Biology subjects

Wu, D.

Publications and source records attributed to Wu, D..

At least 37 records · Page 2Linked to original sources

bioRxiv2026

Targeting NAT10 suppresses TFH responses and alleviates allergic asthma via an ac4C-TOX2 axis

Chemical modifications of RNA, such as N4-acetylcytidine (acC), fine-tune gene expression, but their roles in cell fate determination within the immune system remain poorly understood. Here, we identify the RNA cytidine acetyltransferase NAT10 as a pivotal regulator of T follicular helper (TFH) cell differentiation and the germinal centre response. T cell-specific ablation of Nat10 in mice severely impaired TFH cell development, germinal centre formation, and antibody production following viral infection. Integrated epitranscriptomic and transcriptional profiling revealed that NAT10 deposits acC modifications on a cohort of mRNAs critical for immune function. We pinpointed the transcription factor TOX2 as a key downstream target, showing that acC modification within its mRNA coding sequence enhances both transcript stability and translation efficiency, thereby promoting TOX2 protein expression. Enforced TOX2 expression fully rescued the TFH differentiation defect in NAT10-deficient cells. This NAT10-acC-TOX2 axis is conserved in humans, where its activity correlates with the magnitude of vaccine-elicited TFH responses. Furthermore, we identified R428 as a potent NAT10 inhibitor and demonstrated that its administration could suppress pathogenic TFH responses and alleviate symptoms in a mouse model of allergic asthma. Our work elucidates a central epitranscriptomic mechanism controlling T helper cell fate and humoral immunity, and nominates NAT10 as a potential therapeutic target for TFH-driven diseases.

immunology↗

bioRxiv2026

Deciphering complete archaic introgression sequences in modern human genomes

Genetic introgression from archaic hominins has profoundly reshaped the genetic diversity and adaptive potential of modern humans, yet the full catalog of introgressed sequences, particularly those residing in structurally complex regions has remained elusive. Here, we present ASMaid (ASseMbly-based archaic introgression detector), a Hidden Markov Model-based framework that leverages haplotype-resolved pangenome assemblies to identify archaic-derived sequences with unprecedented completeness. By integrating both single-nucleotide genotype and structural variation (SV) signals, ASMaid captures significantly more intact archaic segments than conventional reference-based approaches. Applying ASMaid to a global panel of 610 phased human genome assemblies, we show that non-African individuals carry approximately 79.8 Mbp of Neanderthal and 8.3 Mbp of Denisovan sequences, representing substantial increases over previous estimates, respectively. Notably, we detected several centromere-spanning archaic segments, including EAS-specific calls on chromosomes 5 and 7. Our assembly-based approach uncovered 1,701 archaic-derived SVs, revealing a previously overlooked layer of archaic functional legacy. High-frequency introgressed loci are enriched in pathways associated with metabolism, immunity, and nervous system (e.g. CTNNA2 linked to early-onset schizophrenia risk), underscoring the fundamental role of introgression in modulating modern human traits. Notably, we identified dozens of loci potentially facilitating local adaptation, such as PRDM16 involved in adipocyte differentiation and cold tolerance, and CSGALNACT2 associated with chondroitin sulfate synthesis. Furthermore, our analysis delineates three distinct Denisovan introgression pulses in Eastern Eurasian genomes, in which the first two pulses are shared across East Eurasian and Oceanian populations, while the third remain primarily exclusive in East Asians. Reflecting these complex introgression events, 31 Denisovan-derived segments, including the TBX15-WARS2 locus, are inferred to have been introduced via at least two events. This comprehensive map of archaic introgression provides a fundamental resource for understanding how ancient gene flow continuously shapes human phenotypic diversity and adaptation.

evolutionary biology↗

bioRxiv2026

Joint analysis of multiply perturbed cells improves statistical power and cost efficiency in Perturb-seq

Perturb-seq measures transcriptomic responses to genetic perturbations at scale, but conventional designs that enrich for one guide RNA per cell remain resource-intensive. Standard analyses discard cells carrying multiple guides, further limiting the usable yield from each experiment. Here, we characterize how incorporating these guide multiplets affects signal recovery, information loss, and cost reduction. At the highest guide burden, cells showed increased stress and suppressed cell-cycle progression. We develop PerturbMatch, a scalable statistical framework to analyze guide multiplets. Among different classes of guide multiplets, doublets and triplets recovered perturbation responses more accurately than higher-order multiplets. Across three 5000-gene Perturb-seq screens with increasing guide loading, per-cell costs decreased by up to 81% while information loss remained within 1.5-fold of the loss observed between technical replicates. In existing genome-wide Perturb-seq data, incorporating previously discarded guide multiplets increased usable cell numbers and improved statistical power. Compared with a singlet holdout set, adding guide multiplets moved signal recovery closer to the theoretical expected reproducibility. Overall, we recommend a design that intentionally includes single-guide cells, guide doublets, and guide triplets to improve cost efficiency while preserving signal recovery.

genomics↗

bioRxiv2026

Environmental complexity reveals memory-guided search as the locus of learning in prey capture

Ethological behavior requires the integration of perception, memory, and decision-making, yet standard laboratory tasks are often too simplified to recruit these processes jointly. Here, mice that were expert hunters in a bare arena were challenged to capture live insect prey among objects that occlude vision and afford opportunities to hide. This revealed an axis of learning absent in an empty arena: mice improved not by refining pursuit but by reorganizing how they searched for concealed prey. An agent-based model showed that short-term memory and learning of object-specific values are sufficient to account for the behavior demonstrated by mice. A compact ethogram of object-directed actions revealed that animals selectively acquired the interactions most likely to expose prey. These results show that environmental complexity recruits memory- and value-guided search strategies. Reproducible with inexpensive materials, the paradigm and its open-source analyses offer a sensitive read-out of cognitive processes largely unobservable in conventional assays.

neuroscience↗

bioRxiv2026

Catalytic rewiring of RuvC-II catalytic site activates trans-cleavage in Fanzor2

Fanzors are eukaryotic RNA-guided endonucleases that mediate programmable cis-cleavage in eukaryotic cells, but their potential target-induced collateral (trans) cleavage activity remains largely unexplored. Here, we show that catalytic rewiring of the RuvC-II catalytic site activates robust trans-cleavage activity in Acanthamoeba polyphaga mimivirus Fanzor2 (ApmFz2). The representative variant ApmFz2-EP displayed DNA- and RNA-triggered trans-cleavage, attenuated cis-cleavage, minimal TAM dependence, and activation by as few as seven nucleotides of guide-target complementarity. Mechanistic analyses indicate that relieving steric constraints surrounding the conserved alternative glutamate within the RuvC-II activates trans-cleavage and reshapes target-recognition specificity. Coupling ApmFz2-EP with nucleic acid amplification enabled FINDER, a Fanzor-based diagnostic platform for sensitive pathogen detection and broad detection across genetically diverse target subtypes. In addition, the mismatch-sensitive ApmFz2-EA variant enabled specific single-nucleotide variant (SNV) genotyping. Together, this work expands the functional scope of Fanzors and identifies catalytic-center engineering as a strategy for developing compact, programmable nucleic acid diagnostics.

bioengineering↗

bioRxiv2026

Multitrophic interaction networks mediate biodiversity effects on ecosystem multifunctionality

Biodiversity loss threatens the multifunctionality of ecosystems on which human well-being ultimately depends. Changes in multitrophic species interactions may be key to explaining the ecological consequences of biodiversity loss, but research explicitly linking species interactions and ecosystem multifunctionality remains rare. To assess interaction-mediated biodiversity effects and underlying mechanisms, characterizing the structure of species interaction networks is invaluable. Using comprehensive species interaction and ecosystem functioning data from a large-scale tree biodiversity experiment, we find consistent effects of the structure of species interaction networks on ecosystem multifunctionality across multiple types of antagonistic and mutualistic interactions. While positive effects of network size align with expected positive effects of multitrophic species diversity, positive effects of niche overlap among interacting species and negative effects of highly connected species (i.e. high linkage density) reveal additional, interaction-mediated drivers of multifunctionality. Specifically, the effects of niche overlap suggest benefits of functionally similar species, and the effects of linkage density underscore the importance of specialized interactions in promoting ecosystem multifunctionality. These findings emphasize that to effectively safeguard ecosystem service provisioning, ecosystem management and biodiversity conservation not only need to account for biodiversity changes at multiple trophic levels, but also explicitly for how species interact among each other.

ecology↗

bioRxiv2026

Nonlinear rheology of laminin-111-modified hyaluronan hydrogels

We experimentally assess the nonlinear rheology of composite biopolymer hydrogels composed of thiolated hyaluronic acid, poly(ethylene glycol) diacrylate (PEGDA), and laminin-111 in varied concentrations. We focus in particular on the influence of laminin on the mechanics of the assembled hydrogels, reporting nonlinear rheological measurements for gels under applied shear and compressive load. We find that increasing the concentration of laminin in the synthesized gels reduces the linear shear modulus and gives rise to a mild strain softening regime at intermediate strains prior to the onset of strain stiffening. In the stiffening regime, we find that all gels exhibit stress-controlled mechanics with K {propto}{sigma} a, with an apparent stiffening exponent of a {approx} 1, in agreement with observations of a variety of other reconstituted biopolymer gels. We discuss the possible implications of this nonlinear mechanical behavior on mechanotransduction and organoid development in biomimetic extracellular matrices.

biophysics↗

bioRxiv2026

Assessing Bioactivity and Biointegration of Engineered Salivary Tissue Constructs in a Preclinical Unilateral Fractionated Irradiated Rat Model

Human salivary stem/progenitor cell (hS/PC)-loaded hyaluronic acid (HA)-based hydrogels, termed 3D-salivary tissue constructs (3D-ST), hold great promise for restoring salivary gland function post-radiation injury. Here, we developed a next-generation 3D-ST using heparin-modified HA and bioactive peptide-modified hydrogels. This new formulation enables controlled pre-loading and localized presentation of heparin-binding growth factors prior to surgical implantation, providing opportunities to enhance in vivo hS/PC bioactivity. To model clinically relevant radiation injury, we established an athymic rat model subjected to computed tomography (CT)-guided fractionated radiation, resulting in hallmark features of radiation-induced salivary dysfunction. Over 60-days post-irradiation, glands exhibited progressive loss of acini, increased fibrosis, and disruption of endothelial, neuronal, and myoepithelial compartments. Within this injured environment, a surgical pocket was created to precisely implant 3D-STs to assess graft performance. Fluorescent labeling of the 3D-STs enabled longitudinal tracking post-implantation. Over 14 days, implanted 3D-STs remained structurally stable within irradiated glands, and hS/PCs remained viable without evidence of local inflammatory responses. Compared to non-injured glands, the irradiated microenvironment suppressed hS/PC proliferation and phenotype, indicating alterations in the irradiated local tissue negatively impact hS/PC bioactivity. In addition, host neurovascular migration into the 3D-ST was majorly restricted in irradiated glands, providing new opportunities to enhance biointegration. Overall, this work establishes a reproducible preclinical framework for assessing hydrogel biocompatibility and stability, cell bioactivity, and host-graft biointegration prior to scale up into preclinical large animal models. This study has successfully established a tractable approach for improving 3D-ST formulations to enhance hS/PC expansion, differentiation, and biointegration following implantation into radiation-injured beds.

bioengineering↗

bioRxiv2026

Optimizing Primary Human Salivary Stem/Progenitor Cells for Tissue Engineering Applications

This work aimed to establish a translationally viable, xeno-free, serum-free platform and protocol for the isolation and expansion of human salivary stem/progenitor cells (hS/PCs) suitable for regulatory qualification and future FDA-approved first-in-human autologous regenerative therapy trials for the treatment of hyposalivation disorders. Parotid gland specimens from non-cancerous regions/tissues were collected from consented surgical patients. Primary hS/PCs were isolated from tissue specimens, cultured in animal-component-free conditions, expanded to produce millions of cells, then enriched for CD44+ stem/progenitor cells by magnetic cell sorting. Normal epithelial purity was assessed using cytokeratins 5/14. Anti-CD133/PROM1 (cancer marker) and anti- fibroblast (clone TE-7) antibodies were used to demonstrate a lack of contaminating cells. Phenotype validation was performed by flow cytometry and immunocytochemistry on both CD44+ sorted and unsorted populations. Senescence-associated beta-galactosidase (SA-{beta}-gal) assays were performed across serial passages (P1-P6). Pluripotency was demonstrated by culture under conditions supporting lineage-specific differentiation. Primary hS/PCs demonstrated consistent expansion and epithelial morphology under serum-free conditions. CD44 expression remained high (>95%) throughout expansion, with negligible detection of CD133 or fibroblast markers, confirming epithelial purity and absence of tumorigenic or stromal contamination. Immunocytochemistry corroborated these expression profiles. SA-{beta}-gal staining revealed only a minor, passage-dependent increase (5-16%) in senescent cells from multiple donors, indicating retention of proliferative potential. Our defined, animal-free culture system supports stable expansion of pure low passage hS/PCs under conditions compatible with good manufacturing practice (GMP).

cell biology↗

bioRxiv2026

A Deep Learning Framework for Spatiotemporal Modeling of Visual Task fMRI

Characterizing the dynamic coordination of distributed brain regions during cognitive tasks remains challenging, as traditional fMRI analysis focuses on localized activations without revealing the underlying information flow that drives them. Here, we propose STREAM (Spatiotemporal Representation for Effective connectivity Analysis Model), a deep-learning framework that learns neural transition functions in task-fMRI to characterize effective connectivity and whole-brain information flow. Applied to visual category processing in 1074 participants, STREAM accurately reconstructs activation maps while further revealing that traditional activation regions are primarily driven by incoming signals. Moreover, the Default Mode Network acts as a high-level regulatory hub with extensive outgoing influence, challenging its passive characterization. Additionally, category-specific communication emerges from dynamic reconfiguration of signaling patterns among key hubs rather than static pathways. These findings establish a novel computational paradigm that uncovers directional signaling mechanisms driving local dynamics in task-fMRI, revealing how the brain flexibly reconfigures functional architecture for complex cognition.

neuroscience↗

bioRxiv2026

Cardiac oxidative stress monitoring enabled by hierarchical mechanical adaptation

Soft bioelectronics have advanced cardiac monitoring through electrophysiological tracking, yet this alone cannot resolve the metabolic pathology essential to surgical decision-making. However, real-time molecular sensing on beating hearts remains unresolved due to deformation-induced sensor failure and stress-induced metabolite artifacts. This challenge is exemplified by ischemia-reperfusion injury (IRI), a major cardiac surgery complication characterized by reactive oxidative species (ROS) bursts, where true pathological ROS signals being confounded by mechanotransduction-induced ROS artifacts. Herein, we propose an enzymatic cardiac oxidative stress biosensor (E-cardiac) with hierarchical mechanical adaptation: macro-scale biofluid-mediated contact, micro-scale fiber reorganization, and nano-scale enzymatic confinement within gold nanoarches dissipate interfacial stress. This produces ultrathin ([~]460 nm), soft (0.79 kPa) E-cardiac with robust electrochemical stability (100% strain), low detection limit (380 nM), rapid adhesion (<3 s), stable biosensing on beating heart, as well as minimal invasive deployment capability. Mechanical analysis and cellular studies confirm mitigated stress-induced ROS and absent PIEZO channel activation. Validated across cardiomyocytes, ex vivo tissues, multi-species ischemia models (mouse, rat, rabbit, pig), rat ischemia-reperfusion injury, and Langendorff hearts simulating graded perfusion deficits, E-cardiac quantitatively differentiates IRI severity (sham < ischemia < reperfusion) as well as detecting the "ECG blind window". The E-cardiac platform provides real-time metabolic feedback for surgical guidance during cardiac procedures, enabling timely intervention before irreversible damage.

bioengineering↗

bioRxiv2026

Molecular dissection of protein complexes isolated from sections of human brain

Molecular studies of brain receptors and transporters have typically relied on recombinant systems, limiting insight into their organization in native tissue. Here, we develop nanobody-based immunoprecipitation coupled with native mass spectrometry to interrogate endogenous protein assemblies from post-mortem mouse and human brain sections. We exemplify our approach by characterizing the synaptic proteins VGluT1 and mGluR2. From a single mouse brain, we discover mGluR2/3 heterodimers, alongside mGluR2 homodimers. Considering regions of human brain heterodimeric mGluR2/3 is highly abundant in the OFC and sgACC ([~]70% and 50%, respectively) and forms regional-specific interactions with additional synaptic proteins. In a modest cohort of biobanked human tissue, associated with depression and suicide, we find increased mGluR2/3 in the OFC. Consistent with this, mice exhibit similar associations between heterodimer levels and stress-susceptibility. Overall, our approach provides a direct means for establishing molecular-behavioural links at the level of receptor organization in brain.

biochemistry↗

bioRxiv2026

Nitrogen fertilization outweighs plant species loss in shaping bacterial belowground diversity in an alpine meadow on the central Tibetan Plateau

Plant species loss and nitrogen fertilization affect grassland biodiversity. However, their interactive effects on plant communities, soil properties, and the soil microbiome remain insufficiently understood. We analyzed how the removal of plant species, with and without urea addition, influenced plant diversity, soil properties, and soil bacterial communities in a Tibetan Plateau grassland. Continuous plant species removal and urea addition over seven years modified plant beta-diversity equally strong, while urea exerted a stronger negative effect on plant alpha-diversity. Both, plant species removal and urea addition caused soil acidification and an increase in NO2-/NO-, while dynamics in TOC, TON and TOC: TON were mainly driven by the growing season. Structural equation modeling identified soil acidification via urea addition as the most important indirect driver that negatively affected bacterial alpha-diversity and shifted bacterial beta-diversity. Urea addition also exerted direct negative effects on bacterial alpha- and beta-diversity, causing repression of oligotrophic (Acidobacteriota, Chloroflexota, Planctomycetota, Gemmatimonadota) and stimulation of copiotrophic (Bacillota, Bacteroidota, Pseudomonadota) bacterial taxa. Plant species removal caused slight increases in bacterial alpha-diversity, paralleled by less diverse but more even plant communities. We show that soil acidification by urea fertilization outweighs plant species loss in its negative effect on bacterial soil biodiversity in Tibetan grasslands.

ecology↗

bioRxiv2026

Estimating chronological and brain age using risk-taking behavior under uncertainty

Risky decision-making under uncertainty reflects complex cognitive processes supported by distributed brain networks that are vulnerable to aging. However, it remains unclear whether risk-taking behavior can serve as a behavioral marker of brain aging. In the present study, we combined behavioral tasks, computational modeling, and structural magnetic resonance imaging to investigate the relationship between risky decision-making, chronological age, and brain age. A total of 55 young adults and 112 healthy older adults completed the Iowa Gambling Task (IGT) and the Balloon Analogue Risk Task (BART), along with neuropsychological assessments and neuroimaging scanning. Decision processes were quantified using computational models, including the Value-Plus-Perseveration model and Exponential-Weight Mean-Variance. Brain age was estimated from gray matter volume. The results showed significant age-related alterations in parameters reflecting feedback sensitivity, learning rate, and loss aversion in both tasks. Within older adults, several decision parameters were significantly associated with both chronological age and brain age. Regression analyses further showed that computational parameters significantly predicted chronological age and brain age, whereas traditional cognitive screening measures did not show significant predictive effect. Structural brain analyses indicated that IGT-related parameters were primarily associated with the basal ganglia, while BART-related parameters were linked to a broader network including prefrontal, cingulate, and temporal regions. These findings suggest that computational markers of risk-taking behavior capture subtle age-related changes in cognitive processes and brain deterioration. Therefore, risk-taking parameters may serve as reliable functional markers of brain aging, providing critical insights into the mechanisms underlying successful aging.

neuroscience↗

bioRxiv2026

Multi-omics definition of the sex-specific glycoproteome of murine tissues

Sex-specific differences in the glycoproteome remain poorly defined despite growing evidence that protein glycosylation is a key determinant of sex biology. Here we present a tissue-resolved glycoproteome atlas of adult male and female C57BL/6J mice, integrating transcriptomics, proteomics and glycoproteomics with sialic acid speciation and lectin microarray profiling across 19 tissues. Quantitative analysis of >26,800 protein- and site-specific N-glycoforms from 1,512 glycoproteins revealed highly distinct tissue glycoproteomes shaped by coordinated regulation of protein abundance and glyco-enzyme expression. Multi-omics integration identified strong glycophenotype-enzyme relationships, including control of tissue sialylation by Cmas and Cmah, suggesting rate-limiting roles in glycosylation. Pronounced sex-linked glycophenotypes were observed in salivary gland, liver and kidney, driven by differences in fucosylation, sialylation and protein abundance, whereas the brain glycome was largely conserved between sexes. An interactive online database (https://igcore.cloud/mta/atlas-viewer/) provides a resource for exploring sex-biased glycosylation across mouse tissues.

cell biology↗

bioRxiv2026

Multi-component functionalized Bifidobacterium longum hydrogel for multi-target integrated therapy of colitis-associated anxiety and depression

Inflammatory bowel diseases (IBDs) are frequently accompanied by anxiety and depression, largely driven by perturbed gut-brain axis signaling. However, current oral therapies remain constrained by the spatial and functional separation between intestinal inflammation and central nervous system dysfunction. Here, we present a comprehensive gut-brain dual region integrated therapeutic strategy based on functionalized Bifidobacterium longum hydrogel (INPs@BL@Gel), in which baicalin and tyrosine are coordinated with Fe(III) to form infinite coordination polymers (ICPs), coated with inulin, assembled onto Bifidobacterium longum (BL), and subsequently encapsulated within a pH- and matrix metalloproteinase-responsive silk fibroin-gelatin hydrogel. INPs@BL@Gel exhibits high drug-loading, effective gastric protection, inflammation-triggered release, and long-term intestinal colonization. Within the inflamed intestine, BL and components synergistically suppress inflammatory responses, restore gut microbiota homeostasis, and promote intestinal barrier repair through multi-target integrated therapy. Importantly, BL combined with components markedly enhances the production of beneficial neuroactive metabolites such as homovanillic acid and short-chain fatty acids, which integrated regulate neuroinflammation, preserve synaptic function, and facilitate blood-brain barrier repair via the gut-brain axis. In vivo studies demonstrate that INPs@BL@Gel not only exert potent therapeutic efficacy against colitis and effectively alleviate associated depression, but also reshape the gut microbiota and restore barrier integrity, achieving an remarkable comprehensive therapeutic effect. O_FIG O_LINKSMALLFIG WIDTH=158 HEIGHT=200 SRC="FIGDIR/small/710940v1_fig1a.gif" ALT="Figure 11"> View larger version (59K): org.highwire.dtl.DTLVardef@1a831f3org.highwire.dtl.DTLVardef@1676617org.highwire.dtl.DTLVardef@15f99c1org.highwire.dtl.DTLVardef@1677cc4_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOScheme 1.C_FLOATNO (a) Schematic diagram of the design and preparation of functionalized Bifidobacterium longum hydrogel. (b) Exploration of the mechanism of INPs@BL@Gel in treating colitis-associated anxiety and depression through a dual-site multi-target synergistic strategy. C_FIG

pathology↗

bioRxiv2026

A Genetic Tool for Specific Tracking of Mature Neutrophils

Tracking mature neutrophils remains challenging due to the lack of reliable cell surface markers. Although CD101 is a promising candidate for mature neutrophils, its stability under pathological conditions is unclear. Using a CD101-tdTomato reporter mouse model, we confirmed that the reporting system does not alter CD101 expression, and tdTomato fluorescence is predominantly expressed in mature neutrophils across peripheral tissues. Further analysis revealed that CD101+ and tdTomato+ neutrophils display identical characteristics of mature neutrophil, including poly-segmented nuclei, cell size, and key functions under homeostasis. By comparing tdTomato fluorescence with CD101 protein levels, we demonstrate that reduced CD101 expression under pathological states was not attributed to shedding or degradation. Our finding enhances CD101 as a robust and reliable marker of neutrophil maturity, providing a foundation for future applications in spatial transcriptomics and lineage tracing studies to dissect neutrophil heterogeneity and function. Highlights of the studyO_LIIn CD101-tdTomato homozygous mice, tdTomato is predominantly expressed in neutrophils and labels nearly 100% of mature neutrophils, aligning with the phenotype of CD101+ mature neutrophils; C_LIO_LIThe CD101-tdTomato reporting system does not interrupt CD101 expression or neutrophil functions; C_LIO_LICD101 remains a stable and reliable cell surface marker for labeling mature neutrophils, even under pathological conditions. C_LI

immunology↗

bioRxiv2026

Active removal of inhibitory components drives the flagellarType III Secretion Specificity Switch

Type III secretion (T3S) systems assemble bacterial nanomachines, including the flagellum and virulence-associated injectisomes, by exporting distinct classes of substrates in a defined temporal order. In both systems, completion of an early assembly intermediate triggers an irreversible switch from early to late substrate secretion. In the flagellar system, this switch is controlled by the secreted molecular ruler FliK acting on the core T3S component FlhB, but the molecular mechanism governing this transition has remained unclear. Here we show that removal of two components, Fluke and the cleaved C-terminal domain of FlhB (FlhBCCD), locks the secretion apparatus in a constitutive late secretion state. In these mutants, secretion specificity no longer requires completion of the hook-basal body or the FliK ruler, indicating that Fluke and FlhBCCD function to maintain the apparatus in early secretion mode. Consistent with this model, synchronized flagellar gene expression experiments reveal that FlhBCCD is retained during early assembly and is lost coincident with hook-basal body completion and activation of {sigma}28-dependent late gene expression of flagellin and chemosensory genes. Structural modeling of the FliK C-terminal switch domain and FlhBCCD supports a mechanism in which secretion of FliK promotes destabilization and ejection of FlhBCCD from the secretion apparatus. Disruption of a folded region within FliK switch domain uncouples secretion from switching, indicating that the timing of FliK unfolding during secretion is critical for activation of the specificity switch. These findings show that secretion specificity switching is driven by FliK-dependent removal of inhibitory components, rather than passive sensing of assembly completion. Significance StatementType III secretion (T3S) systems build complex bacterial nanomachines, including the flagellum and virulence-associated injectisomes, by exporting distinct classes of substrates in a defined temporal order. How these systems switch secretion specificity during assembly has remained a long-standing question. We demonstrate that the flagellar T3S specificity switch requires removal of two inhibitory components that actively maintain the apparatus in an early secretion state. Their FliK-dependent ejection irreversibly triggers the transition to late substrate export, revealing that secretion switching is controlled by active inhibitory regulation rather than passive sensing of assembly completion. These results define a molecular mechanism for hierarchical control in bacterial secretion systems and provide insights likely relevant to other T3S nanomachines.

microbiology↗