bioRxiv : the preprint server for biology最新文献

{"title":"Domain Coupling in Allosteric Regulation of SthK Measured Using Time-Resolved Transition Metal Ion FRET.","authors":"Pierce Eggan, Sharona E Gordon, William N Zagotta","doi":"10.1101/2025.03.31.646362","DOIUrl":"10.1101/2025.03.31.646362","url":null,"abstract":"<p><p>Cyclic nucleotide-binding domain (CNBD) ion channels are vital for cellular signaling and excitability, with activation regulated by cyclic adenosine-or guanosine-monophosphate (cAMP, cGMP) binding. However, the allosteric mechanisms underlying this activation, particularly the energetics that describe conformational changes within individual domains and between domains, remain unclear. The prokaryotic CNBD channel SthK has been a useful model for better understanding these allosteric mechanisms. Here, we applied time-resolved transition metal ion Förster resonance energy transfer (tmFRET) to investigate the conformational dynamics and energetics in the CNBD of SthK in both a soluble C-terminal fragment of the protein, SthK _Cterm , and in the full-length channel, SthK _Full . We incorporated the noncanonical amino acid Acd as a FRET donor and a metal bound to a chelator conjugated to a cysteine as an acceptor. We used time correlated single photon counting (TCSPC) to measure time-resolved FRET and fit the TCSPC data to obtain donor-acceptor distance distributions in the absence and presence of cAMP. The distance distributions allowed us to quantify the energetics of coupling between the C-terminal domains and the transmembrane domains by comparing the donor-acceptor distance distributions for SthK _Cterm and SthK _Full . Our data indicate that the presence of the SthK transmembrane domains makes the activating conformational change in the CNBD more favorable. These findings highlight the power of time-resolved tmFRET to uncover the structural and energetic landscapes of allosteric proteins and of the ligand-mediated mechanism in CNBD channels specifically.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11996379/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144060355","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Two Axes of White Matter Development.","authors":"Audrey C Luo, Steven L Meisler, Valerie J Sydnor, Aaron Alexander-Bloch, Joelle Bagautdinova, Deanna M Barch, Danielle S Bassett, Christos Davatzikos, Alexandre R Franco, Jeff Goldsmith, Raquel E Gur, Ruben C Gur, Fengling Hu, Marc Jaskir, Gregory Kiar, Arielle S Keller, Bart Larsen, Allyson P Mackey, Michael P Milham, David R Roalf, Golia Shafiei, Russell T Shinohara, Leah H Somerville, Sarah M Weinstein, Jason D Yeatman, Matthew Cieslak, Ariel Rokem, Theodore D Satterthwaite","doi":"10.1101/2025.03.19.644049","DOIUrl":"10.1101/2025.03.19.644049","url":null,"abstract":"<p><p>Despite decades of neuroimaging research, how white matter develops along the length of major tracts in humans remains unknown. Here, we identify fundamental patterns of white matter maturation by examining developmental variation along major, long-range cortico-cortical tracts in youth ages 5-23 years using diffusion MRI from three large-scale, cross-sectional datasets (total N = 2,716). Across datasets, we delineate two replicable axes of human white matter development. First, we find a deep-to-superficial axis, in which superficial tract regions near the cortical surface exhibit greater age-related change than deep tract regions. Second, we demonstrate that the development of superficial tract regions aligns with the cortical hierarchy defined by the sensorimotor-association axis, with tract ends adjacent to sensorimotor cortices maturing earlier than those adjacent to association cortices. These results reveal developmental variation along tracts that conventional tract-average analyses have previously obscured, challenging the implicit assumption that white matter tracts mature uniformly along their length. Such developmental variation along tracts may have functional implications, including mitigating ephaptic coupling in densely packed deep tract regions and tuning neural synchrony through hierarchical development in superficial tract regions - ultimately refining neural transmission in youth.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11957034/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143757620","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Neuro-Metabolic and Vascular Dysfunction as an Early Diagnostic for Alzheimer's Disease and Related Dementias.","authors":"Juan Antonio K Chong Chie, Scott A Persohn, Ravi S Pandey, Olivia R Simcox, Paul Salama, Paul R Territo","doi":"10.1101/2025.05.12.653558","DOIUrl":"https://doi.org/10.1101/2025.05.12.653558","url":null,"abstract":"<p><p>Alzheimer's disease (AD) is the most prevalent neurodegenerative condition characterized by significant cognitive decline. Recent studies suggest that the brain undergoes anatomical and functional restructuring, resulting in neuro-metabolic and vascular dysregulation (MVD) prior to amyloid-β accumulation, which begins at an early age and leads to the onset of AD. Using a retrospective clinical population (N=403) of subjects with varying disease stages from the Alzheimer's Disease Neuroimaging Initiative (ADNI), we identified that disease progression follows a stage-dependent MVD pattern, facilitating the identification of at-risk and resilient brain regions. Although each region progresses at a different pace, regions associated with memory, cognitive tasks, and motor function showed significant early dysregulation. These changes aligned with transcriptomics and cognitive signatures. This study underscores that MVD in brain regions varies by sex and disease stage, making it a sensitive tool for early AD diagnosis. Furthermore, this approach could improve patient monitoring, stratification, and therapeutic testing.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12132444/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144218367","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"No facilitatory effects of transcranial random noise stimulation on motion processing: A registered report.","authors":"Grace Edwards, Ryan Ruhde, Mica B Carroll, Chris I Baker","doi":"10.1101/2025.03.18.643903","DOIUrl":"https://doi.org/10.1101/2025.03.18.643903","url":null,"abstract":"<p><p>Non-invasive brain stimulation (NIBS) techniques have the potential to demonstrate the causal impact of targeted brain regions on specific behaviors, and to regulate or facilitate behavior in clinical applications. Transcranial random noise stimulation (tRNS) is one form of transcranial electric stimulation (tES) in which an alternating current is passed between electrodes at random frequencies. High-frequency tRNS (hf-tRNS) is thought to enhance excitability and has been reported to have facilitatory effects on behavior in healthy and clinical populations. Due to the potential application of tRNS, clear demonstrations of the efficacy and replicability of stimulation are critical. Here, we focused on replicating the facilitatory effect of hf-tRNS over the human middle temporal complex (hMT+) on contralateral motion processing, initially demonstrated by Ghin et al. (2018). In this prior study, the improvement in performance was specific to global motion processing in the visual field contralateral to stimulation. The motivation to replicate this effect was reinforced by the well-supported hypothesis that hMT+ is critical for contralateral global motion processing. However, our results indicated that hf-tRNS does not improve motion discrimination. Specifically, we were unable to replicate a contralateral global motion processing facilitation following hf-tRNS to hMT+. In our within-subject controls, we also found no difference between hf-tRNS to hMT+ on contralateral global motion processing in comparison to sham stimulation, or in comparison to hf-tRNS to the forehead. While it remains possible that our lack of replication could be due to minor changes in the protocol from the original Ghin et al., study, for hf-tRNS to become a widely applied method, the modulatory effect of hf-tRNS should be robust to slight adjustments to the procedure.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12132535/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144218383","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Rapid Dopaminergic Signatures in Movement: Reach Vigor Reflects Reward Prediction Error and Learned Expectation.","authors":"Colin C Korbisch, Alaa A Ahmed","doi":"10.1101/2025.03.24.645035","DOIUrl":"10.1101/2025.03.24.645035","url":null,"abstract":"<p><p>Movement vigor across multiple modalities increases with reward, suggesting that the neural circuits that represent value influence the control of movement. Dopaminergic neuron (DAN) activity has been suggested as the potential mediator of this response. If DAN activity is the bridge between value and vigor, then vigor should track canonical mediators of DAN activity, namely learning signals in the form of reward expectation and reward prediction error. Here we ask if a similar time-locked response is present in vigor of reaching movements. We explore this link by leveraging the known phasic dopaminergic response to stochastic rewards, where activity is modulated by both reward expectation at cue and the reward prediction error at feedback. We used probabilistic rewards to create a reaching task rich in reward expectation, reward prediction error, and learning. In one experiment, target reward probabilities were explicitly stated, and in the other, were left unknown and to be learned by the participants. We included two stochastic rewards (probabilities 33% and 66%) and two deterministic ones (probabilities 100% and 0%). In both experiments, outgoing peak velocity increased with increasing reward expectation. Furthermore, we observed a short-latency response in the vigor of the ongoing movement, that tracked reward prediction error: either invigorating or enervating velocity consistent with the sign and magnitude of the error. Reaching kinematics also revealed the value-update process in a trial-to-trial fashion, similar to the effect of prediction error signals typical in dopamine-mediated striatal phasic activity. Lastly, reach vigor increased with reward history over trials, mirroring the motivational effects often linked to fluctuating dopamine levels. Taken together, our results highlight the link between known short-latency dopaminergic learning signals and the invigoration of movement, not only at the time of cue presentation and movement initiation, but during an ongoing movement immediately after feedback is provided.</p><p><strong>New & noteworthy: </strong>Previous research has demonstrated the invigorating effects of reward on movement. Growing evidence suggests this is causally explained by midbrain dopamine transients. Here, we demonstrate that reach vigor tracks canonical variables of learning and motivation across time scales ranging from milliseconds to minutes. Velocity was modulated by reward expectation, reward prediction error and reward rate, key variables that have also been associated with striatal dopaminergic fluctuations. These results point to a potential neural mechanism by which dopamine can influence both decision making and movement control and support the proposition that reward-based invigoration of movement is in part influenced by dopaminergic circuits.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11974846/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143804813","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Low-Strength Type I Interferon Signaling Promotes CAR T-Cell Treatment Efficacy.","authors":"Erting Tang, Yifei Hu, Guoshuai Cao, Nicholas W Asby, Duy-Thuc Nguyen, Nada S Aboelella, Hanna Ruiz, Yu Zhao, Lishi Xie, Xiufen Chen, Michael R Bishop, Peter A Riedell, James L LaBelle, Justin P Kline, Jun Huang","doi":"10.1101/2025.05.13.653878","DOIUrl":"https://doi.org/10.1101/2025.05.13.653878","url":null,"abstract":"<p><p>CD19-directed chimeric antigen receptor (CAR) T-cell therapy has significantly advanced the treatment landscape for relapsed/refractory diffuse large B-cell lymphoma (r/r DLBCL). However, up to 60% of patients do not achieve a complete response. To uncover determinants of therapeutic efficacy, we analyzed the infusion products of eight r/r DLBCL patients with distinct clinical responses to axicabtagene ciloleucel using single-cell transcriptomics. Compared to patients who exhibited progressive disease, infusion products of complete responders demonstrated enriched signatures of type I interferon (IFN-I) signaling. Based on these findings, we developed a novel strategy to improve CD19-directed CAR T-cell treatment efficacy by incorporating IFN-I as an enhancer during the ex vivo manufacturing process. For both CD28- and 4-1BB-costimulated second-generation CARs, we found that low-strength IFN-I signaling enhanced CAR T-cell cytotoxicity and in vivo efficacy. On the other hand, high-strength IFN-I signaling compromised cell viability and in vivo efficacy. Our low-strength IFN-I signaling approach leverages an existing FDA-approved pharmacologic agent and is compatible with current CAR constructs and manufacturing workflows. Together, our results establish IFN-I as a potent and costimulation-independent enhancer of CAR T-cell efficacy and provide a translationally feasible approach to enhance CAR T-cell therapies for r/r DLBCL.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12132530/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144218311","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A mechanism linking dopamine's roles in reinforcement, movement and motivation.","authors":"Allison E Hamilos, Isabella C Wijsman, Qinxin Ding, Pichamon Assawaphadungsit, Zeynep Ozcan, Elias Norri, John A Assad","doi":"10.1101/2025.04.04.647288","DOIUrl":"10.1101/2025.04.04.647288","url":null,"abstract":"<p><p>Dopamine neurons (DANs) play seemingly distinct roles in reinforcement, ^1-3 motivation, ^4,5 and movement, ^6,7 and DA-modulating therapies relieve symptoms across a puzzling spectrum of neurologic and psychiatric symptoms. ⁸ Yet, the mechanistic relationship among these roles is unknown. Here, we show DA's tripartite roles are causally linked by a process in which phasic striatal DA rapidly and persistently recalibrates the propensity to move, a measure of vigor. Using a self-timed movement task, we found that single exposures to reward-related DA transients (both endogenous and exogenously-induced) exerted one-shot updates to movement timing-but in a surprising fashion. Rather than reinforce specific movement times, DA transients quantitatively <i>changed</i> movement timing on the next trial, with larger transients leading to earlier movements (and smaller to later), consistent with a stochastic search process that calibrates the frequency of movement. Both abrupt and gradual changes in external and internal contingencies-such as timing criterion, reward content, and satiety state-caused changes to the amplitude of DA transients that causally altered movement timing. The rapidity and bidirectionality of the one-shot effects are difficult to reconcile with gradual synaptic plasticity, and instead point to more flexible cellular mechanisms, such as DA-dependent modulation of neuronal excitability. Our findings shed light on how natural reinforcement, as well as DA-related disorders such as Parkinson's disease, could affect behavioral vigor.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11996583/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144059547","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Role of channels in the O₂ permeability of murine red blood cells. III. Mathematical modeling and simulations.","authors":"Rossana Occhipinti, Pan Zhao, Fraser J Moss, Walter F Boron","doi":"10.1101/2025.03.05.639964","DOIUrl":"10.1101/2025.03.05.639964","url":null,"abstract":"<p><p>In this third of three papers, we develop a reaction-diffusion model for O ₂ offloading from a red blood cell (RBC), treated as a sphere with diameter approximating RBC thickness. Stopped-flow (SF) analysis (paper #1) of hemoglobin/oxyhemoglobin (Hb/HbO ₂ ) absorbance spectra during O ₂ efflux from intact murine RBCs show that membrane-impermeant inhibitor p-chloromercuribenzenesulfonate (pCMBS) reduces the HbO ₂ -deoxygenation rate constant (k _HbO2 ) by ~61%. SF experiments show that k _HbO2 falls by (1) 9% for aquaporin-1 knockouts (AQP1-KOs), (2) 17% for Rhesus A-glycoprotein knockouts (RhAG-KOs), (3) 30% for double knockouts (dKOs), and (4) ~78% in dKOs/pCMBS. Here, we simulate HbO ₂ dissociation (rate constant, k _HbO2 → Hb); HbO ₂ , Hb, and O ₂ diffusion through RBC cytosol; transmembrane O ₂ diffusion; and O ₂ diffusion through extracellular unconvected fluid (EUF) to bulk extracellular fluid. Informed by automated-hematology data (paper #1) and imaging-flow-cytometry data (paper #2), simulations predict that observed k _HbO2 decreases cannot reflect changes in RBC size/shape or [Hb/HbO ₂ ]. Instead, membrane O ₂ permeability ( <i>P</i> _M,O2 ) must fall by (1) 22% to account for AQP1-KO data, (2) 36% for RhAG-KOs, (3) 55% for dKOs, and (4) 91% for dKOs/pCMBS. Exploring predicted k _HbO2 sensitivities to eight key parameters (e.g., [Hb/HbO ₂ ], diffusion constants, k _HbO2 → Hb, thickness _EUF , diameter _Sphere ) shows that no reasonable changes explain the k _HbO2 data. We introduce a linear-combination approach to accommodate for the presence of poikilocytes. Finally, contrary to common beliefs, the model predicts that, in the absence of inhibitors, the RBC membrane represents >30% of total diffusive \"resistance\" to O ₂ offloading, even for a WT mouse.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-05-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11908236/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143653138","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Long-Distance Trail Running Induces Inflammatory-Associated Protein, Lipid, and Purine Oxidation in Red Blood Cells.","authors":"Travis Nemkov, Emeric Stauffer, Francesca Cendali, Daniel Stephenson, Elie Nader, Mélanie Robert, Sarah Skinner, Monika Dzieciatkowska, Kirk C Hansen, Paul Robach, Guillaume Y Millet, Philippe Connes, Angelo D'Alessandro","doi":"10.1101/2025.04.09.648006","DOIUrl":"10.1101/2025.04.09.648006","url":null,"abstract":"<p><p>Ultra-endurance exercise places extreme physiological demands on oxygen transport, yet its impact on red blood cells (RBCs) remains underexplored. We conducted a multi-omics analysis of plasma and RBCs from endurance athletes before and after a 40-km trail race (MCC) and a 171-km ultramarathon (UTMB ^® ). Ultra-running led to oxidative stress, metabolic shifts, and inflammation-driven RBC damage, including increased acylcarnitines, kynurenine accumulation, oxidative lipid and protein modifications, reduced RBC deformability, enhanced microparticle release, and increased senescence markers such as externalized phosphatidylserine (PS). Post-race interleukin-6 strongly correlated with kynurenine elevation, mirroring inflammatory responses in severe infections. These findings challenge the assumption that RBC damage in endurance exercise is primarily mechanical, revealing systemic inflammation and metabolic remodeling as key drivers. This study underscores RBCs as both mediators and casualties of extreme exercise stress, with implications for optimizing athlete recovery, endurance training, and understanding inflammation-linked RBC dysfunction in clinical settings.</p><p><strong>Teaser: </strong>Marathon running imparts molecular damage to red blood cells, the effects of which are exacerbated by increased distances of ultramarathons.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-05-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12027326/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144063591","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Non-redundant cardiolipin synthases shape membrane composition and support stress resilience in <i>Bacteroides fragilis</i>.","authors":"Matthew K Schnizlein, BongJin Hong, Jennifer N T Nguyen, Katarina Jones, Alyssa I Rodriguez, Aretha Fiebig, Shawn R Campagna, Marcy J Balunas, Thomas V O'Halloran, Sean Crosson","doi":"10.1101/2025.05.12.653583","DOIUrl":"https://doi.org/10.1101/2025.05.12.653583","url":null,"abstract":"<p><p><i>Bacteroides fragilis</i> is an anaerobic resident of the human gut known to tolerate the toxic effects of host-produced and microbially-modified bile acids. Two conserved genes, <i>clsA</i> and <i>clsB</i> , encode putative cardiolipin synthases that have been linked to bile acid tolerance, but their physiological roles remain undefined. Phylogenetic analysis indicates that <i>Bacteroides</i> spp. ClsA and ClsB diverge from the well-characterized cardiolipin synthases of Gammaproteobacteria and Firmicutes. Here, we show that these enzymes have distinct cardiolipin synthase activities and make non-redundant contributions to <i>B. fragilis</i> fitness under gut-relevant stress conditions, including osmotic stress, disruption of membrane potential, and exposure to the bile acid deoxycholate. Although deoxycholate treatment perturbed K⁺/Na⁺ homeostasis in <i>B. fragilis</i> , deletion of <i>clsA</i> or <i>clsB</i> did not significantly alter intracellular ion levels, suggesting that cardiolipin loss does not substantially impact ion balance under standard cultivation conditions. High-resolution lipidomic analyses showed that cardiolipin comprises less than 1% of <i>B. fragilis</i> membranes and that ClsA and ClsB produce distinct cardiolipin products with unique acyl chain lengths and levels of unsaturation. Deletion of either <i>cls</i> gene led to Cls-specific remodeling of <i>B. fragilis</i> envelope lipid content, which was also associated with shifts in non-lipid metabolites indicative of stress-induced metabolic changes. These results define distinct roles for ClsA and ClsB in shaping <i>B. fragilis</i> membrane composition, metabolism, and stress resilience, and highlight cardiolipin as a key determinant of fitness under bile acid stress.</p><p><strong>Importance: </strong>Inflammatory bowel diseases are a growing global health concern, motivating efforts to understand how inflammation-associated microbes, such as <i>B. fragilis</i> , adapt to and persist in stress conditions typical of the intestinal environment. Our targeted genetic analyses reveal that the cardiolipin synthases ClsA and ClsB make non-redundant contributions to <i>B. fragilis</i> fitness under bile acid exposure and other gut-relevant stress conditions. Complementary omics-based approaches show that these enzymes produce chemically distinct cardiolipin species and that their loss leads to specific remodeling of membrane lipid composition and cellular metabolite profiles. Together, our results indicate that cardiolipin synthases contribute to membrane and metabolic adaptation in <i>B. fragilis</i> , supporting stress resilience in the presence of bile acids and other gut-relevant stressors.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-05-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12132519/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144218386","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}