Biology subjects

Xu, Z.

Publications and source records attributed to Xu, Z..

At least 163 records · Page 9Linked to original sources

bioRxiv2025

Brucella proline racemase protein A targets Tpl2 to promote IL-10 secretion for establishment of chronic infection

IL-10, an anti-inflammatory cytokine, plays a crucial role in limiting immune responses to pathogens, preventing host damage. However, the mechanisms underlying Brucella-mediated IL- 10 production remain incompletely understood. In this study, we demonstrate that the proline racemase protein A (PrpA) of Brucella melitensis M5-90 induces macrophages to secrete IL-10 by activating the Tpl2-ERK signaling pathway, thereby promoting chronic infection. Moreover, Tpl2 deletion impairs macrophage bactericidal ability, accompanied by reduced TNF- and IL-1{beta} but unaffected IL-10 levels. Additionally, Trp309, Glu103, and Glu129 of PrpA participate in interaction with Tpl2, but these residues do not influence PrpA-mediated IL-10 production in macrophages. PrpA deletion enhances IFN-{gamma} levels, specific anti-Brucella IgG, and CD4+ and CD8+ T cell numbers in mice. Furthermore, the Brucella melitensis M5-90 prpA mutant provides higher protection than the parental strain against virulent Brucella melitensis M28 infection in mice. Our findings suggest that Brucella PrpA promotes IL-10 secretion by macrophages through Tpl2 activation for bacterial survival and persistent infection, making the Brucella melitensis M5-90 prpA mutant a promising vaccine for enhanced protection. Author SummaryIL-10, an anti-inflammatory cytokine, is exploited by Brucella for survival. We identified Brucella PrpA as a potent IL-10 inducer, activating the Tpl2-ERK signaling pathway. Tpl2 deletion increased Brucella survival in macrophages, accompanied by reduced TNF- and IL-1{beta} but unaffected IL-10 secretion. Residues (Trp309, Glu103, and Glu129) of PrpA were crucial for interaction with Tpl2, yet did not impact PrpA-mediated IL-10 production. The B. melitensis M5-90 prpA mutant induced higher anti-Brucella IgG, IFN-{gamma}, and CD4+ and CD8+ T cell numbers in mice, providing better protection than B. melitensis M5-90. These findings unveil PrpAs role in IL-10 production and highlight the potential of the B. melitensis M5-90 prpA mutant as a promising vaccine for further evaluation.

microbiology↗

bioRxiv2025

Lemur: A Single-Cell Foundation Model with Fine-Tuning-Free Hierarchical Cell-Type Generation for Drosophila melanogaster

Single-cell genomics has revolutionized our understanding of cellular heterogeneity, but automating its analysis remains an open challenge. Cell-type annotation represents a critical bottleneck, particularly as datasets grow in size and complexity. While foundation models have shown promise in addressing this challenge, existing approaches require extensive fine-tuning for effective cell-type annotation. Here, we present Lemur (Large Expression Model for Understanding scRNA-seq), a single-cell foundation model specifically designed for Drosophila melanogaster. Lemur achieves fine-tuning-free cell-type annotation through comprehensive pre-training on an integrated whole-organism atlas with a unified cell-type annotation schema. To leverage this unified schema, we developed a dedicated hierarchical cell-type decoder architecture. This approach enables Lemur to generate consistent cell-type predictions across multiple levels of granularity without requiring additional training on new datasets. The model demonstrates strong performance across diverse tissue types, experimental conditions, and sequencing technologies. It also achieves batch-effect correction without explicitly training for this task. This automated analysis capability positions Lemur as an effective tool for the fly research community. Beyond its immediate applications, Lemur establishes a framework for accelerating biological discovery. It enables rapid iteration between computational predictions and experimental validation in the highly controlled Drosophila melanogaster system, with potential implications for translational research in human biology, particularly in aging and neurodegenerative disease studies.

bioinformatics↗

bioRxiv2025

The CST complex mediates a post-resection non-homologous end-joining repair pathway and promotes local deletions

Repair of a DNA double-strand break (DSB) by non-homologous end-joining (NHEJ) generally leaves an intact or minimally modified DNA sequence. Resection initiation exposes single-stranded DNA and directs repair towards homology-dependent pathways and away from NHEJ. Therefore, NHEJ is not thought to be an available repair pathway once the DSB is resected. Here, we report that the Cdc13/Stn1/Ten1 (CST) complex, well characterized for its telomere-associated functions, acts after resection initiation to mediate a backup NHEJ repair. We found a CST-specific mutation signature after DSB repair, characterized by deletions of 5-85 bp, mostly dependent on NHEJ. In contrast, NHEJ-mediated small deletions of 1-4 bp and insertions are not affected in CST mutants. The interaction between CST and Pol-primase is critical for these intermediate size deletions, suggesting a role for fill-in synthesis. Consistently, in stn1{Delta} and Pol-primase mutant deficient for interaction with CST, resection is increased, leading to larger deletions of several kilobases mediated by microhomologies. Collectively, these results depict a more complex picture of repair pathway choice where CST allows a post-resection NHEJ repair, promoting local deletions but guarding against much larger and potentially more deleterious deletions and rearrangements.

molecular biology↗

bioRxiv2025

Estimation of total mediation effect for a binary trait in a case-control study for high-dimensional omics mediators

Mediation analysis helps uncover how exposures impact outcomes through intermediate variables. Traditional mean-based total mediation effect measures may suffer from the cancellation of opposite component-wise effects, and existing methods often lack the power to capture weak effects in high-dimensional mediators. Additionally, most high-dimensional mediation analysis methods have focused on continuous outcomes, with limited attention to binary outcomes, particularly in case-control studies. To fill this gap, we propose an R2 total mediation effect measure within the liability framework that offers a clear and intuitive causal interpretation, provides additional insights beyond the mean-based measures, and is invariant to disease prevalence. We develop a cross-fitted, modified Haseman-Elston regression-based estimation procedure tailored for mediation analysis in case-control studies, which can also be applied to cohort studies. Our estimator remains consistent in the presence of non-mediators and weak effects, as demonstrated in extensive simulations. Theoretical justification for consistency is provided under mild conditions and without requiring exact mediator selection. In a case-control substudy of the Womens Health Initiative involving 2150 individuals, we found that many metabolites were mediators with weak effects in the path from BMI to coronary heart disease, and we estimated that 89% (95% CI: 57%-100%) of the BMI-explained variation in underlying CHD liability is mediated by the measured metabolomics. The proposed estimation procedure is implemented in the R package "r2MedCausal", available on GitHub.

genomics↗

bioRxiv2025

Mouse Cortical Cellular Diversification Through Lineage Progression of Radial Glia

Cortical radial glia (RGs) sequentially generate pyramidal neurons (PyNs) and glia. In this study, we investigated the cell intrinsic programs underlying cortical cellular diversification using time-series scRNA-seq and snATAC-seq on purified mouse cortical progenitors across embryonic and postnatal stages. Our data revealed that RGs transition from early to late over time, sequentially producing intermediate neuronal progenitors (INPs) and intermediate glial progenitors (IGPs). While INPs expand exclusively to generate PyNs, IGPs progress from young to old, sequentially producing cortical astrocytes, oligodendrocytes, and olfactory bulb interneurons. We constructed comprehensive molecular maps that reflect cell lineage progression. In particularly, we found that chromatin accessibility drives cortical cellular diversification by restricting broadly expressed transcription factors to specific stages and cell types. Developmental changes in chromatin accessibility confine Lhx2-induced neurogenesis to early-stage RGs, leading to the loss of neurogenic competence and the acquisition of gliogenic competence as corticogenesis progresses.

neuroscience↗

bioRxiv2025

Single-Cell Genomics Elucidates Molecular Variations and Regulatory Mechanisms in Circulating Immune Cells

The human peripheral blood displays diverse molecular characteristics across populations, understanding the drivers and underlying mechanisms of which remains challenging. Here, we introduce the Chinese Immune Multi-Omics Atlas (CIMA), elucidating sex-, age-, and genetic-related molecular variations by analyzing multi-omics data from 428 adults with over 10 million immune cells. CIMA generated an enhancer-driven gene regulatory network, identifying 237 high-quality regulons and revealing cell type-specific regulatory mechanisms. Additionally, 11,521 lead cis-expression quantitative trait loci (eQTLs) and 46,339 chromatin accessibility QTLs (caQTLs) were identified at cell type level. CIMA also uncovered pleiotropic associations among immune-related disease risk loci, eQTLs, and caQTLs in a cell type-specific manner. Lastly, a novel cell language model, CIMA-CLM, was developed to predict chromatin accessibility and noncoding variant effects using chromatin sequences and gene expressions. This work represents a population-scale multi-omics resource of human immune cells, providing a valuable reference for future investigation of immune-related diseases.

immunology↗

bioRxiv2025

CPI-Pred: A deep learning framework for predicting functional parameters of compound-protein interactions

Recent advancements in deep learning have enabled functional annotation of genome sequences, facilitating the discovery of new enzymes and metabolites. However, accurately predicting compound-protein interactions (CPI) from sequences remains challenging due to the complexity of these interactions and the sparsity and heterogeneity of available data, which constrain the generalization of patterns across their solution space. In this work, we introduce CPI-Pred, a versatile deep learning model designed to predict compound-protein interaction function. CPI-Pred integrates compound representations derived from a novel message-passing neural network and enzyme representations generated by state-of-the-art protein language models, leveraging innovative sequence pooling and cross-attention mechanisms. To train and evaluate CPI-Pred, we compiled the largest dataset of enzyme kinetic parameters to date, encompassing four key metrics: the Michaelis-Menten constant (KM), enzyme turnover number (kcat), catalytic efficiency (kcat/KM), and inhibition constant (KI).These kinetic parameters are critical for elucidating enzyme function in metabolic contexts and understanding their regulation by compounds within biological networks. We demonstrate that CPI-Pred can predict diverse types of CPI using only the amino acid sequence of enzymes and structural representations of compounds, outperforming state-of-the-art models on unseen compounds and structurally dissimilar enzymes. Over workflow provides a valuable tool for tackling a range of metabolic engineering challenges, including the designing of novel enzyme sequences and compounds, such as enzyme inhibitors. Additionally, the datasets curated in this study offer a valuable resource for the scientific community, serving as a benchmark for machine learning models focused on enzyme activity and promiscuity prediction.

bioinformatics↗

bioRxiv2025

Persistent interferon signaling that causes sensory neuron plasticity and pain in arthritis

While the inflammatory processes in rheumatoid arthritis have been described, mechanisms driving pain are poorly defined. Here, we used a multitude of approaches to uncover the neural basis, mediators, intracellular signaling pathway and the mechanism of inflammatory pain. In cartilage autoantibody-induced arthritis mice, an early immune-activation and a cytokine storm were mainly driven by vascular cells and monocyte/macrophages in the dorsal root ganglion. However, persistently elevated interferons and receptor-activation of the MNK1/2-eIF4E signaling pathway at all disease phases caused sensory-motor dysfunction and pain by inducing hyperexcitability and sensitization of Gfra3+ sensory neurons. Like mice, human sensory neurons expressed interferon receptors and interferons were elevated only in individuals with painful rheumatoid arthritis. Signaling pathway inhibition in vivo reversed pain and restored limb function. The finding that joint pain before and during arthritis is caused by a defined cytokine and signaling pathway holds promise for targeted therapies for pain relief in arthritis.

neuroscience↗

bioRxiv2025

Sequencing-based Spatial Transcriptomics with scRNA-seq Sensitivity

The advent of spatial transcriptomics has dramatically expanded our ability to study the vast network of cell-cell interactions at the molecular level in tissue. Among current methods, sequencing-based approaches have great potential in discovering because of its unbiased capture. In the last couple of years, the spatial resolution for the capture addresses has been significantly improved from 100 m to <1 m, well below the size of a mammalian cell. However, the capture efficiency has always been a pain point, [~]one order of magnitude lower than that of single cell RNA sequencing (scRNA-seq). The low capture efficiency limits the depth and breadth of its applications in the study of complex biological systems and diseases. Here, we introduce Salus Spatial transcriptomic system (Salus-STS), which provides [~]1 m capture resolution and a capture efficiency [~]1 order of magnitude higher than other current methods. Analysis with sub-cellular resolution becomes practical for sequencing-based spatial transcriptomics.

molecular biology↗

bioRxiv2025

Ether Rhodamines with Enhanced Hydrophilicity, Fluorogenicity, and Brightness for Super-Resolution Imaging

Rhodamine dyes are widely used fluorophores in super-resolution fluorescence imaging due to their exceptional optical properties and "aggregation-disaggregation" induced fluorogenic activation. However, their excessive lipophilicity often reduces brightness in aqueous environments and causes off-target staining, limiting their effectiveness in high-resolution imaging. To address these challenges, we introduce an ether-decorated N-terminal modification strategy for rhodamine and silicon-rhodamine (Si-rhodamine), replacing conventional N-alkyl groups. The ether chains enhance water solubility, decrease aggregate size, and improve fluorogenicity across a wide concentration range. Their flexible, hydrophilic structure forms a protective shield around the xanthene core, minimizing dye-water interactions and reducing fluorescence quenching. Additionally, the inductive effect of the ether chains decreases the electron-donating strength of the amino groups, suppressing quenching caused by twisted intramolecular charge transfer (TICT). These modifications collectively increase the quantum yields of ER and ESiR in water from 0.35 and 0.19 (for tetraethyl-substituted analogs) to 0.70 and 0.41, respectively. Probes derived from ER and ESiR exhibit outstanding fluorogenicity, enhanced signal-to-noise ratios, and improved resolution in complex aqueous environments, demonstrating superior performance in advanced super-resolution imaging techniques such as structured illumination microscopy (SIM), stimulated emission depletion (STED) microscopy, and single-molecule localization microscopy (SMLM). This work introduces an innovative strategy for fluorophore design, offering significant advancements for super-resolution imaging applications.

biochemistry↗

bioRxiv2024

RNAGenesis: Foundation Model for Enhanced RNA Sequence Generation and Structural Insights

RNA molecules are central to gene regulation, catalysis, and molecular recognition, and offer broad opportunities for therapeutic applications. However, uncovering their complex sequence, structure, and function relationships, particularly for non-coding RNAs, remains a formidable challenge. Here, we introduce RNAGenesis, a Generalist RNA foundation model that integrates sequence representation, structural prediction, and de novo functional design within a single generative framework. Trained on diverse clustered non-coding RNAs, RNAGenesis leverages a BERT-style encoder, query-based latent compression, and a diffusion-guided decoder enhanced by inference-time alignment with gradient guidance and beam search strategies. Through comprehensive evaluations, RNAGenesis achieves state-of-the-art performance on 11 of 13 tasks in the BEACON benchmark and surpasses structure-aware models in inverse folding, 3D structure prediction, and de novo structure design. We further introduce RNATx-Bench, a dedicated benchmark for RNA therapeutics comprising over 100,000 experimentally validated sequences. RNAGenesis demonstrates strong predictive performance across ASOs, siRNAs, shRNAs, circRNAs, and untranslated region (UTR) variants. Furthermore, RNAGenesis enables functional RNA design, including aptamers targeting IGFBP3 and structurally constrained sgRNA scaffolds. Wet-lab validation confirms aptamer binding with KD values as low as 4.02 nM and up to 2.5-fold improvement in editing efficiency across CRISPR-Cas9, base editing, and prime editing systems. These results position RNAGenesis as a next-generation general-purpose RNA foundation model with broad utility for computational modeling and experimental therapeutic design.

bioinformatics↗

bioRxiv2024

A General Strategy to Endow Dyes with Genic Fluorescence for Wash-Free Imaging of Proteins in Living Cells

Fluorescent dyes are the main fluorophore for protein labeling and fluorescent imaging in living cells. However, due to the inevitable nonspecific binding of the dye to non-targets within the cell, background signals are generated, which severely affect the imaging quality. Here, we endow the dye with genic fluorescence by introducing a fluorescent proteins FRET pair, such that only the dye bound to the target protein emits fluorescence ({lambda}ex = FPs, {lambda}em = Dyes), while nonspecifically bound dye remains non-fluorescent. This significantly improves the signal-to-noise ratio (SNR) in wash-free imaging. This strategy is achieved by fusing the Halo-tag to fluorescent proteins (sfGFP, mCherry) that can generate FRET with Halo dyes O-Rho and Si-Rho. The FPs serve as the donor, and the FRET mechanism imparts genic fluorescence property to O-Rho and Si-Rho. Since the excitation wavelength of the donor ({lambda}ex = FPs) cannot excite the unspecifically dye fluorescence, background fluorescence interference is reduced. We further improved the SNR by increasing the FRET efficiency between the FPs and dyes. The system was used for four-color super-resolution imaging of target proteins and dynamic SIM tracking of mitochondria. In addition, the genic FRET fluorophore constructed with the ultra-stable mStayGold showed significant photobleaching resistance and can be used for long-term dynamic super-resolution imaging. Theoretically, by selecting appropriate FRET pairs, this method can improve the SNR of any fluorophore, providing a new labeling strategy for live-cell wash-free imaging.

biochemistry↗

bioRxiv2024

Development of a novel peripherally acting alpha2A-adrenergic receptor antagonist for anti-diabetic

Yohimbine, a potent alpha2A-adrenergic receptor (2AAR) antagonist, was found therapeutic potential for type 2 diabetes through improving insulin release. However, the adverse side effects mediated by its actions in the brain hampered its use. Here, based on molecular docking analysis and structural modification, we have developed a novel peripherally acting yohimbine derivative (CDS479-2). CryoEM data found that yohimbine and CDS479-2 have similar interactions with the structure of 2AAR. Importantly, CDS479-2 shows similar 2AAR antagonist activity as yohimbine, but with very limited access to the brain, and thus avoiding the unwanted central effects such as hypertension and anxiety. Acute administration of CDS479-2 by injection or gavage lowered blood glucose levels and improved glucose tolerance in the high-fat diet-induced obesity (DIO) mice, an animal model for human type 2 diabetes. Remarkably, DIO mice received 2 weeks of daily administration of CDS479-2, but not yohimbine, exhibited sustained normoglycaemia, and increased density of the insulin-producing beta cells, in which important proliferation genes were found upregulated. Moreover, the overall protein expression levels of their pancreas were more similar to that of the healthy chow-fed mice. Thus, CDS479-2 may indicate a new direction for type 2 diabetes treatment. Importantly, the strategy we employed in this study will inspire the optimization for drugs that with both peripheral and central targets. Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=166 HEIGHT=200 SRC="FIGDIR/small/628818v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@1d3d7corg.highwire.dtl.DTLVardef@1afbad3org.highwire.dtl.DTLVardef@12a1aborg.highwire.dtl.DTLVardef@1341a02_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2024

Mbd4 and MutSα protect cells from spontaneous deamination of 5-methylcytosine.

5-Methylcytosine (5mC) is a common source of somatic mutations. Deamination of 5mC to thymine generates a G/T mismatch, which occurs spontaneously and must be repaired prior to DNA replication to avoid mutation. We generated genetically engineered mice and cell lines to define DNA repair pathways that protect against methylation damage. We observed a low background mutation rate in mouse bone marrow or colon, typically 0.2-0.5 CG>TG mutations/genome/day. This increased 3-7 fold in cells lacking the glycosylase Methyl-binding domain 4 (Mbd4), one of the few glycosylases capable of excising thymine from G/T mismatches. We found no role for Thymine DNA glycosylase (Tdg) in methylation damage repair. Instead, our results support cooperation between Mbd4 and the mismatch repair (MMR) complex MutS (Msh6:Msh2), evident through elevated rates of methylation damage in Msh6-deficient cells; increasing to 2.6-4.8 CG>TG mutations/genome/day in primary cells and up to 13.9 CG>TG mutations/genome/day in cell lines. Our findings support the view that MutS has DNA repair activity outside of replication. While loss of Mbd4 elevates methylation damage selectively, the broader functionality of MutS explains why mutational signatures linked to Msh6-deficiency are variable and reflect the replicative history of the cell.

molecular biology↗

bioRxiv2024

Nucleosome dynamics render heterochromatin accessible in living human cells

The eukaryotic genome is packaged into chromatin, which is composed of a nucleosomal filament that coils up to form more compact structures. Chromatin exists in two main forms: euchromatin, which is relatively decondensed and enriched in transcriptionally active genes, and heterochromatin, which is condensed and transcriptionally repressed 1-10. It is widely accepted that chromatin architecture modulates DNA accessibility, restricting the access of sequence-specific, gene-regulatory, transcription factors to the genome. Here, we measure genome accessibility at all GATC sites in living human MCF7 and MCF10A cells, using an adenovirus vector to express the sequence-specific dam DNA adenine methyltransferase. We find that the human genome is globally accessible in living cells, unlike in isolated nuclei. Active promoters are methylated somewhat faster than gene bodies and inactive promoters. Remarkably, both constitutive and facultative heterochromatic sites are methylated only marginally more slowly than euchromatic sites. In contrast, sites in centromeric chromatin are methylated slowly and are partly inaccessible. We conclude that all nucleosomes in euchromatin and heterochromatin are highly dynamic in living cells, whereas nucleosomes in centromeric -satellite chromatin are static. A dynamic architecture implies that simple occlusion of transcription factor binding sites by chromatin is unlikely to be critical for gene regulation.

genomics↗

bioRxiv2024

Essential genes encoded by the mating-type locus of the human fungal pathogen Cryptococcus neoformans

Fungal sexual reproduction is controlled by the mating-type (MAT) locus. In contrast to a majority of species in the phylum Basidiomycota that have tetrapolar mating-type systems, the opportunistic human pathogen Cryptococcus neoformans employs a bipolar mating-type system, with two mating types (a and ) determined by a single MAT locus that is unusually large ([~]120 kb) and contains more than 20 genes. While several MAT genes are associated with mating and sexual development, others control conserved cellular processes (e.g. cargo transport and protein synthesis), of which five (MYO2, PRT1, RPL22, RPL39, and RPO41) have been hypothesized to be essential. In this study, through genetic analysis involving sporulation of heterozygous diploid deletion mutants, as well as in some cases construction and analyses of conditional expression alleles of these genes, we confirmed that with the exception of MYO2, both alleles of the other four MAT genes are indeed essential for cell viability. We further showed that while MYO2 is not essential, its function is critical for infectious spore production, faithful cytokinesis, adaptation for growth at high temperature, and pathogenicity in vivo. Our results demonstrate the presence of essential genes in the MAT locus that are divergent between cells of opposite mating types. We discuss possible mechanisms to maintain functional alleles of these essential genes in a rapidly-evolving genomic region in the context of fungal sexual reproduction and mating-type evolution. IMPORTANCESexual reproduction is essential for long-term evolutionary success. Fungal cell type identity is governed by the MAT locus, which is typically rapidly evolving and highly divergent between different mating types. In this study, we show that the a and alleles of four genes encoded in the MAT locus of the opportunistic human fungal pathogen C. neoformans are essential. We demonstrate that a fifth gene, MYO2, which had been predicted to be essential, is in fact dispensable for cell viability. However, a functional MYO2 allele is important for cytokinesis and fungal pathogenicity. Our study highlights the need for careful genetic analyses in determining essential genes, which is complementary to high-throughput approaches. Additionally, the presence of essential genes in the MAT locus of C. neoformans provides insights into the function, maintenance, and evolution of these fast-evolving genomic regions.

genetics↗

bioRxiv2024

Cycledesigner Leveraging RFdiffusion and HighFold to Design Cyclic Peptide Binders for Specific Targets

Cyclic peptides are potentially therapeutic in clinical applications, due to their great stability and activity. Yet, designing and identifying potential cyclic peptide binders targeting specific targets remains a formidable challenge, entailing significant time and resources. In this study, we modified the powerful RFdiffusion model to allow the cyclic peptide structure identification and integrated it with ProteinMPNN and HighFold to design binders for specific targets. This innovative approach, termed cycledesigner, was followed by a series of scoring functions that efficiently screen. With the combination of effective cyclic peptide design and screening, our study aims to further broaden the scope of cyclic peptide binder design.

bioinformatics↗

bioRxiv2024

Tetramer formation of CpoS facilitates Inc-Inc interactions during Chlamydia trachomatis infection

Chlamydia trachomatis (C.t.), the leading bacterial cause of sexually transmitted infections, replicates within a unique intracellular compartment called the inclusion, which is modified by secreted proteins known as inclusion membrane (Inc) proteins. Here we further characterize CpoS, an Inc previously shown to be critical for replication and inclusion development. We demonstrate that CpoS directly binds multiple coiled-coil domain-containing Incs while simultaneously engaging Rab GTPases at a separate site. Notably, CpoS-InaC interactions facilitate the recruitment of select Arfs to the inclusion membrane, while Rab recruitment occurs independtly of these interactions. Biochemical and biophysical analyses revealed that Incs self-oligomerize, forming higher-ordered structures, with CpoS adpoting a tetrameric structure resembling eukaryotic SNAREs. We propose these assemblies likely serve as scaffolds to orchestrate vesicle docking, tethering, and fusion. Our findings underscore the intricate interplay between bacterial and host factors, revealing that C.t. leverages both Inc-Inc interactions and host protein engagement to manipulate vesicular trafficking and sustain infection.

microbiology↗