to effectively target this area, specific nucleotides including C1963/U2626, C1965/U2628 and U1940/U2603 of helix 71 (( E.coli /human mtDNA numbering) must undergo substantial movement. However, in bacterial ribosomes, this region contains extensive methylations, constraining the flexibility of modified nucleotides (Fig. S4A). In particular, the m5U1939 modification might sterically hinder the conformational change required for accommodating the flipping of U1940 (Fig. 4 D). To verify this hypothesis, additional mutagenesis studies are warranted. Could the binding of tigecycline to mtLSU site-1 contribute to the inhibition of mitochondrial translation? Due to technical limitations, primarily the lack of efficient methods to manipulate the mitochondrial genome and the absence of a robust in vitro mitochondrial translation assay, it is challenging to design an experiment to determine the specific contributions of different binding sites to translation inhibition. However, comparisons with the translating ribosome suggest that binding to mtLSU site-1 could interfere with mitoribosome recycling and the translocation of tRNA from the A site to the P site (Fig. 4 E and S4B). Further studies are needed to confirm these findings. Together, the efficient binding of tigecycline in all three sites is likely to assure an efficient inhibition of several stages of translation. Our study reports the first structures of mitoribosomes isolated from human lymphoid cells (Jurkat T cells). Given that the features of the binding sites remain conserved with other mammalian mitoribosomes previously characterized (from Hek293 and porcine), it suggests that the interaction is likely to be conserved across mammals. However, our toxicity tests revealed a significantly stronger effect of this antibiotic on Jurkat T cells, compared to Hek293 and Hela cells (Fig. S1 B). This point is particularly important considering the numerous studies modulating mitochondrial function in the context of cancer 42 . Antibiotics that potentially inhibit mitochondrial translation, including tigecycline, have been reported to inhibit survival in glioblastoma cells 5 , leukemia stem cells 43 , ovarian cancer cells 44 , renal cancer cells 45 , and imatinib-resistant chronic myeloid leukemia (CML) 46 cells. Our data encourage the investigation of anti-tumour lymphocyte responses in cancer patients receiving such treatments. In an infectious context, antibiotics need to be delivered to infected lesions to combat the pathogen. The observed IC50 values for tigecycline against T cells in vitro were relevant to drug concentrations observed in soft tissue lesions of patients undergoing treatment for bacterial infections 46 – 48 . Therefore, future studies could also consider how lesion-resident immune cells are affected by tetracycline treatments, as this could influence pathogen clearance and tissue repair. The investigation of myeloid lineages is of similar interest. Nevertheless, these data provide a molecular mechanism to explain the anti-inflammatory effects of tetracyclines and inform antibiotic design. While our manuscript was in preparation, a separate study identified three binding sites for tigecycline on the 55S mitoribosome isolated from HEK293T cells 49 . This finding confirms that tigecycline effectively inhibits mitochondrial translation at clinically relevant concentrations. Methods Ethical declaration Ethical approval for the use of peripheral blood samples from human donors was granted by the Swedish Ethical Review Authority (registration number 2018/1498-31/3). All human studies were carried out in accordance with the guidelines and policies of Karolinska Institutet and EU legislation. Human samples Buffy coats were ordered from anonymous blood donors at Karolinska Universitetslaboratoriet. On the same day as sample collection, PBMCs were isolated by density gradient centrifugation over Lymphoprep (StemCell Technologies), washed with RPMI-1640 (Cytiva HyClone) with 10% FBS, and cryopreservated at -80 o C in FBS with 10% DMSO (Sigma) 50 . Cell viability assays Cells were seeded in 96-well plates ranging from 2,000 to 15,000 cells per well depending on different cell lines and treated with antibiotics in serial dilutions for 72 hours (120 hours for the preliminary assays) in a humidified incubator (37°C, 5% CO 2 ) in 100 µl medium (DMEM with 10% FBS, 2.05 mM L-glutamine (Sigma) for Hek293 and Hela; RPMI-1640 with 10% FBS, 2.05 mM L-glutamine (Sigma), and 55 µM 2-ME (Gibco) for Jurkat T cells and PBMCs). Based on preliminary tests, the number of cells per well was optimized to 2,000 for Hela and Hek293, 6,000 for Jurkat T cells, and 15,000 for PBMCs. The stocks of antibiotics were prepared by dissolving in DMSO. As control, the same amount of DMSO was added to the non-treated groups. Serial drug dilutions were prepared in the corresponding medium to provide a total of 9 concentrations up to 300 µM for tetracyclines and up to 900 µM for additional bacterial protein synthesis inhibitors. After drug treatment, 10% CCK-8 (Cell Counting Kit-8, Sigma Aldrich) (10 µl per well) of the total volume was added to the cells and incubated for 1–4 hours depending on cell lines (1 hour for Hela and Hek293; 3 hours for Jurkat T cells; 4 hours for PBMCs) before the evaluation of the effect of antibiotics on cell viability. The number of viable cells was calculated by measuring the absorbance at 450 nm using a microplate reader and normalized to the non-treated control. Results were visually confirmed under the light microscope. Each treatment was performed in technical repeats. Dose-response curves were plotted, mean ± SEM was shown, and half maximal inhibitory concentration (IC50) values were calculated in GraphPad Prism. De novo mitochondrial translation assay Jurkat T cells were seeded in 6-well plates at a density of 1 million cells/ml and cultured with different concentrations of antibiotics for 18 h in RPMI-1640 medium (with 10% FBS, 2.05 mM L-glutamine (Sigma), and 55 µM 2-ME (Gibco)). After centrifugation, cells were incubated twice for 5 min at 37°C in Cys-/Met-free medium (DMEM, high glucose, no glutamine, no methionine, no cysteine, supplemented with 10% dialyzed fetal bovine serum, 1× GlutaMax, and sodium pyruvate), followed by 20 min incubation at 37°C in Cys-/Met-free medium supplemented with 100 µg/ml emetine to inhibit cytosolic translation (only for mitochondrial translation groups). Subsequently, 1 ml Cys-/Met-free medium supplemented with 200 (for cytosolic translation) or 400µCi (for mitochondrial translation) of EasyTag EXPRESS [ 35 S] protein labeling mix (methionine and cysteine) (Perkin Elmer) were added to each sample and incubated for 20 min at 37°C, 5% CO 2 . Following labeling, cells were washed three times with 1 ml PBS, harvested by centrifugation (400 × g , 10min, 4°C), and stored at − 20°C. Cells were lysed by resuspension in 30 µl PBS supplemented with a Complete EDTA-free protease inhibitor cocktail and 50 U Benzonase nuclease (Sigma) and by application of one freeze-thaw cycle. Protein contents were determined by Pierce BCA assay (Thermo Fisher). 1 × NuPAGE LDS sample buffer (Thermo Fisher) was added to 30 µg lysate for mitochondrial translation and 10 µg lysate for cytosolic translation, respectively, and separated on NuPAGE 12% Bis-Tris gels (Thermo Fisher). Coomassie staining was performed using Imperial Protein Stain (Thermo Fisher) according to manufacturer suggestions. The gel was fixed in fixing solution (20% methanol, 7% acetic acid, 3% glycerol) for 1 h at RT and vacuum-dried at 65°C for 2 h. The resultant gel was exposed to storage Phosphor screens (Fujifilm) and visualized with Typhoon FLA 7000 Phosphorimager (GE Healthcare). Western blotting for OXPHOS complexes Human PBMCs and Jurkat T cells were seeded in 6-well plates at a density of 1 million cells/ml and incubated with or without antibiotics in RPMI-1640 medium (with 10% FBS, 2.05 mM L-glutamine (Sigma), and 55 µM 2-ME (Gibco)) for 6 days. After centrifugation, cells were lysed in RIPA buffer containing protease and phosphatase inhibitors (cOmplete and PhosSTOP, respectively, Roche). Protein concentrations were determined by BCA (Pierce, Thermo Fisher). Protein samples were resuspended with 1× NuPAGE LDS sample buffer (Thermo Fisher) supplemented with 100 mM dithiothreitol, heated for 10 min at 75°C, and separated on NuPAGE 4–12% Bis-Tris mini gels (Thermo Fisher) using NuPAGE 1× MES (Thermo Fisher) running buffer, and transferred to PVDF membranes (0.45 µm, Immobilon-P, Sigma) using the iBlot2 system (Invitrogen). Non-specific binding was blocked with TBST containing 5% non-fat milk, followed by overnight incubation at 4°C with antibodies against Total OXPHOS Human WB Antibody Cocktail (Abcam, Cambridge, UK), followed by HRP-conjugated secondary antibodies for 1 h at room temperature. β-actin, HSP60, and GAPDH (Cell Signaling Technology) were used as loading control. Proteins were detected using Clarity Western ECL Substrate (Bio-Rad, 170–5061). Extracellular Metabolic Flux Assay The Seahorse XFe96 Analyzer (Seahorse Bioscience) was used to measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in human PBMCs. 1.5x10 6 cells/well were seeded in 24-well plates and stimulated using plate-bound anti-CD3 antibody (2 µg/ml, clone OKT3, BD Biosciences), soluble anti-CD28 antibody (0.5 µg/ml, clone CD28.2, Biolegend) and IL-2 (10ng/ml, Proteintech) in RPMI-1640 (with 10% FBS, 2.05 mM L-glutamine (Sigma), and 55 µM 2-ME (Gibco)). Cells were treated with or without 5 and 10 µM Tigecycline for 6 days with a half medium change after 3 days. After treatment, cells were washed with Seahorse XF RPMI assay medium, and then 200,000 cells per well were seeded (in triplicate) in 40 µl assay medium in XF 96-well cell culture microplate coated with poly-D-lysine (Sigma). The plate was centrifuged at 300 g for 5 sec with no brake, rotated 180 ̊, and centrifuged again for 5 sec at 300 g . After centrifugation, 140 µl of assay medium was added per well and the plate was left to stabilize in a 37°C, non-CO 2 incubator for 40 min. During seahorse, wells were sequentially injected with compounds to achieve final concentrations of 1,264 µM oligomycin (Sigma); 2µM FCCP (Sigma); 0.5 µM rotenone (Sigma) together with 0.5 µM antimycin A (Sigma). OCR and ECAR were measured for each well three times, every three minutes, before and after each injection. OCR was normalized to protein concentration using a BCA Protein Assay kit (ThermoFisher Scientific) conducted according to the manufacturer’s instructions. In vitro stimulation of human PBMCs After thawing frozen PBMCs, cells were washed with RPMI complemented with 10% FBS, 2.05 mM L-glutamine (Sigma), and 55 µM 2-ME (Gibco) and rested at 37 o C (5% CO 2 ) for 30 min. 250,000 cells per well were seeded in 96-well U-bottom plates with/without Human T-Activator CD3/CD28 beads (Invitrogen) at a 1:1 ratio and 10 ng/ml IL-2 (Proteintech) in RPMI-1640 medium mentioned above. At the same time, antibiotics were added. For the short-term activation assay, cells were harvested 18 hours post-stimulation and stained with Aqua fixable live/dead dye (Invitrogen) and surface markers. For the proliferation assays, cells were stained with CFSE (eBioscience) or Cell Trace Violet (ThermoFisher Scientific) dyes before seeding and stimulation, according to the manufacturer’s recommendations. After incubation for 6 days with a half medium (complete, containing antibiotics and IL-2) change after 3 days, cells were harvested via centrifugation, stained with live/dead dye and surface markers for analysis by flow cytometry. The Foxp3/Transcription Factor Staining Buffer Set (Invitrogen) was used to fix cells according to the manufacturer’s instructions. Flow cytometry was carried out on a BD Celesta (BD Biosciences) instrument and analyzed in FlowJo (TreeStar) software. Human CD4 + T cell sorting and stimulation Thawed PBMCs were washed with RPMI complemented with 10% FBS, 2.05 mM L-glutamine (Sigma), and 55 µM 2-ME (Gibco) before resting at 37 o C (5% CO 2 ) for 30 min. After counting, cells were resuspended in FACS buffer at a density of 5 x 10 7 cells/ml. CD4 + T cells were isolated using CD4 + T cell negative isolation kits (Miltenyi Biotech) and enriched using CD4 + T cell Enrichment kits (Miltenyi Biotech). Purified CD4 + T cells were counted and stained with CTV dye and live/dead dye. Subsequently, surface makers were stained at 4 o C in FACS buffer (DPBS + 2% FBS). The following panels were used to classify human T CD4 + cells from the live lymphocyte gate: CD4 + T naïve: Aqua − CD4 + CD45RA + CD27 + ; CD4 + T memory: Aqua − CD4 + CD45RA − CD27 + . After staining, cells were washed and resuspended and sorted using a FACSAria Fusion (BD Biosciences). Sorted cells were collected in PBS complemented with 2% FBS at 4 o C. Cells were then harvested via centrifugation and resuspended in RPMI-1640 (+ 10% FBS, 2.05 mM L-glutamine (Sigma), and 55 µM 2-ME (Gibco)) before counting. Sorted CD4 + subsets were counted and 85,000 cells per well seeded onto 96-well U-bottom plates with/without Human T-Activator CD3/CD28 beads (Invitrogen) at a 1:2.8 cell:bead ratio and 10ng/ml IL-2 (Proteintech) in RPMI-1640 (+ 10% FBS, 2.05 mM L-glutamine (Sigma), and 55 µM 2-ME (Gibco)). At the same time, tigecycline in serial concentrations (from 2.5 to 10 µM) was added. With a half medium change after 3 days, cells were harvested after 6 days, and stained and fixed before analysis by flow cytometry. Antibodies used in the study are presented in Table 2. Table 2 Antibodies Total OXPHOS Human WB Antibody Cocktail Abcam Ab110411 HSP60 Enzo Lifesciences AB1-SPA-807-E b-actin Abcam Ab8224 GAPDH Abcam Ab8245 HRP secondary rabbit GE Healthcare NA9340V HRP secondary mouse GE Healthcare NA9310V Human CD3 BD Biosciences Clone 3D12 Human CD4 BD Biosciences Clone RPA-T4 Human CD8 BD Biosciences Clone RPA-T8 Human CD45RA BD Biosciences Clone HI100 Human CD27 BD Biosciences Clone M-T271 Human CD25 BD Biosciences Clone 2A3 Statistical analyses Statistical analyses were carried out in Prism 9 (GraphPad). Differences between groups were analyzed by a Student's t-test or one-way ANOVA with Tukey's multiple comparisons test. Isolation of mitochondria and purification of mitochondrial monosomes Jurkat T cells were grown in RPM1-1640 medium (+ 10% FBS, 2.05 mM L-glutamine (Sigma), and 55 µM 2-ME (Gibco)) in a vented flask shaking at 120 rpm at 37°C under 5% CO 2 . The culture was scaled up by splitting at a cell density of 1.6 × 10 6 cells/ml. A final volume of 1.5-liter cells at a density of 1 × 10 6 cells/ml was harvested by centrifugation at 1000 x g for 10 min at 4°C. After being washed with cold PBS buffer, the cell pellet was resuspended in the cold hypotonic MSE buffer (with 0.6 M mannitol, 10 mM Tris–HCl pH 7.4, 1 mM EDTA, 0.1% BSA), and ruptured on ice by a semi-automatic homogenizer (Schuett-biotech). The lysate was clarified by centrifugation at 400 × g and 4°C for 10 min. The pellet was resuspended and subsequently homogenized. After 3 cycles of homogenization-centrifugation, the cell lysates were combined and the mitochondria were pelleted by additional centrifugation at 11,000 × g and 4°C for 10 min. The crude mitochondria were loaded onto the sucrose cushion (1.0 M and 1.5 M sucrose in, 20 mM Tris–HCl pH 7.4, 1 mM EDTA) and centrifuged for 1 hr at 77,000 × g (25,000 rpm) in a SW41 Ti rotor (Beckman Coulter). The band formed by the mitochondria in the middle between 1 and 1.5 M sucrose was collected carefully and resuspended in 10 mM Tris–HCl pH 7.4 in a 1:1 ratio, The pure mitochondrial pellet was collected after centrifugation at 11,000 × g and 4°C for 15 min and then resuspended in mitochondrial freezing buffer (300 mM trehalose, 10 mM Tris–HCl pH 7.4, 10 mM KCl, 0.1% BSA, 1 mM EDTA), flash-frozen in liquid N2 and stored at − 80°C. The purified mitochondria were thawed and lysed by incubating at 4°C for 30 min in the lysis buffer (25 mM HEPES–KOH pH 7.5, 20 mM Mg(OAc)2, 50 mM KCl, 2% (vol/vol) Triton X-100, 2 mM Dithiothreitol (DTT), 1× cOmplete EDTA-free protease inhibitor cocktail (Roche), 40 U/µl RNase inhibitor (Invitrogen)). The mitochondrial lysate was centrifuged at 19,000 × g (13000 rpm) for 12 min at 4°C, and subsequently overlayed on top of a 10–30% sucrose gradient in the ribosome buffer (25 mM HEPES/KOH pH 7.5, 50 mM KCl, 20 mM Mg(OAc)2, 2 mM DTT). After centrifugation for 21 hr at 54,331 × g (21,000 rpm) in a SW41 Ti rotor (Beckman Coulter), the gradients were fractionated with a Biocomp Fractionator. Fractions corresponding to the monosomes were pooled and pelleted at 135,520 × g (55,000 rpm) for 16 hr at 4°C using a TLA55 rotor (Beckman Coulter). The pellet was gently washed 3 times and dissolved in ribosome buffer. The solution was kept on ice for 15 min and the soluble mitoribosomes were collected by centrifugation at 20,000 x g for 15 hr at 4°C to get rid of aggregation. The concentration of the purified monosomes was quantified by nanodrop. CryoEM data collection and analysis In vitro reconstitution of the mitoribosomal monosome-tigecycline complex was performed using 30 µM of tigecycline incubated with 100 nM of monosome in a ratio of 1:300. Holey carbon grids (Quantifoil R2/2, copper, 300 mesh) coated with a layer of continuous carbon (~ 3 nm thickness) were subjected to a glow discharge of 25 mA for 120 s. The sample was applied to the grids at 4°C with 100% humidity and incubated for 30 s using a Vitrobot MKIV (Thermo Fisher Scientific), followed by 3 s blotting with blot force 3 and plunge-freezing in liquified ethane. The dataset was acquired on Titan Krios G3i transmission electron microscope (Thermo Fisher Scientific) operated at 300 kV in the Karolinska Institutet’s 3D-EM facility using a slit width of 20 eV with GIF quantum energy filter (Gatan). For imaging, a K3 detector (Gatan) was used capturing micrographs at magnification of 165kX yielding a pixel size of 0.505 Å. A dose of 45 electrons per Å 2 in 50 frames was used with defocus values ranging from − 0.4 to − 1.6 µm. Motion correction followed by CTF estimation, Fourier cropping (to 1.01 Å/px), particle picking, and extraction in 512-pixel boxes (size threshold 300 Å, distance threshold 20 Å, using the pre-trained BoxNet2Mask_20180918 model) were performed on the fly using Warp 51 . Only particles from micrographs with an estimated resolution of 5 Å were retained for further processing. Detailed parameters are given in the Supplementary Table 1. A total of 12,972 micrographs from 2 datasets were selected based on an estimated resolution cut-off of 5 Å and defocus below 2 µm as estimated by Warp. A total of 519,237 picked particles from Warp were imported to CryoSPARC (v4.2.1) 52 to perform further processing. 2D classification was carried out followed by ab initio reconstructions of clean or good classes with high-resolution features and junk classes. These ab initio reconstructions were used for the heterogeneous refinements of all picked particles. After several round of heterogeneous refinements, 266,550 clean particles comprised the mitoribosomal monosome with high-resolution features were retained and used for further processing. Homogenous refinement of these clean particle stacks was performed which yielded a resolution of 2.4 Å. A recent model of human mitoribosomal monosome (PDB:7QI4 32 ) purified from the Hek293 T cells bound to A-tRNA, P-tRNA, and mRNA was fitted in our reconstruction of the monosome, which also comprises these features with additional densities in both mtSSU and mtLSU that could be attributed to the antibiotic tigecycline. Mask targeting the P-tRNA was generated to perform 3D variability analysis, and subsequently classified into three particle clusters with each representing different features of the monosome. Particles with clear density for the P-tRNA were subjected to a second round of 3D variability analysis using the A-tRNA as a mask. We could eventually obtain 3 major classes, class 1 with the majority of the particles (‘Empty’: 130,443 particles) lacked densities for the A-site and P-site tRNA on the monosome, class 2 (‘P-tRNA only’: 87,076 particles) lacked the occupancy for the A-tRNA and is represented by the monosome bound to P-tRNA only and mRNA, and class 3 (‘A- and P-tRNA’: 36,974 particles) contained monosome bound A-tRNA, P-tRNA, and mRNA. Each class/particle set was subjected to a homogenous refinement which yielded a corresponding reconstruction at 2.5 Å, 2.7 Å, and 2.8 Å resolution, respectively. To improve the local resolution of the tigecycline-bound regions on the monosome, 3D refined classes were subjected to CTF refinement (global and local refinement). Furthermore, two masks covering the region of the tigecycline-binding sites on the mtSSU (SSU-head) and mtLSU (LSU-body) were prepared (Supplementary Fig. 2), and local-masked 3D refinement was performed. Reported resolutions are based on gold-standard, applying the 0.143 criterion on the FSC between the reconstructed half-maps (Supplementary Fig. 2). Maps underwent local-resolution filtering, superposed to the consensus map, and combined via Phenix 53 for model building and refinement. Model building and refinement Model building of the tigecycline-bound monosome was carried out using Coot 54 . The starting model for the monosome was Protein Data Bank (PDB) ID 7QI4 32 . This model was fitted as a rigid body into the map of the ‘P-tRNA only’ monosome class, and further adjustments were made manually. Three active sites for tigecycline were identified on the monosome, which agreed with the density: one at the mtSSU and two in the mtLSU. Water molecules were picked by Coot automatically around the tigecycline-binding region, and adjusted manually. Metal ions, cofactors (2Fe-S), and modifications were placed based on map densities. Geometrical restraints of modified residues and ligands were calculated by Grade Web Server ( http://grade.globalphasing.org ) or obtained from the CCP4 library 55 . Hydrogens were added to all molecules except water by REFMAC5 56 . The final model was refined using the composite map via Phenix.real_space_refine v1.18 53 . The refined model was validated with MolProbity 57 , 58 and the Phenix suite 50 . Model statistics are listed in Table 1. UCSF ChimeraX 1.6.1 59 was used to make the figures. Declarations Conflict of interest The authors declare no competing interests. Acknowledgments The work was funded by Max Planck Institute-Karolinska Institute, the Knut & Alice Wallenberg Foundation (WAF2017, KAW 2018.0080, to JR), Swedish Research Council (VR2022-02179, to JR), and EMBO (STF 7213, to XCD; LTF 2020 − 606, to MDN). Data availability. Cryo-EM maps have been deposited at the Electron Microscopy Data Bank as follows: Class 1 (empty class), EMD-19544 (consensus map), EMD-19545 (SSU-head), EMD-19546 (LSU-body); Class 2 (P-site tRNA), EMD-19493 (consensus map), EMD-19490 (SSU-head), EMD-19491 (LSU-body), EMD-19460 (composite); Class 3 (A- and P-site tRNA), EMD-19526 (consensus map), EMD-19539 (SSU-head), EMD-19542 (LSU-body), EMD-19460 (composite map). Associated molecular model has been deposited at the PDB: 8RRI (tigecycline bound human mitoribosome containing P-site tRNA and mRNA). The map and the model are available at https://figshare.com/ ; Login: [email protected] ; Password: AntibioticTig12##. References Chukwudi CU (2016) rRNA binding sites and the molecular mechanism of action of the tetracyclines. Antimicrobial Agents and Chemotherapy vol. 60 4433–4441 Preprint at https://doi.org/10.1128/AAC.00594-16 Nagiec EE et al (2005) Oxazolidinones inhibit cellular proliferation via inhibition of mitochondrial protein synthesis. Antimicrob Agents Chemother 49:3896–3902 McKee EE, Ferguson M, Bentley AT, Marks TA (2006) Inhibition of mammalian mitochondrial protein synthesis by oxazolidinones. Antimicrob Agents Chemother 50:2042–2049 Itoh Y et al (2022) Structure of the mitoribosomal small subunit with streptomycin reveals Fe-S clusters and physiological molecules. Elife 11 Sighel D et al (2021) Inhibition of mitochondrial translation suppresses glioblastoma stem cell growth. Cell Rep 35:109024 Scharping NE et al (2016) The Tumor Microenvironment Represses T Cell Mitochondrial Biogenesis to Drive Intratumoral T Cell Metabolic Insufficiency and Dysfunction. Immunity 45:374–388 O’sullivan D et al Fever supports CD8 + effector T cell responses by promoting mitochondrial translation. 10.1073/pnas.2023752118/-/DCSupplemental Tan H et al (2017) Integrative Proteomics and Phosphoproteomics Profiling Reveals Dynamic Signaling Networks and Bioenergetics Pathways Underlying T Cell Activation. Immunity 46:488–503 West AP et al (2015) Mitochondrial DNA stress primes the antiviral innate immune response. Nature 520:553–557 Colaço HG et al (2021) Tetracycline Antibiotics Induce Host-Dependent Disease Tolerance to Infection. Immunity 54:53–67e7 Patel CH, Leone RD, Horton MR, Powell JD (2019) Targeting metabolism to regulate immune responses in autoimmunity and cancer. Nature Reviews Drug Discovery vol. 18 669–688 Preprint at https://doi.org/10.1038/s41573-019-0032-5 Almeida L et al (2021) Ribosome-Targeting Antibiotics Impair T Cell Effector Function and Ameliorate Autoimmunity by Blocking Mitochondrial Protein Synthesis. Immunity 54:68–83e6 Franz T et al (2022) Pleiotropic effects of antibiotics on T cell metabolism and T cell-mediated immunity. Frontiers in Microbiology vol. 13 Preprint at https://doi.org/10.3389/fmicb.2022.975436 Kroon AM, Taanman JW (2015) Clonal expansion of T cells in abdominal aortic aneurysm: A role for doxycycline as drug of choice? International Journal of Molecular Sciences vol. 16 11178–11195 Preprint at https://doi.org/10.3390/ijms160511178 Moullan N et al (2015) Tetracyclines disturb mitochondrial function across eukaryotic models: A call for caution in biomedical research. Cell Rep 10:1681–1691 Kloppenburg M et al (1995) The influence of tetracyclines on T cell activation. Clin Exp Immunol 102:635–641 Mottis A et al (2022) Tetracycline-induced mitohormesis mediates disease tolerance against influenza. J Clin Invest 132 McKee EE, Ferguson M, Bentley AT, Marks TA (2006) Inhibition of mammalian mitochondrial protein synthesis by oxazolidinones. Antimicrob Agents Chemother 50:2042–2049 Barnhill AE, Brewer MT, Carlson SA (2012) Adverse effects of antimicrobials via predictable or idiosyncratic inhibition of host mitochondrial components. Antimicrobial Agents and Chemotherapy vol. 56 4046–4051 Preprint at https://doi.org/10.1128/AAC.00678-12 Moullan N et al (2015) Tetracyclines disturb mitochondrial function across eukaryotic models: A call for caution in biomedical research. Cell Rep 10:1681–1691 Perry EA et al (2021) Tetracyclines promote survival and fitness in mitochondrial disease models. Nature Metabolism vol. 3 33–42 Preprint at https://doi.org/10.1038/s42255-020-00334-y Chopra I, Roberts M, Tetracycline Antibiotics (2001) Mode of Action, Applications, Molecular Biology, and Epidemiology of Bacterial Resistance. Microbiol Mol Biol Rev 65:232–260 Rusu A, Buta EL (2021) The development of third-generation tetracycline antibiotics and new perspectives. Pharmaceutics vol. 13 Preprint at https://doi.org/10.3390/pharmaceutics13122085 Nicholson D, Edwards TA, O’Neill AJ, Ranson NA (2020) Structure of the 70S Ribosome from the Human Pathogen Acinetobacter baumannii in Complex with Clinically Relevant Antibiotics. Structure 28:1087–1100e3 Jenner L et al (2013) Structural basis for potent inhibitory activity of the antibiotic tigecycline during protein synthesis. Proc Natl Acad Sci U S A 110:3812–3816 Schedlbauer A et al (2015) Structural characterization of an alternative mode of tigecycline binding to the bacterial ribosome. Antimicrob Agents Chemother 59:2849–2854 Zhang Z, Morgan CE, Bonomo RA, Yu EW (2021) Cryo-em determination of eravacycline-bound structures of the ribosome and the multidrug efflux pump adej of acinetobacter baumannii. mBio 12 Lomakin IB, Devarkar SC, Patel S, Grada A, Bunick CG (2023) Sarecycline inhibits protein translation in Cutibacterium acnes 70S ribosome using a two-site mechanism. Nucleic Acids Res 51:2915–2930 Batool Z, Lomakin IB, Polikanov YS, Bunick CG, Jacobson A Sarecycline interferes with tRNA accommodation and tethers mRNA to the 70S ribosome. 10.1073/pnas.2008671117/-/DCSupplemental Cocozaki AI et al (2016) Resistance mutations generate divergent antibiotic susceptibility profiles against translation inhibitors. Proc Natl Acad Sci U S A 113:8188–8193 Bowman JC, Petrov AS, Frenkel-Pinter M, Penev PI, Williams LD (2020) Root of the Tree: The Significance, Evolution, and Origins of the Ribosome. Chemical Reviews vol. 120 4848–4878 Preprint at https://doi.org/10.1021/acs.chemrev.9b00742 Singh V et al Structure of mitoribosome reveals mechanism of mRNA binding, tRNA 1 interactions with L1 stalk, roles of cofactors and rRNA modifications 2 3. 10.1101/2023.05.24.542018 Koripella RK et al (2021) Distinct mechanisms of the human mitoribosome recycling and antibiotic resistance. Nat Commun 12 Kummer E, Schubert KN, Schoenhut T, Scaiola A, Ban N (2021) Structural basis of translation termination, rescue, and recycling in mammalian mitochondria. Mol Cell 81:2566–2582e6 Osterman IA et al (2020) Tetracenomycin X inhibits translation by binding within the ribosomal exit tunnel. Nat Chem Biol 16:1071–1077 Arenz S et al (2016) A combined cryo-EM and molecular dynamics approach reveals the mechanism of ErmBL-mediated translation arrest. Nat Commun 7 Leroy EC, Perry TN, Renault TT, Innis CA (2023) Tetracenomycin X sequesters peptidyl-tRNA during translation of QK motifs. Nat Chem Biol 19:1091–1096 Kummer E et al (2018) Unique features of mammalian mitochondrial translation initiation revealed by cryo-EM. Nature 560:263–267 Itoh Y et al (2021) Mechanism of membrane-tethered mitochondrial protein synthesis. Sci (1979) 371:846–849 Paternoga H et al (2023) Structural conservation of antibiotic interaction with ribosomes. Nat Struct Mol Biol 30:1380–1392 Barrientos A, Fontanesi F The Mitoribosome Methods and Protocols Methods in Molecular Biology 2661. http://www.springer.com/series/7651 Klein K et al (2020) Role of Mitochondria in Cancer Immune Evasion and Potential Therapeutic Approaches. Frontiers in Immunology vol. 11 Preprint at https://doi.org/10.3389/fimmu.2020.573326 Škrtić M et al (2011) Inhibition of Mitochondrial Translation as a Therapeutic Strategy for Human Acute Myeloid Leukemia. Cancer Cell 20:674–688 Hu B, Guo Y (2019) Inhibition of mitochondrial translation as a therapeutic strategy for human ovarian cancer to overcome chemoresistance. Biochem Biophys Res Commun 509:373–378 Wang B et al (2017) Inhibition of mitochondrial translation effectively sensitizes renal cell carcinoma to chemotherapy. Biochem Biophys Res Commun 490:767–773 Kuntz EM et al (2017) Targeting mitochondrial oxidative phosphorylation eradicates therapy-resistant chronic myeloid leukemia stem cells. Nat Med 23:1234–1240 Meagher AK, Ambrose PG, Grasela TH, Ellis-Grosse EJ (2005) The Pharmacokinetic and Pharmacodynamic Profile of Tigecycline. Clin Infect Dis 41 https://academic.oup.com/cid/article/41/Supplement_5/S333/289231 Stein GE et al (2011) Tigecycline penetration into skin and soft tissue. Surg Infect (Larchmt) 12:465–467 Li X et al (2024) Structural basis for differential inhibition of eukaryotic ribosomes by tigecycline. Nat Commun 15:5481 Zhang J et al (2023) Antigen receptor stimulation induces purifying selection against pathogenic mitochondrial tRNA mutations. 10.1172/jci Tegunov D, Cramer P (2020) Real-time cryo-EM data pre-processing with Warp Europe PMC Funders Group. 10.6019/EMPIAR-10078 Punjani A, Rubinstein JL, Fleet DJ, Brubaker MA (2017) CryoSPARC: Algorithms for rapid unsupervised cryo-EM structure determination. Nat Methods 14:290–296 Liebschner D et al (2019) Macromolecular structure determination using X-rays, neutrons and electrons: Recent developments in Phenix. Acta Crystallogr D Struct Biol 75:861–877 Emsley P, Lohkamp B, Scott WG, Cowtan K (2010) Features and development of Coot. Acta Crystallogr D Biol Crystallogr 66:486–501 Lebedev AA et al (2012) JLigand: A graphical tool for the CCP4 template-restraint library. Acta Crystallogr D Biol Crystallogr 68:431–440 Murshudov GN et al (2011) REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr D Biol Crystallogr 67:355–367 Williams CJ et al (2018) MolProbity: More and better reference data for improved all-atom structure validation. Protein Sci 27:293–315 Chen VB et al (2010) MolProbity: All-atom structure validation for macromolecular crystallography. Acta Crystallogr D Biol Crystallogr 66:12–21 Pettersen EF et al (2021) UCSF ChimeraX: Structure visualization for researchers, educators, and developers. Protein Sci 30:70–82 Tables Table 1 is available in the Supplementary Files section. Additional Declarations There is NO Competing Interest. Supplementary Files 2JulyTigSupplementarymaterial.pdf PDBvalreportfullP1.pdf Cite Share Download PDF Status: Published Journal Publication published 01 May, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4671643","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":327688018,"identity":"3d5b1699-045b-4690-866b-d09f08cd23ef","order_by":0,"name":"Joanna Rorbach","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4klEQVRIiWNgGAWjYBACxgY4kwfEszFgI1VLGmEtSACs5bABQXXM7c3PHhfUMOTzS589+OHnjvPGfAzsDx/gdVjPMXPjGccYLGf25SVL9p65bcbGwGOM1yrGGQlm0jxsDAYGZ3gMpBnbbtsAtbBJ4NeS/k2a5x9Yi/FvxrZzQC3sz3/g15JjJs3bBtZiBrTlANBhDGb4dAD9cqZMmrdPwkCyhy/Nsrct2ZiNmccYr8MM29u3SfN8szHg5+E9fONnm53h/Pb2hx/wamkAU8jGMuN1FgODPAH5UTAKRsEoGAUMDAA30zzn/ZLrfgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-2891-2840","institution":"Karolinska Institutet","correspondingAuthor":true,"prefix":"","firstName":"Joanna","middleName":"","lastName":"Rorbach","suffix":""},{"id":327688019,"identity":"ff18b27f-f260-4501-a9ca-63d1cb085cb2","order_by":1,"name":"Qiuya Shao","email":"","orcid":"","institution":"Karolinska Institutet","correspondingAuthor":false,"prefix":"","firstName":"Qiuya","middleName":"","lastName":"Shao","suffix":""},{"id":327688020,"identity":"da2e99ee-b97d-4c4d-8f0c-8645d751014a","order_by":2,"name":"Anas Khawaja","email":"","orcid":"","institution":"Karolinska Institutet","correspondingAuthor":false,"prefix":"","firstName":"Anas","middleName":"","lastName":"Khawaja","suffix":""},{"id":327688021,"identity":"75eed227-eddc-4456-ae05-84d88c4117a4","order_by":3,"name":"Minh Nguyen","email":"","orcid":"","institution":"Karolinska Institutet","correspondingAuthor":false,"prefix":"","firstName":"Minh","middleName":"","lastName":"Nguyen","suffix":""},{"id":327688022,"identity":"c284d36a-49d1-465b-9184-b55c0b16abda","order_by":4,"name":"Vivek Singh","email":"","orcid":"","institution":"University of Stockholm","correspondingAuthor":false,"prefix":"","firstName":"Vivek","middleName":"","lastName":"Singh","suffix":""},{"id":327688023,"identity":"1b5d2e22-8c0d-493e-91e4-3161475be0fa","order_by":5,"name":"Jingdian Zhang","email":"","orcid":"","institution":"Karolinska Institutet","correspondingAuthor":false,"prefix":"","firstName":"Jingdian","middleName":"","lastName":"Zhang","suffix":""},{"id":327688024,"identity":"93a45fcf-7579-4907-ae06-2eb73aa616d6","order_by":6,"name":"Monica Adori","email":"","orcid":"","institution":"Karolinska Institutet","correspondingAuthor":false,"prefix":"","firstName":"Monica","middleName":"","lastName":"Adori","suffix":""},{"id":327688025,"identity":"603688d0-acdd-4a46-b744-aed9ee96ef14","order_by":7,"name":"C. Axel Innis","email":"","orcid":"https://orcid.org/0000-0003-3153-9490","institution":"INSERM U1212, CNRS UMR 5320","correspondingAuthor":false,"prefix":"","firstName":"C.","middleName":"Axel","lastName":"Innis","suffix":""},{"id":327688026,"identity":"bb4522c0-4f2b-49f9-8f82-ca43aa02d45c","order_by":8,"name":"Xaquin Castro Dopico","email":"","orcid":"https://orcid.org/0000-0002-9005-6774","institution":"Karolinska Institutet","correspondingAuthor":false,"prefix":"","firstName":"Xaquin","middleName":"Castro","lastName":"Dopico","suffix":""}],"badges":[],"createdAt":"2024-07-02 05:40:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4671643/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4671643/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-025-59388-9","type":"published","date":"2025-05-01T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":60517638,"identity":"920674d6-6ebd-4f61-b9d6-3509a0d281c6","added_by":"auto","created_at":"2024-07-17 15:48:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":900780,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTigecycline compromises human T cell activation and proliferation by inhibiting mitochondrial translation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e 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.\u003cstrong\u003e (B)\u003c/strong\u003e Representative dose-response curves for additional bacterial protein synthesis inhibitors on PBMCs for 72 hours. \u003cem\u003en\u003c/em\u003e = 6 biological replicates, mean ± SEM. Data are shown relative to DMSO treatment without antibiotics. \u003cstrong\u003e(C)\u003c/strong\u003e \u003csup\u003e35\u003c/sup\u003eS metabolic labeling assay of mitochondrial (left) and cytosolic (right) translation on Jurkat T cells after 18 h treatment with Q/D, tigecycline, or doxycycline. One representative result from \u003cem\u003en\u003c/em\u003e = 3 is shown. \u003cstrong\u003e(D)\u003c/strong\u003e 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. \u003cem\u003en\u003c/em\u003e = 2 biological replicates, one shown. \u003cstrong\u003e(E)\u003c/strong\u003e 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 minute and normalized according to protein amount/well. \u003cem\u003en\u003c/em\u003e = 4 biological replicates. Oligo, oligomycin; FCCP, carbonyl cyanide-p-tri-fluoromethoxyphenylhydrazone; Rot, rotenone; AA, antimycin A. \u003cstrong\u003e(F)\u003c/strong\u003e CD25 expression (MFI) on CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells 18 h after anti-CD3/CD28 activation IL-2 (10ng/ml) in the presence or absence of tigecycline (\u003cem\u003en\u003c/em\u003e = 3 blood donor PBMC samples). An RM one-way ANOVA with Tukey's multiple comparisons test was used to analyze the data. \u003cstrong\u003e(G) \u003c/strong\u003eT cell proliferation assessed by flow cytometry. PBMC’s T cells were stimulated with anti-CD3/CD28 beads + IL-2 (10ng/ml) in the presence or absence of tigecycline, and proliferation was assessed by CTV dilution in live cells after 6 days (\u003cem\u003en\u003c/em\u003e = 5 blood donor PBMC samples). PBMCs were labelled with CTV prior to stimulation. \u003cstrong\u003e(H)\u003c/strong\u003e 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. \u003cstrong\u003e(I)\u003c/strong\u003e Proliferation of FACS-isolated CD45RA\u003csup\u003e+\u003c/sup\u003eCD27\u003csup\u003e+\u003c/sup\u003e naïve and CD45RA\u003csup\u003e-\u003c/sup\u003eCD27\u003csup\u003e+\u003c/sup\u003e memory CD4\u003csup\u003e+\u003c/sup\u003e T cells after 6 days of stimulation with anti-CD3/CD28 beads and IL-2 (10ng/ml). Representative CTV dot plots from non-treated and 10 μM tigecycline-treated samples after 6 days of treatment are shown. FACS-isolated cells were seeded in the presence or absence of tigecycline (from 2.5 to 10 μM) and proliferation was assessed by CTV dilution in live cells (\u003cem\u003en\u003c/em\u003e = 3 blood donor PBMC samples). The gating strategy is shown in Fig. S1E. \u003cstrong\u003e(J)\u003c/strong\u003e Percentage of live CD4\u003csup\u003e+\u003c/sup\u003e T naïve cells and memory cells in each division, data from (I), mean ± SEM. Tn: naïve T cells; Tm: memory T cells.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-4671643/v1/df2e19b54803ecec8b625c78.png"},{"id":60517083,"identity":"6026e093-d1dc-4562-99e6-2d3c806dcd59","added_by":"auto","created_at":"2024-07-17 15:40:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":648362,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverview of the interactions of tigecycline with the human mitoribosome.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Chemical structure of tigecycline. \u003cstrong\u003e(B)\u003c/strong\u003eOverview 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. \u003cstrong\u003e(C) \u003c/strong\u003emtSSU 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.\u003cstrong\u003e (D) \u003c/strong\u003emtLSU site-1: a novel tigecycline binding site located at helix 71 of the 16S rRNA, close to the acceptor stem of P-tRNA. \u003cstrong\u003e(E)\u003c/strong\u003e 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.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-4671643/v1/0ff1c07abdea78c666ebdb78.png"},{"id":60517088,"identity":"8ca5f709-f380-4cf3-82c3-fef780315334","added_by":"auto","created_at":"2024-07-17 15:40:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":878804,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe conserved binding site of tigecycline on the mtSSU. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Tigecycline establishes canonical interactions with the 12S rRNA of the mtSSU accompanied by coordinated Mg\u003csup\u003e2+\u003c/sup\u003e ions (lime) and waters (HOH) (red). Hydrogen bond interactions are indicated as dashed lines. \u003cstrong\u003e(B)\u003c/strong\u003e Structure of tigecycline (purple) (PDB: 5J91) \u003cstrong\u003e(C)\u003c/strong\u003e and sarecycline (yellow) (PDB: 6XQD), a tetracycline-derivative, in complex with the SSU of 70S ribosome from \u003cem\u003eE.coli\u003c/em\u003e and \u003cem\u003eT. thermophilis, \u003c/em\u003erespectively\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-4671643/v1/dd757c3828ddf0e8064e48b2.png"},{"id":60517087,"identity":"660b65f0-f552-4092-a194-4824b7da2b5f","added_by":"auto","created_at":"2024-07-17 15:40:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1011343,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInteraction of tigecycline with helix 71 of the mtLSU (mtLSU site-1) and its functional implications.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Tigecycline stabilizes in the binding pocket through multiple interactions either directly with the neighbouring nucleotides or indirectly through Mg\u003csup\u003e2+\u003c/sup\u003e ion coordination as compared to the non-treated mitoribosomes (PDB:7QI4\u003csup\u003e32\u003c/sup\u003e) (\u003cstrong\u003eB\u003c/strong\u003e). (\u003cstrong\u003eC\u003c/strong\u003e) The superposition shows the large displacement of H71 upon the binding of tigecycline. \u003cstrong\u003e(D)\u003c/strong\u003e 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 \u003cem\u003eE. coli\u003c/em\u003e (PDB: 5J91), the flexible movement of this region is likely restricted by the presence of methylation of U1939.\u0026nbsp; \u003cstrong\u003e(E) \u003c/strong\u003eThe binding of tigecycline to the mtLSU site-1 might inhibit mitoribosome recycling. The RRF1-bound state (PDB: 7NSI\u003csup\u003e34\u003c/sup\u003e) 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).\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-4671643/v1/45f982f91ce91603dee11f4e.png"},{"id":60517637,"identity":"20b2a019-3246-4a0c-8d35-f3b378e0ae13","added_by":"auto","created_at":"2024-07-17 15:48:35","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":683710,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInteractions of tigecycline with the PTC (mtLSU site-2).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTigecycline 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\u003csup\u003e32\u003c/sup\u003e). G2992, U2993, and G3063 in the PTC rearranges upon binding of the A-tRNA (as also observed for the untreated mitoribosomes).\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-4671643/v1/b8076ea14060b9b7edf502bd.png"},{"id":81805138,"identity":"bcfd7b70-2b14-40df-8359-1420aaa4f8fd","added_by":"auto","created_at":"2025-05-02 07:06:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5579487,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4671643/v1/870d134c-4060-45a4-9e12-53dc7c7773f2.pdf"},{"id":60517089,"identity":"b69bc6db-2928-49a1-96da-d674ec4107fc","added_by":"auto","created_at":"2024-07-17 15:40:36","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":15100747,"visible":true,"origin":"","legend":"","description":"","filename":"2JulyTigSupplementarymaterial.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4671643/v1/4fdde40f15716261925149a8.pdf"},{"id":60517086,"identity":"77e2b436-d723-4641-974a-e84a1cd5c050","added_by":"auto","created_at":"2024-07-17 15:40:35","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2486840,"visible":true,"origin":"","legend":"","description":"","filename":"PDBvalreportfullP1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4671643/v1/f25f9eb584aa80f229011c1e.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"\u003cp\u003eStructural Basis of T Cell Toxicity Induced by Tigecycline Binding to the Mitochondrial Ribosome\u003c/p\u003e","fulltext":[{"header":"Highlights","content":"\u003cp\u003e\u0026bull; Mitoribosome inhibition by tetracyclines curtails T cell function.\u003c/p\u003e\n\u003cp\u003e\u0026bull; First cryo-EM analysis of human lymphoid mitoribosomes.\u003c/p\u003e\n\u003cp\u003e\u0026bull; Tigecycline blocks A-site tRNA binding and binds to the PTC.\u003c/p\u003e\n\u003cp\u003e\u0026bull; A human-specific, third binding site of tigecycline serves as a guide for antibiotic design.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eAntibiotic resistance is a major One Health problem that has led to an intensive search for new antibacterials, as well as the modification of existing entities to broaden their spectrum of activity. Many antibiotics on the WHO\u0026rsquo;s list of essential medicines (such as tetracycline and doxycycline) are bacterial protein synthesis inhibitors, which mediate their effects by binding to bacterial ribosomes and halting translation. Although these entities have been of clinical importance for many decades, serious complications are known to arise due to therapy. For example, tetracyclines (which bind both 30S and 50S subunits and are used to treat plague, brucellosis, and Lyme disease) have long been noted for nephro- and hepato-toxicity, together with anti-inflammatory effects that have shown benefits in patients with chronic inflammatory skin, autoimmune and neurodegenerative diseases\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Similarly, chloramphenicol - which binds the bacterial 50S ribosomal subunit and is used to treat meningitis, cholera, and typhoid fever - is known to induce bone marrow suppression\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. As antibiotic resistance mechanisms and drugs evolve, so too will the need for efforts to dissect treatment effects on different foreign organisms, as well as host cells.\u003c/p\u003e \u003cp\u003eMitochondria possess their own genome that encodes core components of the oxidative phosphorylation (OXPHOS) machinery - translated by the organelle\u0026rsquo;s specialized ribosomes, mitoribosomes, in proximity to the inner mitochondrial membrane to efficiently generate ATP. Evolutionarily descended from an α-proteobacterial ancestor, mitoribosomes share several features with their bacterial counterparts, and it has been long recognized that bacterial protein synthesis inhibitors can interfere with mitochondrial translation and cellular aerobic capacity, although the molecular bases of such effects have only been described in a handful of cases. Unlike interactions between bacterial or eukaryotic cytosolic ribosomes and antibiotics, which have been well characterized at atomic resolution, only more recently have insights emerged concerning the mitoribosome, the function of which is increasingly implicated in a range of clinical conditions. For example, streptomycin was recently shown to bind the mitoribosomal small subunit (mtSSU)\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, while dalfopristin/quinupristin (Q/D) binds the large subunit (mt-LSU), effectively suppressing glioblastoma stem cell growth\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. The interactions between these antibiotics and mitoribosomes closely resemble that of bacterial ribosome-antibiotic interactions.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eIn recent years, the multifaceted and central role mitochondria play in the immune system has become increasingly clear. Via dynamic rearrangements in specialized immune lineages, the organelle determines cellular fate via metabolic reprogramming and innate signalling. Naive lymphocytes fundamentally depend upon OXPHOS to power clonal expansion \u003csup\u003e\u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, something which if inhibited can affect the effector-memory response\u003csup\u003e\u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. On the other hand, such inhibition could explain the beneficial effects of mitochondria-targeting drugs in other clinical contexts\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, such as autoimmunity\u003csup\u003e\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, although the concomitant disruption of the metagenome and increased risk of antibiotic resistance may limit their application to these conditions.\u003c/p\u003e\u003cp\u003eTo this end, we explored the effects of several important antibiotics targeting protein synthesis on human T cells. For the most potent inhibitor of mitochondrial translation and proliferation identified, tigecycline, we then sought to determine the mechanism of action at atomic resolution, guiding the design of tetracyclines with reduced off-target binding.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003e\u003cstrong\u003eTetracyclines compromise lymphocyte survival\u003c/strong\u003e \u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eWe first compared the survival of immortalized Jurkat T cells and HeLa cells in the presence of chloramphenicol or doxycycline, two important bacterial ribosome-targeting antibiotics associated with white blood cell phenotypes\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. We found chloramphenicol to reduce the survival of both cell lines after 3 and 5 days of treatment (Jurkat IC50\u0026thinsp;=\u0026thinsp;25.85 \u0026micro;M; HeLa IC50\u0026thinsp;=\u0026thinsp;41.71 \u0026micro;M, after 5 days). However, Jurkat T cells were uniquely sensitive to lower concentrations of doxycycline (IC50\u0026thinsp;=\u0026thinsp;6.93 \u0026micro;M after 5 days) (Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003eA).\u003c/p\u003e\n\u003c/div\u003e\n\u003cp\u003eTetracyclines (such as doxycycline) are broad-spectrum protein synthesis inhibitor agents characterized by a four-ring core structure\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Their mechanism of action involves reversible binding to the 30S bacterial ribosomal subunit, thereby preventing the attachment of aminoacyl-tRNA to the mRNA-ribosome complex\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Given our results, we chose to investigate six additional tetracyclines, including newer derivates reserved for difficult to treat infections (medocycline, methacycline, minocycline, oxycycline, tetracycline, tigecycline). In both Jurkat T cells and peripheral blood mononuclear cells (PBMCs) isolated from healthy blood donor samples, we found the third-generation tigecycline to have the greatest negative effect on cell survival (IC50\u0026thinsp;=\u0026thinsp;2.94\u0026ndash;3.08 \u0026micro;M for Jurkat T cells; 2.02\u0026ndash;9.42 \u0026micro;M for PMBCs after 3 days of treatment (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). Tigecycline is a glycylcycline with a N, N-dimethyglycylamido (DMG) moiety attached to position 9 of tetracycline ring D that confers enhanced activity against tetracycline-resistant bacteria.\u003c/p\u003e\n\u003cp\u003eWe then compared the effect of tigecycline to an additional set of widely used bacterial protein synthesis inhibitors (spanning different drug classes) described in the literature (dalfopristin/quinupristin (Q/D), azithromycin, tiamulin, linezolid, and clindamycin) (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). In this comparison, tigecycline again showed the greatest toxicity to PBMCs, followed by Q/D, which was previously reported to bind the mitochondrial large subunit\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eGiven our prior observations with doxycycline, it was interesting to observe that HeLa cells were also relatively resistant to 10 \u0026micro;M tigecycline, although Q/D did have a potent effect on their survival (IC50\u0026thinsp;=\u0026thinsp;16.66 \u0026micro;M) (Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003eB). Q/D also had a greater influence on Hek293 cell survival than did tigecycline and doxycycline (Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003eB), together illustrating that different cell lines have differential susceptibility to the same compounds, likely due to differences in their metabolism.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eTigecycline inhibits oxidative phosphorylation and primary T cell expansion\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eTo ascertain whether tigecycline affected mitoribosomal function, we profiled mitochondrial and cytosolic translation with/without antibiotic treatment in Jurkat T cells. Metabolic labelling using \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003eS-methionine was performed after 18 h of treatment with tigecycline; doxycycline and Q/D were used as comparators. We found tigecycline and Q/D inhibited mitochondrial translation at 5 \u0026micro;M, while weaker inhibition was observed for doxycycline (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC). On the other hand, 10 \u0026micro;M of these compounds did not have profound effects on cytosolic translation, suggesting mitochondrial protein synthesis is more greatly affected than cytosolic in these rapidly dividing cells (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e\n \u003cp\u003eThe defect in translation could be confirmed at the protein level. OXPHOS subunit profiling of Jurkat T cells and human PBMCs by western blot revealed the levels of subunits of complex I (NADH: ubiquinone oxidoreductase subunit B (NDUFB8)), complex III (ubiquinol-cytochrome c reductase core protein 2 (UQCRCII)) and complex IV (cytochrome c oxidase subunit 2 (COX2)), which contain mitochondrially-encoded molecules, to drop in response to 5 \u0026micro;M tigecycline (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD). In contrast, subunits of complex II (succinate dehydrogenase complex iron sulphur subunit B (SDHB)) and complex V (ATP Synthase F1 subunit alpha (ATP5A)) were much less affected, indicating treatment-induced nuclear-mitochondrial protein imbalance in the electron transport chain.\u003c/p\u003e\n \u003cp\u003eGiven the pivotal role OXPHOS plays in T cell clonal expansion and memory generation, we wanted to determine whether these deficits correlated with reduced OXPHOS capacity at the cellular level. To do this, we activated T cells within PBMCs via plate-bound anti-CD3/CD28 antibodies and analyzed oxygen consumption using the Seahorse MitoStress test 6 days after stimulation. 5 and 10 \u0026micro;M tigecycline reduced basal, ATP-coupled, maximal and spare oxygen consumption rates (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE, S1C). In the shorter term (18 h post-stimulation), the drug reduced expression of the T cell activation marker, CD25 (\u003cem\u003eIL2RA\u003c/em\u003e), by stimulated CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells, suggesting cells are affected prior to cell division (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eF).\u003c/p\u003e\n \u003c/div\u003e\n \u003cp\u003eTo determine whether treatment inhibited T cell expansion, we assessed proliferation using flow cytometry. After 6 days of anti-CD3/CD28 stimulation in the presence of IL-2, both CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells in healthy donor PBMCs showed dose-dependent reductions in proliferation after tigecycline treatment (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eG-H). The effects on T cell proliferation by other antibiotics included in the study are presented in Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003eD. Given our observations in cell lines (Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003eB) and previous research indicating differential susceptibility to antibiotics by specialized T cell subsets\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, we FACS-isolated na\u0026iuml;ve (CD45RA\u003csup\u003e+\u003c/sup\u003eCD27\u003csup\u003e+\u003c/sup\u003e) and memory (CD45RA\u003csup\u003e-\u003c/sup\u003eCD27\u003csup\u003e+\u003c/sup\u003e) CD4\u003csup\u003e+\u003c/sup\u003e T cells and compared proliferation in the presence or absence of tigecycline (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eI-J). As observed for total T cells, dose-dependent inhibition was observed for both subsets between 2.5\u0026ndash;10 \u0026micro;M.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003eStructure determination of tigecycline-mitoribosome complexes\u003c/h2\u003e\n \u003cp\u003eTo explore the molecular basis of the off-target effects of tigecycline, we isolated 55S mitochondrial ribosomes (mitoribosomes) from Jurkat T cells, incubated them with tigecycline, and analyzed the resulting complexes using single-particle cryo-EM (see \u0026ldquo;Methods\u0026rdquo;).\u003c/p\u003e\n \u003cp\u003eThe initial reconstruction of the mitoribosome-tigecycline complex yielded an initial cryo-EM density map with an overall resolution of 2.4 \u0026Aring; (Fig. \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003e and Table 1). The particles underwent 3D classification with a solvent mask to get rid of poorly aligned particles, which yielded three major classes of monosomes: (class 1) mitoribosomes with no tRNA (\u0026lsquo;empty class\u0026rsquo;), (class 2) with tRNA in the P-site only, (class 3) with A- and P- tRNAs (Fig. \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003e). Further local refinements led to a resolution of 2.2 \u0026Aring; for the core region of the monosome (Fig. \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003e and Table 1), detecting all known methylations of 12S and 16S rRNAs, along with 2 iron-sulphur (2Fe-S) clusters in the mtSSU, and 1 Fe-S in the mtLSU, previously identified in the mitoribosomes isolated from Hek293 cells\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Interestingly, other reported cofactors of the mitoribosome such as NAD, spermine, and spermidine were not found in any of the classes of tigecycline-bound monosomes. The absence of these cofactors might result from the preferential occupancy of these cofactors in specific cell types (T cells versus embryonic kidney cells).\u003c/p\u003e\n \u003cp\u003eThird-generation tetracyclines are based on a common naphthacene-carboxamide core comprising four rings (A, B, C, and D). The optimization of tigecycline consists of structural modifications of ring D at positions C7 (dimethyl-amino group) and C9 (\u003cem\u003etert-\u003c/em\u003ebutylglycylamide)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). Our analyses unambiguously identified three densities corresponding to tigecycline molecules (Fig. S3A-C); one on the mtSSU (\u0026lsquo;mtSSU site\u0026rsquo;) and two on the mtLSU (mtLSU site-1 and site-2) (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB-E). This is in contrast to previous structural data from bacteria, which reported a single tigecycline binding site on the SSU\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. The antibiotic\u0026apos;s relative occupancy in the three sites was notably high, particularly in the mtSSU and mtLSU site-1, where the P-site-only class of mitoribosomes showed an occupancy exceeding 80% (Fig. S3D).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003eTigecycline blocks aminoacyl-tRNA binding to the mtSSU A site\u003c/h2\u003e\n \u003cp\u003eTigecycline density on the mtSSU was detected in two mitoribosomal classes, \u0026lsquo;P-site tRNA\u0026rsquo; and \u0026lsquo;empty monosome\u0026rsquo;, but not in the class containing A-site tRNA. Based on bacterial studies, it is established that tigecycline specifically targets the head region of the SSU and competes with the binding of A-site tRNA, in a similar manner observed for other tetracycline derivatives\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n \u003cp\u003eThe interaction between tigecycline and the mtSSU is analogous to how tetracyclines bind to the small ribosomal subunits of various bacterial species (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA-C)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. In the mtSSU, tigecycline interacts with helix 34 of the 12S rRNA through its polar edge and through ring A, which establish potential hydrogen bonds with the sugar-phosphate backbone of several nucleotides (A1258, U1259, U1325, A1326, G1327, and G1328) or make indirect contact via coordinated Mg\u003csup\u003e2+\u003c/sup\u003e ions (G1328) (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC and S3A). Ring D stacks on top of nucleobase U1259, which further stacks on A1326, stabilizing the binding of tigecycline to the mtSSU A-site. Tigecycline coordinates a Mg\u003csup\u003e2+\u003c/sup\u003e ion (denoted as Mg-1) via its keto-enol system (C11 and C12) and a second one (denoted as Mg-2) via hydroxyl and amide groups of ring A (O3 and O21, respectively). This coordination of the two Mg\u003csup\u003e2+\u003c/sup\u003e ions is a conserved feature of the tetracycline-ribosome interaction (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA-C). We detected the density for an additional Mg\u003csup\u003e2+\u003c/sup\u003e ion (Mg-3) in close proximity to tigecycline, likely interacting with the oxygen atom from the 9-t\u003cem\u003e-\u003c/em\u003ebutylglycylamido moiety adopting an extended conformation. Furthermore, the sugar phosphate of U1259 is within hydrogen-bonding distance from nitrogen atoms of the C9 butylglycylamido substituent (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). These extensive interactions stabilize the overall binding of tigecycline to the decoding centre of mtSSU. Overall, the interactions observed for the tigecycline-bound mtSSU are comparable to tetracycline binding with the bacterial SSU, since the functional core of the ribosome including the decoding centre is universally conserved\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003eA unique tigecycline binding site on helix 71 of the mtLSU\u003c/h2\u003e\n \u003cp\u003eIn all our mitoribosome classes, we observed additional density in the region of mtLSU comprising helix 69 and 71 (denoted as mtLSU site-1). We identified tigecycline in this density and observed multiple stabilizing interactions of the antibiotic with the residues forming its binding pocket (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA and S3B). In this position, the polar edge of the tigecycline molecule is directed towards the 16S rRNA where it forms multiple interactions, while its C7 extension is near the acceptor stem of the P-tRNA (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e\n \u003cp\u003eTo form a tigecycline binding site that consists primarily of a hydrophobic cavity between the base of U2628 and the nucleoside of A2604, helix 71 must undergo large conformational changes (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA and Fig S4B). The rearrangement of nucleotides U2626 and U2628 contributes to opening-up a space where the drug can bind. Nucleotide U2626 shifts outward and forms stacking interactions with A2598 and C2625. U2628 changes conformation such that its nucleobase becomes intercalated between ring D of tigecycline and the nucleobase of A2629. These conformational changes are accompanied by a downward shift of helix 71 (~\u0026thinsp;4\u0026ndash;5 \u0026Aring;) as compared to the untreated monosome (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Additionally, nucleotide C2603 flips inward together with A2600, with which it forms stacking interactions (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC). This rearrangement positions the nucleobase of A2604 such that it stacks against ring C of tigecycline. Overall, stacking interactions between rings C and D of tigecycline and residues U2628 and A2604 of the 16S rRNA help to stabilize the binding of the drug to this region (Fig. S3B). Moreover, these conformational changes result in the formation of a non-canonical U-U pair between nucleotides 2599 and 2606, which further contributes to stabilizing the drug-bound conformation of helix 71 (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e\n \u003cp\u003eAs observed for the tigecycline molecule in the mtSSU, the drug molecule bound to mtLSU site-1 forms metal ion complexes with the phenol-ketone system of rings B and C, and via the oxygen atom of ring A\u0026rsquo;s amide group and the hydroxyl oxygen at position C3. Interestingly, similarly to the mtSSU, we located an additional Mg\u003csup\u003e2+\u003c/sup\u003e ion density, which coordinates with the hydroxyl of ring D and facilitates indirect interaction with the backbone phosphate of A2604 (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD and S3B). Through its polar edge, tigecycline forms potential hydrogen bonds with the phosphate groups of adjacent nucleotide G2627 of 16S rRNA and indirectly through Mg\u003csup\u003e2+\u003c/sup\u003e ion coordination. The 9-t-butylglycylamido moiety of tigecycline extends towards the PTC center of the mitoribosome, causing nucleotide A3089 to shift (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e\n \u003cp\u003eAs of now, there is no documentation of antibiotics binding to a comparable location within the bacterial LSU. Interestingly, helix 71 from multiple bacterial species comprises conserved methylations of 23S rRNA surrounding the region where tigecycline binds to the 16S rRNA of the mitoribosome (Fig. S4A). In \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eT. thermophilus\u003c/em\u003e these correspond to positions m\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003eU1939 and m\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003eC1962, and in \u003cem\u003eC. acnes\u003c/em\u003e, positions m\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003eU2122 and m\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003eC2145. There is additional methylation of cytosine m\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003eC1942 in \u003cem\u003eT. thermophilus\u003c/em\u003e (Fig. S4A). In contrast, these modifications are absent in the mitochondrial LSU\u0026rsquo;s rRNA. The methylations in helix 71 of 23S rRNA in bacteria are not expected to directly clash with tigecycline; nevertheless, they may confer rigidity of the region, preventing antibiotic binding. Especially, m\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003eC1942 is likely to prevent the conformational change needed for U1940 to flip out and the drug to bind to the bacterial ribosome (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e\n \u003cp\u003eTo understand the potential consequences of the binding of tigecycline to the mtLSU site-1 on mitochondrial translation, we compared our structure with previously reported structures of translating mitoribosome. As depicted in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eE, the tigecycline binding site likely hinders the interaction of mitoribosome with the ribosomal recycling factor 1 (RRF1)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. This obstruction may impede mitoribosome recycling, leading to the accumulation of non-functional mitoribosomes prone to aggregation. Furthermore, the mtLSU site-1 is positioned between the P and A site tRNA, and binding of the drug to this site may interfere with the translocation of a tRNA from the A site to the P site during the elongation phase of translation. Indeed, the dimethylamine group at C4 of tigecycline might interfere with the phosphate backbone of C71, as well as the OH group of the ribose ring of C70 in the hybrid (A/P site) tRNA (Fig. S4B).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003eTigecycline binds to the peptidyl transferase center of the mtLSU\u003c/h2\u003e\n \u003cp\u003eThe third density corresponding to a tigecycline molecule was identified at the catalytic peptidyl transferase center (PTC). There have been previous reports of tetracycline-derivative such as sarecycline, anthracycline-like tetracenomycin X (TcmX), as well as the macrolide erythromycin, binding to the PTC and mediating translation arrest in bacteria\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Though the occupancy of tigecycline at the PTC in our study is the lowest, as compared to the other two sites in the mtLSU and mtSSU (Fig. S3D), it represents a unique occurrence for tigecycline (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE). In this position, the ring A of tigecycline faces the lumen of the nascent polypeptide exit tunnel while the 9-t-butylglycylamido extension is in proximity of the terminal adenosine A76 of the P-site tRNA, potentially hindering the progression of the elongating nascent chain along the tunnel (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). As observed for the mtSSU and mtLSU site-1, tigecycline mediates canonical metal Mg\u003csup\u003e2+\u003c/sup\u003e ion complexes through its polar groups at rings B and C, and via the oxygen atoms of the carboxamide group and C3. Further stabilization of tigecycline at the PTC occurs via nucleobase C3073 which stacks on ring D and forms a non-canonical base-pair with C2502 (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE and S3C). In the PTC of \u003cem\u003eC. acnes\u003c/em\u003e 70S, similar non-canonical C1965-C2768 base pairing is observed, contributing to sarecycline binding, whereas corresponding U-U (U1782-U2586) base-pairing in the \u003cem\u003eE. coli\u003c/em\u003e 23S rRNA is proposed to be crucial for TcmX accommodation (Fig. S6)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. In the absence of a nascent chain, the exit tunnel in mitoribosomes is occupied by the mito-specific N-terminal extension of mL45\u003csup\u003e38\u003c/sup\u003e. A2725 interacts with either the mL45 N-terminal extension or the nascent chain in the exit tunnel, which may contribute to the stabilization of the mL45 or the nascent chain\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. However, in the tigecycline-bound mtLSU, the A2725 nucleobase is rotated 90\u0026deg; and stacks onto ring D, further securing the binding of tigecycline to the PTC (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). The binding sites for tigecycline and sarecycline at the PTC overlap, but surprisingly sarecycline is rotated 180\u0026deg; and shifted laterally relative to the tigecycline, resulting in its polar edge arranged in the opposite direction (Fig. S6). These differences can be either species-specific or antibiotic-specific, requiring further investigations. Ring A of tigecycline is placed away from the N-terminal extension of mL45 in the exit tunnel, and it coordinates with the Mg\u003csup\u003e2+\u003c/sup\u003e ion through the oxygen atom of the carboxamide group and hydroxyl group at C3 (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e and S3C). Additionally, the oxygen atom at C1 of ring A forms a hydrogen bond with C2502, which forms a \u003cem\u003ecis\u003c/em\u003e Watson-Crick/Watson-Crick base-pair with C3073 and stacks against the ring D (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). The overall positioning of tigecycline allows 9-t-butylglycylamido moiety to extend towards the acceptor arm of the P-tRNA.\u003c/p\u003e\n \u003cp\u003eTigecycline 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\u003csup\u003e32\u003c/sup\u003e). G2992, U2993, and G3063 in the PTC rearranges upon binding of the A-tRNA (as also observed for the untreated mitoribosomes).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we investigated the off-target effects of bacterial ribosome-binding antibiotics on OXPHOS-dependent lymphocytes. Our screening pinpointed tigecycline as an antibiotic exhibiting toxicity towards human T cell lines and primary cells, although other members of this class (such as doxycycline) showed similar phenotypes at the cellular level.\u003c/p\u003e \u003cp\u003eWhile our experiments were not designed to assess antibiotic toxicity in a comparative manner between bacteria and eukaryotic cells, the observation of three binding sites for tigecycline on the mitochondrial ribosome, as opposed to one on the bacterial ribosome, implies that this antibiotic is a potent inhibitor of mitochondrial translation. Extensive studies in bacterial systems have shown that high concentrations of antibiotics may result in secondary binding sites in \u003cem\u003ein vitro\u003c/em\u003e experiments, which may not be physiologically relevant\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. To avoid this issue, we employed a notably lower concentration of tigecycline (30 \u0026micro;M) than that previously used to investigate its interactions with ribosomes isolated from various bacterial species (concentrations ranging from ~\u0026thinsp;60 to 300 \u0026micro;M)\u003csup\u003e\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Nevertheless, unlike in previous studies on bacterial ribosomes, where the tigecycline molecule was located only on the SSU, we found two additional binding sites on the mtLSU (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), with relatively high occupancy for identified new sites (Fig. S3D). The binding to the PTC (mtLSU site-2) overlapped with the recently identified binding site for tetracenomycin X (TcmX) and sarecycline, both with a very similar architecture to tigecycline (Fig. S5).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eIncreasing evidence indicates that many ribosome-targeting antibiotics act in a context-dependent manner, influenced by the nature of the nascent protein. This was recently demonstrated for TcmX, where it was shown to sequester the 3\u0026prime; adenosine of peptidyl-tRNALys in the nascent polypeptide exit tunnel of the ribosome upon translation of a QK motif\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Analysis of our tigecycline-PTC site did not detect any changes in the conformation of P-tRNA in either of the mitoribosomal classes comprising P-tRNA only or A- and P-tRNA. This could be due to the absence of the nascent polypeptide in our structures. Unfortunately, current mitoribosome purification methods often do not retain the nascent polypeptide, instead, the exit tunnel in mitoribosomes is occupied by the mito-specific N-terminal extension of mL45, which enters the tunnel once mitoribosomes are detached from the inner mitochondrial membrane. Therefore, the context specificity of tigecycline binding to the PTC needs to be addressed in the future using other methods, for example, mitoribosome profiling\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOne of the most intriguing observations arising from our structural studies was the identification of the novel tigecycline binding site on the mtLSU (mtLSU site-1). This specific region has not previously been recognized as a prominent site for antibiotic binding on bacterial ribosomes, raising the question of its mitochondrial specificity. To create sufficient space for the drug to effectively target this area, specific nucleotides including C1963/U2626, C1965/U2628 and U1940/U2603 of helix 71 ((\u003cem\u003eE.coli\u003c/em\u003e/human mtDNA numbering) must undergo substantial movement. However, in bacterial ribosomes, this region contains extensive methylations, constraining the flexibility of modified nucleotides (Fig. S4A). In particular, the m5U1939 modification might sterically hinder the conformational change required for accommodating the flipping of U1940 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). To verify this hypothesis, additional mutagenesis studies are warranted.\u003c/p\u003e \u003cp\u003eCould the binding of tigecycline to mtLSU site-1 contribute to the inhibition of mitochondrial translation? Due to technical limitations, primarily the lack of efficient methods to manipulate the mitochondrial genome and the absence of a robust in vitro mitochondrial translation assay, it is challenging to design an experiment to determine the specific contributions of different binding sites to translation inhibition. However, comparisons with the translating ribosome suggest that binding to mtLSU site-1 could interfere with mitoribosome recycling and the translocation of tRNA from the A site to the P site (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE and S4B). Further studies are needed to confirm these findings. Together, the efficient binding of tigecycline in all three sites is likely to assure an efficient inhibition of several stages of translation.\u003c/p\u003e \u003cp\u003eOur study reports the first structures of mitoribosomes isolated from human lymphoid cells (Jurkat T cells). Given that the features of the binding sites remain conserved with other mammalian mitoribosomes previously characterized (from Hek293 and porcine), it suggests that the interaction is likely to be conserved across mammals. However, our toxicity tests revealed a significantly stronger effect of this antibiotic on Jurkat T cells, compared to Hek293 and Hela cells (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). This point is particularly important considering the numerous studies modulating mitochondrial function in the context of cancer\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Antibiotics that potentially inhibit mitochondrial translation, including tigecycline, have been reported to inhibit survival in glioblastoma cells\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, leukemia stem cells\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, ovarian cancer cells\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, renal cancer cells\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e, and imatinib-resistant chronic myeloid leukemia (CML)\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e cells. Our data encourage the investigation of anti-tumour lymphocyte responses in cancer patients receiving such treatments.\u003c/p\u003e \u003cp\u003eIn an infectious context, antibiotics need to be delivered to infected lesions to combat the pathogen. The observed IC50 values for tigecycline against T cells \u003cem\u003ein vitro\u003c/em\u003e were relevant to drug concentrations observed in soft tissue lesions of patients undergoing treatment for bacterial infections\u003csup\u003e\u003cspan additionalcitationids=\"CR47\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Therefore, future studies could also consider how lesion-resident immune cells are affected by tetracycline treatments, as this could influence pathogen clearance and tissue repair. The investigation of myeloid lineages is of similar interest. Nevertheless, these data provide a molecular mechanism to explain the anti-inflammatory effects of tetracyclines and inform antibiotic design. While our manuscript was in preparation, a separate study identified three binding sites for tigecycline on the 55S mitoribosome isolated from HEK293T cells\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. This finding confirms that tigecycline effectively inhibits mitochondrial translation at clinically relevant concentrations.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eEthical declaration\u003c/h2\u003e \u003cp\u003eEthical approval for the use of peripheral blood samples from human donors was granted by the Swedish Ethical Review Authority (registration number 2018/1498-31/3). All human studies were carried out in accordance with the guidelines and policies of Karolinska Institutet and EU legislation.\u003c/p\u003e\u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eHuman samples\u003c/h2\u003e \u003cp\u003eBuffy coats were ordered from anonymous blood donors at Karolinska Universitetslaboratoriet. On the same day as sample collection, PBMCs were isolated by density gradient centrifugation over Lymphoprep (StemCell Technologies), washed with RPMI-1640 (Cytiva HyClone) with 10% FBS, and cryopreservated at -80\u003csup\u003eo\u003c/sup\u003eC in FBS with 10% DMSO (Sigma)\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCell viability assays\u003c/h2\u003e \u003cp\u003eCells were seeded in 96-well plates ranging from 2,000 to 15,000 cells per well depending on different cell lines and treated with antibiotics in serial dilutions for 72 hours (120 hours for the preliminary assays) in a humidified incubator (37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e) in 100 \u0026micro;l medium (DMEM with 10% FBS, 2.05 mM L-glutamine (Sigma) for Hek293 and Hela; RPMI-1640 with 10% FBS, 2.05 mM L-glutamine (Sigma), and 55 \u0026micro;M 2-ME (Gibco) for Jurkat T cells and PBMCs). Based on preliminary tests, the number of cells per well was optimized to 2,000 for Hela and Hek293, 6,000 for Jurkat T cells, and 15,000 for PBMCs. The stocks of antibiotics were prepared by dissolving in DMSO. As control, the same amount of DMSO was added to the non-treated groups. Serial drug dilutions were prepared in the corresponding medium to provide a total of 9 concentrations up to 300 \u0026micro;M for tetracyclines and up to 900 \u0026micro;M for additional bacterial protein synthesis inhibitors. After drug treatment, 10% CCK-8 (Cell Counting Kit-8, Sigma Aldrich) (10 \u0026micro;l per well) of the total volume was added to the cells and incubated for 1\u0026ndash;4 hours depending on cell lines (1 hour for Hela and Hek293; 3 hours for Jurkat T cells; 4 hours for PBMCs) before the evaluation of the effect of antibiotics on cell viability. The number of viable cells was calculated by measuring the absorbance at 450 nm using a microplate reader and normalized to the non-treated control. Results were visually confirmed under the light microscope. Each treatment was performed in technical repeats. Dose-response curves were plotted, mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM was shown, and half maximal inhibitory concentration (IC50) values were calculated in GraphPad Prism.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eDe novo mitochondrial translation assay\u003c/h2\u003e \u003cp\u003eJurkat T cells were seeded in 6-well plates at a density of 1\u0026nbsp;million cells/ml and cultured with different concentrations of antibiotics for 18 h in RPMI-1640 medium (with 10% FBS, 2.05 mM L-glutamine (Sigma), and 55 \u0026micro;M 2-ME (Gibco)). After centrifugation, cells were incubated twice for 5 min at 37\u0026deg;C in Cys-/Met-free medium (DMEM, high glucose, no glutamine, no methionine, no cysteine, supplemented with 10% dialyzed fetal bovine serum, 1\u0026times; GlutaMax, and sodium pyruvate), followed by 20 min incubation at 37\u0026deg;C in Cys-/Met-free medium supplemented with 100 \u0026micro;g/ml emetine to inhibit cytosolic translation (only for mitochondrial translation groups). Subsequently, 1 ml Cys-/Met-free medium supplemented with 200 (for cytosolic translation) or 400\u0026micro;Ci (for mitochondrial translation) of EasyTag EXPRESS [\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003eS] protein labeling mix (methionine and cysteine) (Perkin Elmer) were added to each sample and incubated for 20 min at 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e. Following labeling, cells were washed three times with 1 ml PBS, harvested by centrifugation (400 \u0026times; \u003cem\u003eg\u003c/em\u003e, 10min, 4\u0026deg;C), and stored at \u0026minus;\u0026thinsp;20\u0026deg;C. Cells were lysed by resuspension in 30 \u0026micro;l PBS supplemented with a Complete EDTA-free protease inhibitor cocktail and 50 U Benzonase nuclease (Sigma) and by application of one freeze-thaw cycle. Protein contents were determined by Pierce BCA assay (Thermo Fisher). 1 \u0026times; NuPAGE LDS sample buffer (Thermo Fisher) was added to 30 \u0026micro;g lysate for mitochondrial translation and 10 \u0026micro;g lysate for cytosolic translation, respectively, and separated on NuPAGE 12% Bis-Tris gels (Thermo Fisher). Coomassie staining was performed using Imperial Protein Stain (Thermo Fisher) according to manufacturer suggestions. The gel was fixed in fixing solution (20% methanol, 7% acetic acid, 3% glycerol) for 1 h at RT and vacuum-dried at 65\u0026deg;C for 2 h. The resultant gel was exposed to storage Phosphor screens (Fujifilm) and visualized with Typhoon FLA 7000 Phosphorimager (GE Healthcare).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting for OXPHOS complexes\u003c/h2\u003e \u003cp\u003eHuman PBMCs and Jurkat T cells were seeded in 6-well plates at a density of 1\u0026nbsp;million cells/ml and incubated with or without antibiotics in RPMI-1640 medium (with 10% FBS, 2.05 mM L-glutamine (Sigma), and 55 \u0026micro;M 2-ME (Gibco)) for 6 days. After centrifugation, cells were lysed in RIPA buffer containing protease and phosphatase inhibitors (cOmplete and PhosSTOP, respectively, Roche). Protein concentrations were determined by BCA (Pierce, Thermo Fisher). Protein samples were resuspended with 1\u0026times; NuPAGE LDS sample buffer (Thermo Fisher) supplemented with 100 mM dithiothreitol, heated for 10 min at 75\u0026deg;C, and separated on NuPAGE 4\u0026ndash;12% Bis-Tris mini gels (Thermo Fisher) using NuPAGE 1\u0026times; MES (Thermo Fisher) running buffer, and transferred to PVDF membranes (0.45 \u0026micro;m, Immobilon-P, Sigma) using the iBlot2 system (Invitrogen). Non-specific binding was blocked with TBST containing 5% non-fat milk, followed by overnight incubation at 4\u0026deg;C with antibodies against Total OXPHOS Human WB Antibody Cocktail (Abcam, Cambridge, UK), followed by HRP-conjugated secondary antibodies for 1 h at room temperature. β-actin, HSP60, and GAPDH (Cell Signaling Technology) were used as loading control. Proteins were detected using Clarity Western ECL Substrate (Bio-Rad, 170\u0026ndash;5061).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eExtracellular Metabolic Flux Assay\u003c/h2\u003e \u003cp\u003eThe Seahorse XFe96 Analyzer (Seahorse Bioscience) was used to measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in human PBMCs. 1.5x10\u003csup\u003e6\u003c/sup\u003e cells/well were seeded in 24-well plates and stimulated using plate-bound anti-CD3 antibody (2 \u0026micro;g/ml, clone OKT3, BD Biosciences), soluble anti-CD28 antibody (0.5 \u0026micro;g/ml, clone CD28.2, Biolegend) and IL-2 (10ng/ml, Proteintech) in RPMI-1640 (with 10% FBS, 2.05 mM L-glutamine (Sigma), and 55 \u0026micro;M 2-ME (Gibco)). Cells were treated with or without 5 and 10 \u0026micro;M Tigecycline for 6 days with a half medium change after 3 days. After treatment, cells were washed with Seahorse XF RPMI assay medium, and then 200,000 cells per well were seeded (in triplicate) in 40 \u0026micro;l assay medium in XF 96-well cell culture microplate coated with poly-D-lysine (Sigma). The plate was centrifuged at 300 \u003cem\u003eg\u003c/em\u003e for 5 sec with no brake, rotated 180 ̊, and centrifuged again for 5 sec at 300 \u003cem\u003eg\u003c/em\u003e. After centrifugation, 140 \u0026micro;l of assay medium was added per well and the plate was left to stabilize in a 37\u0026deg;C, non-CO\u003csub\u003e2\u003c/sub\u003e incubator for 40 min. During seahorse, wells were sequentially injected with compounds to achieve final concentrations of 1,264 \u0026micro;M oligomycin (Sigma); 2\u0026micro;M FCCP (Sigma); 0.5 \u0026micro;M rotenone (Sigma) together with 0.5 \u0026micro;M antimycin A (Sigma). OCR and ECAR were measured for each well three times, every three minutes, before and after each injection. OCR was normalized to protein concentration using a BCA Protein Assay kit (ThermoFisher Scientific) conducted according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eIn vitro stimulation of human PBMCs\u003c/h2\u003e \u003cp\u003eAfter thawing frozen PBMCs, cells were washed with RPMI complemented with 10% FBS, 2.05 mM L-glutamine (Sigma), and 55 \u0026micro;M 2-ME (Gibco) and rested at 37\u003csup\u003eo\u003c/sup\u003eC (5% CO\u003csub\u003e2\u003c/sub\u003e) for 30 min. 250,000 cells per well were seeded in 96-well U-bottom plates with/without Human T-Activator CD3/CD28 beads (Invitrogen) at a 1:1 ratio and 10 ng/ml IL-2 (Proteintech) in RPMI-1640 medium mentioned above. At the same time, antibiotics were added. For the short-term activation assay, cells were harvested 18 hours post-stimulation and stained with Aqua fixable live/dead dye (Invitrogen) and surface markers. For the proliferation assays, cells were stained with CFSE (eBioscience) or Cell Trace Violet (ThermoFisher Scientific) dyes before seeding and stimulation, according to the manufacturer\u0026rsquo;s recommendations. After incubation for 6 days with a half medium (complete, containing antibiotics and IL-2) change after 3 days, cells were harvested via centrifugation, stained with live/dead dye and surface markers for analysis by flow cytometry. The Foxp3/Transcription Factor Staining Buffer Set (Invitrogen) was used to fix cells according to the manufacturer\u0026rsquo;s instructions. Flow cytometry was carried out on a BD Celesta (BD Biosciences) instrument and analyzed in FlowJo (TreeStar) software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eHuman CD4\u003csup\u003e+\u003c/sup\u003e T cell sorting and stimulation\u003c/h2\u003e \u003cp\u003eThawed PBMCs were washed with RPMI complemented with 10% FBS, 2.05 mM L-glutamine (Sigma), and 55 \u0026micro;M 2-ME (Gibco) before resting at 37\u003csup\u003eo\u003c/sup\u003eC (5% CO\u003csub\u003e2\u003c/sub\u003e) for 30 min. After counting, cells were resuspended in FACS buffer at a density of 5 x 10\u003csup\u003e7\u003c/sup\u003e cells/ml. CD4\u003csup\u003e+\u003c/sup\u003e T cells were isolated using CD4\u003csup\u003e+\u003c/sup\u003e T cell negative isolation kits (Miltenyi Biotech) and enriched using CD4\u003csup\u003e+\u003c/sup\u003e T cell Enrichment kits (Miltenyi Biotech). Purified CD4\u003csup\u003e+\u003c/sup\u003e T cells were counted and stained with CTV dye and live/dead dye. Subsequently, surface makers were stained at 4\u003csup\u003eo\u003c/sup\u003eC in FACS buffer (DPBS\u0026thinsp;+\u0026thinsp;2% FBS). The following panels were used to classify human T CD4\u003csup\u003e+\u003c/sup\u003e cells from the live lymphocyte gate: CD4\u003csup\u003e+\u003c/sup\u003e T na\u0026iuml;ve: Aqua\u003csup\u003e\u0026minus;\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003eCD45RA\u003csup\u003e+\u003c/sup\u003e CD27\u003csup\u003e+\u003c/sup\u003e; CD4\u003csup\u003e+\u003c/sup\u003e T memory: Aqua\u003csup\u003e\u0026minus;\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003eCD45RA\u003csup\u003e\u0026minus;\u003c/sup\u003eCD27\u003csup\u003e+\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAfter staining, cells were washed and resuspended and sorted using a FACSAria Fusion (BD Biosciences). Sorted cells were collected in PBS complemented with 2% FBS at 4\u003csup\u003eo\u003c/sup\u003eC. Cells were then harvested via centrifugation and resuspended in RPMI-1640 (+\u0026thinsp;10% FBS, 2.05 mM L-glutamine (Sigma), and 55 \u0026micro;M 2-ME (Gibco)) before counting. Sorted CD4\u003csup\u003e+\u003c/sup\u003e subsets were counted and 85,000 cells per well seeded onto 96-well U-bottom plates with/without Human T-Activator CD3/CD28 beads (Invitrogen) at a 1:2.8 cell:bead ratio and 10ng/ml IL-2 (Proteintech) in RPMI-1640 (+\u0026thinsp;10% FBS, 2.05 mM L-glutamine (Sigma), and 55 \u0026micro;M 2-ME (Gibco)). At the same time, tigecycline in serial concentrations (from 2.5 to 10 \u0026micro;M) was added. With a half medium change after 3 days, cells were harvested after 6 days, and stained and fixed before analysis by flow cytometry. Antibodies used in the study are presented in Table\u0026nbsp;2.\u003c/p\u003e \u003cp\u003eTable 2\u003c/p\u003e\u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003eAntibodies\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal OXPHOS Human WB Antibody Cocktail\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAbcam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAb110411\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHSP60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEnzo Lifesciences\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAB1-SPA-807-E\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eb-actin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAbcam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAb8224\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGAPDH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAbcam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAb8245\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHRP secondary rabbit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGE Healthcare\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNA9340V\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHRP secondary mouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGE Healthcare\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNA9310V\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHuman CD3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBD Biosciences\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eClone 3D12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHuman CD4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBD Biosciences\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eClone RPA-T4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHuman CD8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBD Biosciences\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eClone RPA-T8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHuman CD45RA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBD Biosciences\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eClone HI100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHuman CD27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBD Biosciences\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eClone M-T271\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHuman CD25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBD Biosciences\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eClone 2A3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eStatistical analyses were carried out in Prism 9 (GraphPad). Differences between groups were analyzed by a Student's t-test or one-way ANOVA with Tukey's multiple comparisons test.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eIsolation of mitochondria and purification of mitochondrial monosomes\u003c/h2\u003e \u003cp\u003eJurkat T cells were grown in RPM1-1640 medium (+\u0026thinsp;10% FBS, 2.05 mM L-glutamine (Sigma), and 55 \u0026micro;M 2-ME (Gibco)) in a vented flask shaking at 120 rpm at 37\u0026deg;C under 5% CO\u003csub\u003e2\u003c/sub\u003e. The culture was scaled up by splitting at a cell density of 1.6 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/ml. A final volume of 1.5-liter cells at a density of 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/ml was harvested by centrifugation at 1000 x g for 10 min at 4\u0026deg;C. After being washed with cold PBS buffer, the cell pellet was resuspended in the cold hypotonic MSE buffer (with 0.6 M mannitol, 10 mM Tris\u0026ndash;HCl pH 7.4, 1 mM EDTA, 0.1% BSA), and ruptured on ice by a semi-automatic homogenizer (Schuett-biotech). The lysate was clarified by centrifugation at 400 \u0026times; g and 4\u0026deg;C for 10 min. The pellet was resuspended and subsequently homogenized. After 3 cycles of homogenization-centrifugation, the cell lysates were combined and the mitochondria were pelleted by additional centrifugation at 11,000 \u0026times; g and 4\u0026deg;C for 10 min. The crude mitochondria were loaded onto the sucrose cushion (1.0 M and 1.5 M sucrose in, 20 mM Tris\u0026ndash;HCl pH 7.4, 1 mM EDTA) and centrifuged for 1 hr at 77,000 \u0026times; g (25,000 rpm) in a SW41 Ti rotor (Beckman Coulter). The band formed by the mitochondria in the middle between 1 and 1.5 M sucrose was collected carefully and resuspended in 10 mM Tris\u0026ndash;HCl pH 7.4 in a 1:1 ratio, The pure mitochondrial pellet was collected after centrifugation at 11,000 \u0026times; g and 4\u0026deg;C for 15 min and then resuspended in mitochondrial freezing buffer (300 mM trehalose, 10 mM Tris\u0026ndash;HCl pH 7.4, 10 mM KCl, 0.1% BSA, 1 mM EDTA), flash-frozen in liquid N2 and stored at \u0026minus;\u0026thinsp;80\u0026deg;C.\u003c/p\u003e \u003cp\u003eThe purified mitochondria were thawed and lysed by incubating at 4\u0026deg;C for 30 min in the lysis buffer (25 mM HEPES\u0026ndash;KOH pH 7.5, 20 mM Mg(OAc)2, 50 mM KCl, 2% (vol/vol) Triton X-100, 2 mM Dithiothreitol (DTT), 1\u0026times; cOmplete EDTA-free protease inhibitor cocktail (Roche), 40 U/\u0026micro;l RNase inhibitor (Invitrogen)). The mitochondrial lysate was centrifuged at 19,000 \u0026times; g (13000 rpm) for 12 min at 4\u0026deg;C, and subsequently overlayed on top of a 10\u0026ndash;30% sucrose gradient in the ribosome buffer (25 mM HEPES/KOH pH 7.5, 50 mM KCl, 20 mM Mg(OAc)2, 2 mM DTT). After centrifugation for 21 hr at 54,331 \u0026times; g (21,000 rpm) in a SW41 Ti rotor (Beckman Coulter), the gradients were fractionated with a Biocomp Fractionator. Fractions corresponding to the monosomes were pooled and pelleted at 135,520 \u0026times; g (55,000 rpm) for 16 hr at 4\u0026deg;C using a TLA55 rotor (Beckman Coulter). The pellet was gently washed 3 times and dissolved in ribosome buffer. The solution was kept on ice for 15 min and the soluble mitoribosomes were collected by centrifugation at 20,000 x g for 15 hr at 4\u0026deg;C to get rid of aggregation. The concentration of the purified monosomes was quantified by nanodrop.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eCryoEM data collection and analysis\u003c/h2\u003e \u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e reconstitution of the mitoribosomal monosome-tigecycline complex was performed using 30 \u0026micro;M of tigecycline incubated with 100 nM of monosome in a ratio of 1:300. Holey carbon grids (Quantifoil R2/2, copper, 300 mesh) coated with a layer of continuous carbon (~\u0026thinsp;3 nm thickness) were subjected to a glow discharge of 25 mA for 120 s. The sample was applied to the grids at 4\u0026deg;C with 100% humidity and incubated for 30 s using a Vitrobot MKIV (Thermo Fisher Scientific), followed by 3 s blotting with blot force 3 and plunge-freezing in liquified ethane. The dataset was acquired on Titan Krios G3i transmission electron microscope (Thermo Fisher Scientific) operated at 300 kV in the Karolinska Institutet\u0026rsquo;s 3D-EM facility using a slit width of 20 eV with GIF quantum energy filter (Gatan). For imaging, a K3 detector (Gatan) was used capturing micrographs at magnification of 165kX yielding a pixel size of 0.505 \u0026Aring;. A dose of 45 electrons per \u0026Aring;\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e in 50 frames was used with defocus values ranging from \u0026minus;\u0026thinsp;0.4 to \u0026minus;\u0026thinsp;1.6 \u0026micro;m. Motion correction followed by CTF estimation, Fourier cropping (to 1.01 \u0026Aring;/px), particle picking, and extraction in 512-pixel boxes (size threshold 300 \u0026Aring;, distance threshold 20 \u0026Aring;, using the pre-trained BoxNet2Mask_20180918 model) were performed on the fly using Warp\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Only particles from micrographs with an estimated resolution of 5 \u0026Aring; were retained for further processing. Detailed parameters are given in the Supplementary Table\u0026nbsp;1.\u003c/p\u003e \u003cp\u003eA total of 12,972 micrographs from 2 datasets were selected based on an estimated resolution cut-off of 5 \u0026Aring; and defocus below 2 \u0026micro;m as estimated by Warp. A total of 519,237 picked particles from Warp were imported to CryoSPARC (v4.2.1)\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e to perform further processing. 2D classification was carried out followed by \u003cem\u003eab initio\u003c/em\u003e reconstructions of clean or good classes with high-resolution features and junk classes. These ab initio reconstructions were used for the heterogeneous refinements of all picked particles. After several round of heterogeneous refinements, 266,550 clean particles comprised the mitoribosomal monosome with high-resolution features were retained and used for further processing. Homogenous refinement of these clean particle stacks was performed which yielded a resolution of 2.4 \u0026Aring;. A recent model of human mitoribosomal monosome (PDB:7QI4\u003csup\u003e32\u003c/sup\u003e) purified from the Hek293 T cells bound to A-tRNA, P-tRNA, and mRNA was fitted in our reconstruction of the monosome, which also comprises these features with additional densities in both mtSSU and mtLSU that could be attributed to the antibiotic tigecycline. Mask targeting the P-tRNA was generated to perform 3D variability analysis, and subsequently classified into three particle clusters with each representing different features of the monosome. Particles with clear density for the P-tRNA were subjected to a second round of 3D variability analysis using the A-tRNA as a mask. We could eventually obtain 3 major classes, class 1 with the majority of the particles (\u0026lsquo;Empty\u0026rsquo;: 130,443 particles) lacked densities for the A-site and P-site tRNA on the monosome, class 2 (\u0026lsquo;P-tRNA only\u0026rsquo;: 87,076 particles) lacked the occupancy for the A-tRNA and is represented by the monosome bound to P-tRNA only and mRNA, and class 3 (\u0026lsquo;A- and P-tRNA\u0026rsquo;: 36,974 particles) contained monosome bound A-tRNA, P-tRNA, and mRNA. Each class/particle set was subjected to a homogenous refinement which yielded a corresponding reconstruction at 2.5 \u0026Aring;, 2.7 \u0026Aring;, and 2.8 \u0026Aring; resolution, respectively. To improve the local resolution of the tigecycline-bound regions on the monosome, 3D refined classes were subjected to CTF refinement (global and local refinement). Furthermore, two masks covering the region of the tigecycline-binding sites on the mtSSU (SSU-head) and mtLSU (LSU-body) were prepared (Supplementary Fig.\u0026nbsp;2), and local-masked 3D refinement was performed. Reported resolutions are based on gold-standard, applying the 0.143 criterion on the FSC between the reconstructed half-maps (Supplementary Fig.\u0026nbsp;2). Maps underwent local-resolution filtering, superposed to the consensus map, and combined via Phenix\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e for model building and refinement.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eModel building and refinement\u003c/h2\u003e \u003cp\u003eModel building of the tigecycline-bound monosome was carried out using \u003cem\u003eCoot\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. The starting model for the monosome was Protein Data Bank (PDB) ID 7QI4\u003csup\u003e32\u003c/sup\u003e. This model was fitted as a rigid body into the map of the \u0026lsquo;P-tRNA only\u0026rsquo; monosome class, and further adjustments were made manually. Three active sites for tigecycline were identified on the monosome, which agreed with the density: one at the mtSSU and two in the mtLSU. Water molecules were picked by \u003cem\u003eCoot\u003c/em\u003e automatically around the tigecycline-binding region, and adjusted manually. Metal ions, cofactors (2Fe-S), and modifications were placed based on map densities. Geometrical restraints of modified residues and ligands were calculated by Grade Web Server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://grade.globalphasing.org\u003c/span\u003e\u003cspan address=\"http://grade.globalphasing.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) or obtained from the CCP4 library\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Hydrogens were added to all molecules except water by REFMAC5\u003csup\u003e56\u003c/sup\u003e. The final model was refined using the composite map via Phenix.real_space_refine v1.18\u003csup\u003e53\u003c/sup\u003e. The refined model was validated with MolProbity\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e and the Phenix suite\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Model statistics are listed in Table\u0026nbsp;1. UCSF ChimeraX 1.6.1\u003csup\u003e59\u003c/sup\u003ewas used to make the figures.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflict of interest\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThe work was funded by Max Planck Institute-Karolinska Institute, the Knut \u0026amp; Alice Wallenberg Foundation (WAF2017, KAW 2018.0080, to JR), Swedish Research Council (VR2022-02179, to JR), and EMBO (STF 7213, to XCD; LTF 2020\u0026thinsp;\u0026minus;\u0026thinsp;606, to MDN).\u003c/p\u003e\u003ch2\u003eData availability.\u003c/h2\u003e \u003cp\u003eCryo-EM maps have been deposited at the Electron Microscopy Data Bank as follows: Class 1 (empty class), EMD-19544 (consensus map), EMD-19545 (SSU-head), EMD-19546 (LSU-body); Class 2 (P-site tRNA), EMD-19493 (consensus map), EMD-19490 (SSU-head), EMD-19491 (LSU-body), EMD-19460 (composite); Class 3 (A- and P-site tRNA), EMD-19526 (consensus map), EMD-19539 (SSU-head), EMD-19542 (LSU-body), EMD-19460 (composite map). Associated molecular model has been deposited at the PDB: 8RRI (tigecycline bound human mitoribosome containing P-site tRNA and mRNA). The map and the model are available at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://figshare.com/\u003c/span\u003e\u003cspan address=\"https://figshare.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; Login: [email protected]; Password: AntibioticTig12##.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eChukwudi CU (2016) rRNA binding sites and the molecular mechanism of action of the tetracyclines. Antimicrobial Agents and Chemotherapy vol. 60 4433\u0026ndash;4441 Preprint at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/AAC.00594-16\u003c/span\u003e\u003cspan address=\"10.1128/AAC.00594-16\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNagiec EE et al (2005) Oxazolidinones inhibit cellular proliferation via inhibition of mitochondrial protein synthesis. Antimicrob Agents Chemother 49:3896\u0026ndash;3902\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcKee EE, Ferguson M, Bentley AT, Marks TA (2006) Inhibition of mammalian mitochondrial protein synthesis by oxazolidinones. Antimicrob Agents Chemother 50:2042\u0026ndash;2049\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eItoh Y et al (2022) Structure of the mitoribosomal small subunit with streptomycin reveals Fe-S clusters and physiological molecules. Elife 11\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSighel D et al (2021) Inhibition of mitochondrial translation suppresses glioblastoma stem cell growth. Cell Rep 35:109024\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eScharping NE et al (2016) The Tumor Microenvironment Represses T Cell Mitochondrial Biogenesis to Drive Intratumoral T Cell Metabolic Insufficiency and Dysfunction. Immunity 45:374\u0026ndash;388\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eO\u0026rsquo;sullivan D et al Fever supports CD8\u0026thinsp;+\u0026thinsp;effector T cell responses by promoting mitochondrial translation. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1073/pnas.2023752118/-/DCSupplemental\u003c/span\u003e\u003cspan address=\"10.1073/pnas.2023752118/-/DCSupplemental\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTan H et al (2017) Integrative Proteomics and Phosphoproteomics Profiling Reveals Dynamic Signaling Networks and Bioenergetics Pathways Underlying T Cell Activation. Immunity 46:488\u0026ndash;503\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWest AP et al (2015) Mitochondrial DNA stress primes the antiviral innate immune response. Nature 520:553\u0026ndash;557\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCola\u0026ccedil;o HG et al (2021) Tetracycline Antibiotics Induce Host-Dependent Disease Tolerance to Infection. Immunity 54:53\u0026ndash;67e7\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePatel CH, Leone RD, Horton MR, Powell JD (2019) Targeting metabolism to regulate immune responses in autoimmunity and cancer. Nature Reviews Drug Discovery vol. 18 669\u0026ndash;688 Preprint at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41573-019-0032-5\u003c/span\u003e\u003cspan address=\"10.1038/s41573-019-0032-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlmeida L et al (2021) Ribosome-Targeting Antibiotics Impair T Cell Effector Function and Ameliorate Autoimmunity by Blocking Mitochondrial Protein Synthesis. Immunity 54:68\u0026ndash;83e6\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFranz T et al (2022) Pleiotropic effects of antibiotics on T cell metabolism and T cell-mediated immunity. Frontiers in Microbiology vol. 13 Preprint at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fmicb.2022.975436\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2022.975436\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKroon AM, Taanman JW (2015) Clonal expansion of T cells in abdominal aortic aneurysm: A role for doxycycline as drug of choice? International Journal of Molecular Sciences vol. 16 11178\u0026ndash;11195 Preprint at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ijms160511178\u003c/span\u003e\u003cspan address=\"10.3390/ijms160511178\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoullan N et al (2015) Tetracyclines disturb mitochondrial function across eukaryotic models: A call for caution in biomedical research. Cell Rep 10:1681\u0026ndash;1691\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKloppenburg M et al (1995) The influence of tetracyclines on T cell activation. Clin Exp Immunol 102:635\u0026ndash;641\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMottis A et al (2022) Tetracycline-induced mitohormesis mediates disease tolerance against influenza. J Clin Invest 132\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcKee EE, Ferguson M, Bentley AT, Marks TA (2006) Inhibition of mammalian mitochondrial protein synthesis by oxazolidinones. Antimicrob Agents Chemother 50:2042\u0026ndash;2049\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarnhill AE, Brewer MT, Carlson SA (2012) Adverse effects of antimicrobials via predictable or idiosyncratic inhibition of host mitochondrial components. Antimicrobial Agents and Chemotherapy vol. 56 4046\u0026ndash;4051 Preprint at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/AAC.00678-12\u003c/span\u003e\u003cspan address=\"10.1128/AAC.00678-12\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoullan N et al (2015) Tetracyclines disturb mitochondrial function across eukaryotic models: A call for caution in biomedical research. Cell Rep 10:1681\u0026ndash;1691\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePerry EA et al (2021) Tetracyclines promote survival and fitness in mitochondrial disease models. Nature Metabolism vol. 3 33\u0026ndash;42 Preprint at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s42255-020-00334-y\u003c/span\u003e\u003cspan address=\"10.1038/s42255-020-00334-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChopra I, Roberts M, Tetracycline Antibiotics (2001) Mode of Action, Applications, Molecular Biology, and Epidemiology of Bacterial Resistance. Microbiol Mol Biol Rev 65:232\u0026ndash;260\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRusu A, Buta EL (2021) The development of third-generation tetracycline antibiotics and new perspectives. Pharmaceutics vol. 13 Preprint at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/pharmaceutics13122085\u003c/span\u003e\u003cspan address=\"10.3390/pharmaceutics13122085\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNicholson D, Edwards TA, O\u0026rsquo;Neill AJ, Ranson NA (2020) Structure of the 70S Ribosome from the Human Pathogen Acinetobacter baumannii in Complex with Clinically Relevant Antibiotics. Structure 28:1087\u0026ndash;1100e3\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJenner L et al (2013) Structural basis for potent inhibitory activity of the antibiotic tigecycline during protein synthesis. Proc Natl Acad Sci U S A 110:3812\u0026ndash;3816\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchedlbauer A et al (2015) Structural characterization of an alternative mode of tigecycline binding to the bacterial ribosome. Antimicrob Agents Chemother 59:2849\u0026ndash;2854\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Z, Morgan CE, Bonomo RA, Yu EW (2021) Cryo-em determination of eravacycline-bound structures of the ribosome and the multidrug efflux pump adej of acinetobacter baumannii. \u003cem\u003emBio\u003c/em\u003e 12\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLomakin IB, Devarkar SC, Patel S, Grada A, Bunick CG (2023) Sarecycline inhibits protein translation in Cutibacterium acnes 70S ribosome using a two-site mechanism. Nucleic Acids Res 51:2915\u0026ndash;2930\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBatool Z, Lomakin IB, Polikanov YS, Bunick CG, Jacobson A Sarecycline interferes with tRNA accommodation and tethers mRNA to the 70S ribosome. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1073/pnas.2008671117/-/DCSupplemental\u003c/span\u003e\u003cspan address=\"10.1073/pnas.2008671117/-/DCSupplemental\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCocozaki AI et al (2016) Resistance mutations generate divergent antibiotic susceptibility profiles against translation inhibitors. Proc Natl Acad Sci U S A 113:8188\u0026ndash;8193\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBowman JC, Petrov AS, Frenkel-Pinter M, Penev PI, Williams LD (2020) Root of the Tree: The Significance, Evolution, and Origins of the Ribosome. Chemical Reviews vol. 120 4848\u0026ndash;4878 Preprint at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acs.chemrev.9b00742\u003c/span\u003e\u003cspan address=\"10.1021/acs.chemrev.9b00742\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSingh V et al Structure of mitoribosome reveals mechanism of mRNA binding, tRNA 1 interactions with L1 stalk, roles of cofactors and rRNA modifications 2 3. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1101/2023.05.24.542018\u003c/span\u003e\u003cspan address=\"10.1101/2023.05.24.542018\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoripella RK et al (2021) Distinct mechanisms of the human mitoribosome recycling and antibiotic resistance. Nat Commun 12\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKummer E, Schubert KN, Schoenhut T, Scaiola A, Ban N (2021) Structural basis of translation termination, rescue, and recycling in mammalian mitochondria. Mol Cell 81:2566\u0026ndash;2582e6\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOsterman IA et al (2020) Tetracenomycin X inhibits translation by binding within the ribosomal exit tunnel. Nat Chem Biol 16:1071\u0026ndash;1077\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArenz S et al (2016) A combined cryo-EM and molecular dynamics approach reveals the mechanism of ErmBL-mediated translation arrest. Nat Commun 7\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeroy EC, Perry TN, Renault TT, Innis CA (2023) Tetracenomycin X sequesters peptidyl-tRNA during translation of QK motifs. Nat Chem Biol 19:1091\u0026ndash;1096\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKummer E et al (2018) Unique features of mammalian mitochondrial translation initiation revealed by cryo-EM. Nature 560:263\u0026ndash;267\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eItoh Y et al (2021) Mechanism of membrane-tethered mitochondrial protein synthesis. Sci (1979) 371:846\u0026ndash;849\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePaternoga H et al (2023) Structural conservation of antibiotic interaction with ribosomes. Nat Struct Mol Biol 30:1380\u0026ndash;1392\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarrientos A, Fontanesi F The Mitoribosome Methods and Protocols Methods in Molecular Biology 2661. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.springer.com/series/7651\u003c/span\u003e\u003cspan address=\"http://www.springer.com/series/7651\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKlein K et al (2020) Role of Mitochondria in Cancer Immune Evasion and Potential Therapeutic Approaches. Frontiers in Immunology vol. 11 Preprint at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fimmu.2020.573326\u003c/span\u003e\u003cspan address=\"10.3389/fimmu.2020.573326\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eŠkrtić M et al (2011) Inhibition of Mitochondrial Translation as a Therapeutic Strategy for Human Acute Myeloid Leukemia. Cancer Cell 20:674\u0026ndash;688\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu B, Guo Y (2019) Inhibition of mitochondrial translation as a therapeutic strategy for human ovarian cancer to overcome chemoresistance. Biochem Biophys Res Commun 509:373\u0026ndash;378\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang B et al (2017) Inhibition of mitochondrial translation effectively sensitizes renal cell carcinoma to chemotherapy. Biochem Biophys Res Commun 490:767\u0026ndash;773\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKuntz EM et al (2017) Targeting mitochondrial oxidative phosphorylation eradicates therapy-resistant chronic myeloid leukemia stem cells. Nat Med 23:1234\u0026ndash;1240\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeagher AK, Ambrose PG, Grasela TH, Ellis-Grosse EJ (2005) The Pharmacokinetic and Pharmacodynamic Profile of Tigecycline. Clin Infect Dis 41 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://academic.oup.com/cid/article/41/Supplement_5/S333/289231\u003c/span\u003e\u003cspan address=\"https://academic.oup.com/cid/article/41/Supplement_5/S333/289231\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStein GE et al (2011) Tigecycline penetration into skin and soft tissue. Surg Infect (Larchmt) 12:465\u0026ndash;467\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi X et al (2024) Structural basis for differential inhibition of eukaryotic ribosomes by tigecycline. Nat Commun 15:5481\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang J et al (2023) Antigen receptor stimulation induces purifying selection against pathogenic mitochondrial tRNA mutations. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1172/jci\u003c/span\u003e\u003cspan address=\"10.1172/jci\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTegunov D, Cramer P (2020) Real-time cryo-EM data pre-processing with Warp Europe PMC Funders Group. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.6019/EMPIAR-10078\u003c/span\u003e\u003cspan address=\"10.6019/EMPIAR-10078\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePunjani A, Rubinstein JL, Fleet DJ, Brubaker MA (2017) CryoSPARC: Algorithms for rapid unsupervised cryo-EM structure determination. Nat Methods 14:290\u0026ndash;296\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiebschner D et al (2019) Macromolecular structure determination using X-rays, neutrons and electrons: Recent developments in Phenix. Acta Crystallogr D Struct Biol 75:861\u0026ndash;877\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEmsley P, Lohkamp B, Scott WG, Cowtan K (2010) Features and development of Coot. Acta Crystallogr D Biol Crystallogr 66:486\u0026ndash;501\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLebedev AA et al (2012) JLigand: A graphical tool for the CCP4 template-restraint library. Acta Crystallogr D Biol Crystallogr 68:431\u0026ndash;440\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMurshudov GN et al (2011) REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr D Biol Crystallogr 67:355\u0026ndash;367\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilliams CJ et al (2018) MolProbity: More and better reference data for improved all-atom structure validation. Protein Sci 27:293\u0026ndash;315\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen VB et al (2010) MolProbity: All-atom structure validation for macromolecular crystallography. Acta Crystallogr D Biol Crystallogr 66:12\u0026ndash;21\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePettersen EF et al (2021) UCSF ChimeraX: Structure visualization for researchers, educators, and developers. Protein Sci 30:70\u0026ndash;82\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"antibiotics, immunometabolism, mitochondrial ribosomes, tetracyclines, T cells","lastPublishedDoi":"10.21203/rs.3.rs-4671643/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4671643/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTetracyclines 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 \u003cem\u003ein vitro\u003c/em\u003e, 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.\u003c/p\u003e","manuscriptTitle":"Structural Basis of T Cell Toxicity Induced by Tigecycline Binding to the Mitochondrial Ribosome","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-17 15:40:30","doi":"10.21203/rs.3.rs-4671643/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0652b97a-b4b2-42ec-aa6c-9726d75ed379","owner":[],"postedDate":"July 17th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":34747822,"name":"Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy"},{"id":34747823,"name":"Biological sciences/Immunology/Lymphocytes"}],"tags":[],"updatedAt":"2025-05-02T07:06:35+00:00","versionOfRecord":{"articleIdentity":"rs-4671643","link":"https://doi.org/10.1038/s41467-025-59388-9","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-05-01 04:00:00","publishedOnDateReadable":"May 1st, 2025"},"versionCreatedAt":"2024-07-17 15:40:30","video":"","vorDoi":"10.1038/s41467-025-59388-9","vorDoiUrl":"https://doi.org/10.1038/s41467-025-59388-9","workflowStages":[]},"version":"v1","identity":"rs-4671643","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4671643","identity":"rs-4671643","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}