Biology subjects

Zhou, Y.

Publications and source records attributed to Zhou, Y..

At least 55 records · Page 3Linked to original sources

bioRxiv2026

Bacterial Peptidoglycan Extends Lifespan by Activating Lysosomal Activity through V-ATPase Binding

Lysosomal dysfunction is a hallmark of aging, yet whether microbial components actively regulate this organelle to influence longevity remains unknown. Here, we identify bacterial peptidoglycan (PGN), a major cell wall component degraded by host lysozyme, as an evolutionarily conserved activator of lysosomal function that extends lifespan in both C. elegans and mice. We show that aging leads to an intestinal decline in lysozyme expression, which impairs bacterial cell-wall digestion and results in systemic PGN deficiency. Late-life PGN supplementation (starting at 18 months of age) significantly prolongs mouse lifespan and improves healthspan. Mechanistically, PGN localizes to lysosomes and directly binds V-ATPase subunits, enhancing ATP hydrolysis activity and promoting lysosomal acidification. This effect is abolished by V-ATPase inhibition (bafilomycin A1) or genetic disruption of lysosomal components (cup-5 and vha-12 mutants), confirming that functional V-ATPase is strictly required for lysosomal function and the longevity benefit. Importantly, PGN restores lysosomal acidification in aged cells, alleviates cellular senescence markers, and improves multiple hallmarks of aging including locomotion and muscle integrity. Collectively, these findings reveal an evolutionarily conserved mechanism whereby hosts exploit bacterial cell wall components to maintain cellular homeostasis, establishing a gut microbiome-lysosome-longevity axis with implications for microbiome-based anti-aging interventions.

physiology↗

bioRxiv2026

Multi-omic characterization of axolotl perilymph-cerebrospinal fluid reveals shifts in composition during limb regeneration

The axolotl salamander can fully regenerate amputated limbs, yet the systemic consequences underlying this process remain largely understudied. Cerebrospinal fluid is an emerging signaling medium capable of communicating with both the central and peripheral nervous systems, but its composition and potential role in salamander limb regeneration have not yet been examined using modern multi-omics techniques. Here, we developed a protocol for extracting mixed perilymph-cerebrospinal fluid (P-CSF) from axolotl and provided the first proteomic and metabolomic characterization of this biological fluid. We identified 2,626 unique proteins and 173 high-confidence metabolites and quantified them across four time points of early limb regeneration. We demonstrated that limb amputation drives progressive shifts in P-CSF proteins, including an elevation of sarcomeric muscle proteins, regeneration-associated factors, and protease/extracellular matrix proteins. We observed shifts in metabolites involved in oxidative stress, polyunsaturated fatty acid oxidation, and histamine metabolism. Injury-comparison experiments revealed that the observed proteomic changes as a result of limb amputation are different than crush injury, denervation, or tail amputation. This study proposes axolotl P-CSF as a reservoir for limb amputation-associated systemic signaling and as a potential conduit of signals involved in limb regeneration.

systems biology↗

bioRxiv2026

The MOS4-associated complex subunit MAC5A maintains meristem development by regulating transcription elongation in Arabidopsis

The precise coordination of co-transcriptional RNA processing with transcriptional elongation is essential for eukaryotic gene regulation, yet the machineries coupling these processes remain largely elusive. Here, we reveal a role for the spliceosome-associated MOS4-associated complex (MAC) in regulating plant development through the direct regulation of transcriptional elongation. We demonstrate that the MAC subunit MAC5A physically interacts with Elongator Protein 6 (ELP6), a core component of the transcriptional Elongator complex. Genetic analyses reveal that MAC5A and ELP6 function synergistically to regulate apical meristem activity. Importantly, the MAC5A-Elongator module is required for the efficient expression of the critical auxin efflux carrier PIN-FORMED1 (PIN1) by enhancing RNA Polymerase II (RNAPII) occupancy across the PIN1 locus. Furthermore, we show that this elongation-promoting function is not unique to MAC5A, as other core MAC components are similarly required for efficient transcription progression. Together, our findings uncover a splicing-elongation nexus where MAC5A likely acts as a molecular bridge between the spliceosome and the elongation polymerase. This functional coupling ensures the efficient transcription of key developmental regulators, providing a mechanistic framework for the coupling of RNA processing to transcription and suggesting a conserved principle of gene expression control across eukaryotes.

plant biology↗

bioRxiv2026

NeuroGraphBench: Interacting with Drosophila Connectomes at Scale for Exploring the Functional Logic of Neural Circuits

Drosophila connectomic datasets provide increasingly comprehensive maps of neuronal morphology and synaptic connectivity, offering an unprecedented opportunity to explore the structural organization of its neural circuits. This calls for designing automated tools to interact with connectomic datasets at scale for efficiently exploring structural features embedded in the vast amount of data. Yet the central challenge remains the understanding of the functional logic of neural circuits. In order to understand how elements of the functional logic may emerge from this structural organization, it is critical to (i) characterize the objects in the natural environment in which brain circuits operate, and (ii) formulate how brain circuits represent and process the defined objects in the natural environment. To develop and demonstrate a methodology for these requirements, we focus on the Drosophila looming-evoked escape pathway. We modeled the trajectory of looming objects that are on a collision course (direct-hits) or pass-by the fly (near-misses): their projected images on the retina can be characterized by the solid angle (angular size) and elevation. We then analyzed the pathway's morphology across the OpticLobe, Hemibrain, and FlyWire connectome datasets. By abstracting their sub-neuronal structure and retinotopic organization, we constructed an executable circuit model that maps each structural element to a processing block. We demonstrate that this model separates direct hits from near misses well before the angular size could tell them apart. To accelerate the connectomic analysis step, we developed a Python toolset with an agentic, code-free workspace interface called NeuroGraphBench (NGB). NGB provides four composable morphology-analysis primitives and an AI agent that composes them to interactively respond to natural-language queries aided by visualization on an interactive 3D canvas. Thus, NGB automates tedious and repetitive tasks to enable faster and scalable connectomic exploration, keeping human reasoning, instead of writing code, at the center of an open-ended research inquiry.

neuroscience↗

bioRxiv2026

Chromatin-Assisted Targeting Enables Precise DNA Methylation Editing in Plants

Precise installation of DNA methylation at selected loci offers a powerful strategy for regulating gene expression without altering DNA sequence, but existing plant epigenome editors are constrained by limited efficiency, locus dependence, and genome-wide off-target methylation. Here, we developed SunTag-MQ1v variants incorporating TRBIP1, which promotes removal of the antagonistic H3K4me3 mark, and CHLAMY, an oligomerizing alpha crystalline domain protein from Chlamydomonas reinhardtii. TRBIP1 enhanced methylation and silencing at the Arabidopsis FWA promoter but caused widespread off-target methylation and severe developmental defects. Adding CHLAMY produced SunTag-CHLAMY-TRBIP1-MQ1v (designated as SunTag-NOVA), which successfully overcame the lethality and widespread off-target effects associated with direct TRBIP1-MQ1v fusions. We demonstrate that CHLAMY drives higher-order oligomerization of the editing complex, which enhances target specificity and mitigates off-target accumulation. SunTag-NOVA robustly installed DNA methylation and repressed transcription at the endogenous FWA, FT and TMM genes with minimal genome-wide off-target consequences. These results show that combining local chromatin modification with controlled effector assembly can improve targeted DNA methylation, and establish SunTag-NOVA as a specific epigenome-editing platform for plants.

plant biology↗

bioRxiv2026

Multiplexed Quantification of Variant Abundance in the Globin Gene Family: Integrating Saturation Mutagenesis with Cross-Paralog Prediction

Widespread genetic testing has expanded variant identification, yet functional characterization remains a bottleneck in genome guided medicine. Here, we present a modified Variant Abundance by Massively Parallel Sequencing (VAMP-seq) platform integrating experimental and computational approaches for high-resolution abundance profiling of protein variants. Utilizing a lentiviral integration system, we systematically assessed the stability effects of 2,696 amino acid substitutions in {zeta}-globin (HBZ) via saturation mutagenesis in human cells, achieving complete variant coverage with high reproducibility. Representative variants showed strong concordance with orthogonal low-throughput validation assays. We further developed a deep learning framework leveraging VAMP-seq derived HBZ data to predict variant abundance across thalassemia-associated globin paralogs (HBA, HBB, and HBG1) not experimentally tractable. Our hybrid framework demonstrates how targeted experimental profiling combined with AI-driven extrapolation can accelerate variant interpretation across protein family members.

genetics↗

bioRxiv2026

VEGFA-Positive Macrophages Regulate Aqueous Humor Outflow in Aged Mice and Humans

Elevated intraocular pressure (IOP) and aging are major risk factors for primary open-angle glaucoma (POAG), but how aging affects IOP regulation remains poorly understood. IOP remains within a narrow range despite age-associated changes predicted to increase aqueous humor outflow (AHO) resistance at the interface between the trabecular meshwork and Schlemm's canal (SC), suggesting compensatory mechanisms preserve AHO homeostasis during aging. Single-cell RNA sequencing of mouse ocular angle tissues revealed immunomodulatory transcriptional reprogramming of SC endothelial cells in older mice, while mouse and human imaging showed reduced SC size and increased peri-SC macrophage accumulation with aging. Ligand-receptor analysis predicted enhanced macrophage-to-SC VEGFA-VEGFR signaling in aged and Tie2-haploinsufficient mice, an independent model of vascular stress and glaucoma risk. Deletion of Vegfa in CX3CR1+ macrophages increased IOP and reduced AHO facility in 9-month-old mice, demonstrating that macrophage-derived VEGFA supports AHO homeostasis. Tie2 haploinsufficiency recapitulated key age-associated SC niche changes, including peri-SC macrophage accumulation, whereas gene therapy boosting TIE2 activity protected wild-type mice against age-related changes. Together, these findings identify peri-SC macrophage-derived VEGFA as a compensatory mechanism maintaining AHO homeostasis during aging and vascular stress and support TIE2 activation as a therapeutic strategy to preserve SC function and IOP regulation.

physiology↗

bioRxiv2026

Mechanosignaling Promotes Macrophage Apoptosis Resistance in Pulmonary Fibrosis via Metabolic Reprogramming

The mechanisms underlying the progression of pulmonary fibrosis in idiopathic pulmonary fibrosis (IPF) and other interstitial lung diseases remain unclear. Increased extracellular matrix stiffness is a hallmark of fibrotic lung diseases. Monocyte-derived macrophages can promote fibrosis progression. However, there is limited understanding of how the mechanical properties of the fibrotic microenvironment influence macrophage phenotypes. Profibrotic macrophages are apoptosis-resistant, and this phenotype is modulated by enhanced mitochondrial bioenergetics. The objective of the study was to determine how lung tissue stiffness impacts macrophage phenotypes and fibrotic progression. We demonstrate that mechanoactivated macrophages exhibit apoptosis-resistance, increased expression of the antiapoptotic protein Bcl-xL and increased mitochondrial oxidative phosphorylation. Critically, the metabolic reprogramming observed in mechanoactivated macrophages is dependent on increased glutaminolysis. Inhibition of glutaminolysis attenuated apoptosis resistance in mechanoactivated macrophages. Moreover, inhibition of Bcl-xL in vivo protected mice against experimental pulmonary fibrosis. Lastly, mechanoactivated primary IPF macrophages produce more profibrotic cytokines and promote extracellular matrix production in precision-cut lung slices. We describe a mechanism for acquired macrophage apoptosis resistance dependent on metabolic reprogramming regulated by extracellular matrix stiffness. Our results identify mechanoactivated apoptosis-resistant macrophages as pro-fibrotic mediators, suggesting a novel therapeutic target in IPF and related fibrotic disorders.

molecular biology↗

bioRxiv2026

Within-Subject Optogenetic Model Reveals Spatiotemporal Cortical Reorganization in Artificial Vision

Visual prostheses can elicit simple percepts such as letter forms through dynamically patterned stimulation, yet naturalistic dynamic vision remains out of reach despite hardware fully capable of high-resolution temporal control. This persistent gap suggests the bottleneck lies in how artificial input is represented by the cortex itself. A fundamental question remains unresolved: when artificial vision bypasses natural retinal encoding, what is lost in cortical representation? We address this question in this paper using a retinal optogenetic mouse model that isolates the consequence of bypassing retinal encoding while preserving downstream pathways. Using within-subject V1 cortex electrophysiology and CNN-based decoding under identical dynamic stimuli, we reveal dissociable, dimension-specific gaps. Temporally, artificial vision bypassed the frequency-dependent compression imposed by natural vision, extending stimulus-locked entrainment bandwidth. Spatially, direction-selective representations underwent systematic remapping, with disrupted phase organization and reduced encoding regularity. These findings show that bypassing natural retinal encoding reorganizes cortical representation along dissociable spatiotemporal dimensions, establishing a within-subject framework that renders such structural divergences easy to locate and quantifiable.

bioengineering↗

bioRxiv2026

An interoperable research agent network for scientific discovery

Recent advances in agentic systems have enabled the autonomous execution of research tasks across scientific domains. However, the rapid emergence of specialized scientific agents for areas such as computational pathology, microbiome research, gene editing, materials science, organic chemistry, and drug discovery has created a fragmented ecosystem of scientific capabilities. While these agents often demonstrate strong performance within their respective domains, limited interoperability makes it difficult to combine expertise across platforms and coordinate complex interdisciplinary workflows. Here we introduce GUIA (Guided-research Utilizing Intelligent Agents), an interoperable research-agent network built upon a flexible Agent-to-Agent (A2A) communication architecture. GUIA enables both in-house and third-party agents to collaborate within shared workflows, allowing scientific capabilities to accumulate through the integration of complementary expertise. We evaluated GUIA through four assessments spanning baseline benchmarking, third-party single-agent integration, third-party multi-agent integration, and cross-server agent collaboration. Furthermore, we demonstrate its practical utility through real-world applications involving therapeutic target discovery, drug discovery, and spatial proteomics analysis. Together, our results show that interoperable research-agent networks can coordinate specialized expertise across independently developed systems, providing a scalable framework for expanding scientific capabilities through collaboration.

bioinformatics↗

bioRxiv2026

Base-and-Sugar Dual-Frame Flow Matching for RNA Co-Design

Current frame-based RNA generative models represent each nucleotide with a single rigid coordinate frame. However, our analysis of seven nucleotide frame constructions across 11,497 static RNA chains and 31,432 multi-state relation groups reveals a clear trade-off: base-centered frames better preserve base-mediated relations, whereas sugar-centered frames better support sugar-phosphate backbone reconstruction. Motivated by this complementarity, we introduce DuetRNA, a joint sequence-structure generation model for RNA co-design that performs SE(3) flow matching over coupled base-and-sugar frames. Empirically, we evaluate DuetRNA under both the inverse-folded (IF) backbone-design protocol and generated-sequence (GS) co-design protocol. DuetRNA improves scTM-validity over the strongest matched baselines by 7.67 and 4.33 % under IF and GS, respectively. Controlled ablations further support the necessity of the dual-frame modeling in DuetRNA. The official DuetRNA repository is https://github.com/XjunLi/DuetRNA.

bioinformatics↗

bioRxiv2026

A CRISPR-Cas9 platform for primary human hepatocytes enables arrayed screening and in vivo validation of HBV host factors

More than two million deaths annually are attributed to liver-related conditions, making primary human hepatocytes (PHH) an invaluable in vitro model for studying liver pathophysiology and the molecular mechanisms underlying hepatic diseases. However, because PHH do not proliferate in culture, CRISPR gene editing has been highly inefficient. Here, we report lipofection- and lentivirus-mediated protocols for CRISPR-Cas9 delivery in mouse-passaged primary human hepatocytes (mpPHH), a system that enables PHH expansion in liver-humanized mice. We achieve robust gene editing efficiencies exceeding 90% in mpPHH while maintaining cell viability. We demonstrate the utility of these protocols by disrupting CYP3A4 to impair xenobiotic metabolism and by showing that edited mpPHH efficiently engraft and expand in mice, generating liver-humanized animals. We establish the feasibility of arrayed CRISPR screening in mpPHH using an 85-gene screen to identify host factors influencing hepatitis B virus (HBV) infection, and validate key findings in humanized mice by targeting the HBV entry receptor SLC10A1 (NTCP), which reduced viral infection in vivo. Our methodology enables scalable genetic manipulation of mpPHH, opening new avenues for HBV research and liver disease modeling.

genomics↗

bioRxiv2026

Pi-Ensemble: Sequence-guided generation of interpolated protein conformational ensembles

Proteins are critical biomolecular machines that populate ensembles of interconverting conformations. Many biological processes depend on transitions between metastable states. Although molecular dynamics (MD) simulations provide a physically grounded route to characterize these motions, routine sampling of large-scale conformational transitions remains computationally demanding. Recent advances in protein structure prediction have created new opportunities for ensemble generation, but many existing approaches require noising inputs, task-specific training, supervised fitting on extensive MD data, or experimentally-informed restraints. Here, we introduce Pi-Ensemble (Predicting Interpolated Ensemble), a sequence-guided framework for generating protein conformational ensembles interpolating between two structural anchor states. Unlike previous methods, Pi-Ensemble alternately leverages inverse-folding and structure-prediction models to propose intermediate conformations between known protein states, generating diverse ensembles without additional training. We evaluate Pi-Ensemble across diverse protein systems, including enzymes, transporters, receptors, and benchmark cases with reference MD simulations or experimental Double Electron-Electron Resonance (DEER) data. Pi-Ensemble recovers physically plausible intermediate conformations, captures transition pathways observed in large-scale MD simulations, and generates structures consistent with experimental distance distributions. Furthermore, Pi-Ensemble-generated conformations provide effective starting seeds for parallel MD simulations, improving conformational exploration and accelerating convergence relative to simulations initiated only from endpoint structures. These results establish sequence-guided structural interpolation as a practical strategy for probing protein conformational landscapes. By generating diverse and physically reasonable conformational proposals without long-timescale MD or model retraining, Pi-Ensemble provides an extensible framework for studying protein flexibility, guiding adaptive sampling, and accelerating mechanistic investigations of protein function.

biophysics↗

bioRxiv2026

Antibiotic condensates activate magnesium signaling to drive drug tolerance

The spatial distribution of small molecules within cells shapes their biological activity, yet these distributions are generally assumed to be governed passively by reaction-driven electrochemical gradients. Here we show that aminoglycoside antibiotics actively control their own subcellular organization by undergoing phase separation with RNAs. Combining in vitro reconstitution, bacterial assays, and molecular dynamics simulations, we discovered that aminoglycosides coacervate with RNA through multivalent electrostatic interactions, displacing and releasing RNA-bound Mg2+. This condensate-dependent Mg2+ release remodels the cytosolic labile Mg2+ pool and activates magnesium signaling. This effect dampens the magnesium-starvation regulation, sustains ribosome activity, and shifts the cellular electrochemical state, promoting bacterial fitness. Because condensation occurs only above a defined concentration threshold, it generates a non-monotonic dose-response in which higher antibiotic concentrations paradoxically enhance bacterial survival. This antibiotic condensate-dependent Mg2+ signaling confers tolerance to multiple ribosome-targeting antibiotics simultaneously even in cells lacking resistance gene, while condensate dissolution restores antibiotic efficacy. Our findings establish antibiotic-driven phase separation as a previously unrecognized mechanism to encode cellular signaling and identify antibiotic condensates as a distinct functional unit underlying drug tolerance.

microbiology↗

bioRxiv2026

TBpop: an open-access genomic portal integrating genomic variation, population genetic statistics, phylogeny, pangenome composition, and strain metadata of epidemic Mycobacterium tuberculosis strains from China

Tuberculosis remains a major global public health threat. While whole-genome sequencing has transformed our understanding of the causative agent, Mycobacterium tuberculosis (MTB), existing genomic databases are highly fragmented and often underrepresent structural variations (SVs). Furthermore, critical population-genetic statistics are rarely integrated with phylogenetic and geographic context, forcing researchers to reconcile separate datasets manually. To address this gap, we developed TBpop (https://tbpop.chinacdc.cn), an open-access, integrated population genomics portal. TBpop is built from 420 clinical MTB isolates selected from the first national drug resistance baseline survey in China. The portal integrates isolate metadata, pangenome categories, SNPs, SVs, IS6110 insertion sites, strain phylogeny, and gene-level statistics, and provides three interactive explorer modules: the Population Explorer, the Statistics Explorer, and the Variation Explorer. Additionally, a User Analysis module allows researchers to run population genetic workflows on their own alignments. TBpop provides an integrated platform for exploring genome plasticity, signatures of positive selection, and conservation patterns of functionally important genes in MTB.

bioinformatics↗

bioRxiv2026

Ischemia-reperfusion and acidosis induce retrotransposon derepression in the mouse brain

Retrotransposons are repetitive DNA elements normally suppressed through epigenetic mechanisms. In aging and neurodegenerative diseases, abnormal retrotransposon activation occurs and leads to neurotoxicity. However, whether ischemic stroke induces retrotransposon activation remains unclear. Here, we investigated whether cerebral ischemia triggers dysregulation of retrotransposon in the brain. Since brain ischemia leads to tissue acidosis, we further examined whether acidosis contributes to this response. We performed 45 min of transient middle cerebral artery occlusion (tMCAO) followed by reperfusion on adult wild-type mice, then measured expression of retrotransposons LINE-1, IAP, ETn2 and SINE B2 in ipsilateral brain tissue using RT-qPCR. Retroviral GAG protein expression was examined by Western blotting. In vitro, we exposed neuro-2a (N2A) cells to acidic extracellular pH (6.4 or 6.0) and retrotransposon transcripts were analyzed. We found that ischemia-reperfusion increased expression of IAPEz-gag and ETn2 in ipsilateral ischemic brain tissue at 24 h, with stronger induction of LINE-1 Orf1, IAPEz-gag and B2 in infarct than peri-infarct brain regions. Western blotting analysis revealed dynamic changes in GAG proteins, with no detectable changes at 6 h of reperfusion followed by reduced levels of the precursor Pr65 and mature capsid p30 products at 24 h. In N2A cells, extracellular acidosis induced time-dependent increases in LINE-1 Orf1, IAPEz-gag and B2 transcripts. These findings identify retrotransposon dysregulation as a molecular feature of ischemic brain injury and provide a framework for further investigating its role in stroke pathology.

neuroscience↗

bioRxiv2026

IGF2BP3 amplifies antiviral innate immunity with implications for autoimmune diseases

The insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) is a known N6-methyladenosine (m6A) reader, but its role in antiviral innate immunity is unknown. Here, we identify IGF2BP3 as a critical positive regulator of antiviral responses. Viral infection and interferon (IFN) stimulation upregulate IGF2BP3, establishing a feedforward loop that potentiates virus-induced activation of the TBK1-IRF3 and NF-{kappa}B pathways, thereby amplifying type I interferon (IFN-I) production, and restricting viral replication in human and murine cells and in vivo. Mechanistically, IGF2BP3 directly binds and stabilizes MAVS and TBK1 mRNAs and promotes their translation by facilitating recruitment to the eIF4F/PABP-associated initiation complex. Upon infection, IGF2BP3 relocalizes to antiviral stress granules (avSGs), where it scaffolds the RIG-I-G3BP1 complex to enhance viral RNA sensing. Notably, IGF2BP3 is aberrantly upregulated in patients with systemic lupus erythematosus (SLE) and in Trex1 knockout (KO) mice, and pharmacological inhibition by curcumol suppresses IFN-I-driven pathology and improves survival. Collectively, our findings establish IGF2BP3 as a central feedforward circuit that couples viral RNA sensing to the control of RNA stability and translation of key signaling molecules, and reveals its potential as a therapeutic target in interferon-associated autoimmune diseases. Significance StatementAntiviral immunity demands rapid and coordinated gene expression, yet how RNA-binding proteins link viral recognition to downstream signaling is poorly understood. We reveal that the m6A reader IGF2BP3 acts as a central amplifier of antiviral innate immunity. Induced by both viruses and interferons, IGF2BP3 enhances viral RNA sensing, stabilizes key signaling transcripts, and boosts their translation, creating a self-reinforcing feedforward circuit that strengthens interferon responses. Beyond host defense, IGF2BP3 is aberrantly elevated in interferon-driven autoimmunity, and its pharmacological inhibition reduce disease pathology in vivo. Our findings uncover a previously unrecognized mechanism that integrates RNA metabolism, stress-granule signaling, and translational control to regulate innate immunity, offering new therapeutic perspectives for both infectious and autoimmune disease.

immunology↗

bioRxiv2026

Conserved copper(I)-binding auxiliary domains link virulence-associated LPMOs to copper homeostasis in the intestinal environment

Multidomain lytic polysaccharide monooxygenases (LPMOs) are widely distributed in human pathogenic bacteria and increasingly recognized for promoting host interactions and virulence. Recent work has shown that immunizing mice with CbpD, a trimodular LPMO from Pseudomonas aeruginosa, provides protection against lethal P. aeruginosa infection. In several human intestinal pathogenic bacteria, LPMOs occur as tetra- and pentamodular proteins and their ability to interact with the host and facilitate virulence has primarily been ascribed to their ability to bind to N-acetylglucosamine containing glycans. Recently, AlphaFold predictions and substrate-binding studies of the Vibrio cholerae colonization factor GbpA suggested the presence of a copper-binding site in its non-catalytic third domain. In light of the recent demonstration that MUC2, the primary intestinal mucin, harbors two conserved and distinct copper binding sites for Cu(II) and Cu(I), we hypothesized that the auxiliary non-catalytic copper-binding domains of intestinal LPMOs evolved as a response to the intestinal environment. Here, we examine GbpA and homologous multidomain LPMOs from food-borne intestinal disease-causing Gram-positive bacteria from the genera Bacillus and Listeria. By combining mutagenesis with biochemical, spectroscopic, and computational approaches, we demonstrate that these multidomain LPMOs contain conserved, yet structurally distinct copper(I)-binding motifs on their non-catalytic third domain. A comprehensive review and reassessment of the literature spanning the past two decades reveals consistent links between the copper-binding ability of these LPMOs and host-pathogen interactions. Thus, our findings link these LPMOs to copper management in the intestine, offering an explanation for why their unique architecture is preserved across different bacterial lineages causing intestinal disease.

biochemistry↗