Biology subjects

Feng, X.

Publications and source records attributed to Feng, X..

At least 55 records · Page 3Linked to original sources

bioRxiv2025

Dengue Virus NS1 Binds Ephrin B1 to Trigger Endothelial Dysfunction

Dengue virus (DENV), a member of the Flaviviridae family, is the most prevalent and medically important mosquito-borne viral pathogen. Infected cells secrete the viral non-structural protein 1 (NS1) into the bloodstream, where it can interact with endothelial cells to induce vascular leak. Host factors involved in NS1-mediated endothelial dysfunction are incompletely understood, leading us to investigate the host interactome of DENV NS1 in endothelial cells via a comparative mass spectrometry approach. We identified ephrin B1 (EFNB1) as a critical host factor in NS1-mediated endothelial barrier dysfunction and show that phosphorylation of EFNB1 is necessary for induction of barrier dysfunction. Further, we map the interface of the EFNB1-NS1 complex through biochemical and computational approaches, and we show that EFNB1-Fc-fusion proteins can act as decoys to block NS1-induced barrier dysfunction in vitro and in vivo. This study provides insights into the mechanism of flavivirus NS1-mediated endothelial barrier dysfunction and new avenues to target vascular leak.

microbiology↗

bioRxiv2025

Microcompartments in Hodarchaeales: a bioenergetic engine that could have fuelled eukaryogenesis

Eukaryotic intracellular compartmentalization is a key innovation in the evolution of complex cellular life. While microcompartments enable metabolic specialization in many bacteria, to our knowledge, no analogous systems have been identified in Archaea. Here, we report the discovery of archaeal microcompartments (AMCs) in Hodarchaeales, an order within the phylum Promethearchaeati (Asgard archaea) that includes the closest known archaeal relatives of eukaryotes. Phylogenetic and structural analyses indicate that these catabolic AMCs, which are specialized for sugar-phosphate metabolism, were acquired by horizontal gene transfer from deep-rooted bacteria of the phylum Myxococcota. The shell pentamers of AMCs are fused to lysine/arginine-rich intrinsically disordered regions that capture cytosolic DNA, facilitating nutrient scavenging. Reaction-diffusion modelling predicts that enzyme colocalization and substrate channelling within AMCs can increase the NADH flux approximately 100-fold. Thus, the AMCs substantially boost energy production in the cell and might have primed the archaeal host for eukaryogenesis.

microbiology↗

bioRxiv2025

Development of an efficient PEG-Mediated protoplast transformation system for the medicinal fungus Ophiocordyceps xuefengensis

Ophiocordyceps xuefengensis is a newly identified medicinal fungus with significant pharmacological and economic value, but its genetic manipulation has been impeded by the lack of an efficient transformation system. Here, we established the first stable and highly efficient polyethylene glycol (PEG)-mediated protoplast transformation platform for O. xuefengensis using hygromycin B as a selectable marker. Through systematic optimization of critical parameters such as enzyme composition, enzyme concentration, mycelial age, and digestion conditions, we developed an optimized protocol for protoplast preparation. A high protoplast yield of 9.42x107 CFU/mL was achieved using 4-day-old mycelia with 1.5% lywallzyme 1 and 1.5% snailase digested at 34{whitebullet}C with shaking at 130 rpm for 3.5 h. PY medium containing 0.6 M mannitol significantly enhanced protoplast regeneration. Stable plasmid integration and robust hygR gene expression were confirmed through PCR detection and sustained antibiotic resistance over four successive generations. Furthermore, a controllable expression system was established by using the endogenous promoter that drove the stable expression of the glycoside hydrolase gene cbhI. The result of enzymatic assay confirmed the functional production of CBHI enzyme, demonstrating the credibility of this system. This work provides a reliable genetic toolbox for functional genomics studies, targeted gene manipulation, and strain engineering in O. xuefengensis, facilitating fundamental research and sustainable utilization of this valuable medicinal species. IMPORTANCEThe conservation and sustainable utilization of medicinal fungi represents a critical challenge in biotechnology and natural product research. Ophiocordyceps xuefengensis, a newly identified species with significant pharmacological potential, exemplifies this challenge: its wild resources are diminishing while artificial cultivation remains restricted by limited understanding of its fundamental biology. The lack of genetic tools has impeded progress in elucidating its biosynthetic pathways, regulating fruiting body development, and enhancing metabolite production. We established a highly efficient PEG-mediated transformation system that directly addresses this technological gap. In addition, we demonstrated that this transformation system can support heterologous gene expression using endogenous promoters. This platform enables reliable genetic manipulation in O. xuefengensis, permitting functional genomics studies and targeted bioactive compounds synthesis.

microbiology↗

bioRxiv2025

Reprogrammable bacterial nanosyringes for RNA and gene editors delivery

The engineered Photorhabdus virulence cassette (PVC) has not yet achieved the RNA packaging and injection delivery, although it can accurately deliver diverse proteins. Fortunately, RNA-binding proteins have a high affinity for binding to specific sequences and are excellent anchoring components between RNA and the inner tube of PVC. By combining the U1A RNA-binding domain (U1A RBD) and PVC, we developed a convenient, RNA non-degradable, and universal PVC-based RNA delivery tool, establishing DART (PVC Docker-based All-purpose RNA Injection Delivery Tool). DART successfully achieved the delivery of diverse RNAs, including Pepper RNA, gRNA, siRNA, and miRNA. We also demonstrated that the DART enabled effective knockout of GFP in vivo and in vitro. Our DART system provided a powerful platform for RNA and gene editing therapeutics in vivo.

molecular biology↗

bioRxiv2025

Methane Cycling Microbes are Important Predictors of Methylmercury Accumulation in Rice Paddies

Microbial production of methylmercury from inorganic mercury in rice paddies poses health risks to consumers of this essential dietary staple. Although mercury-methylating communities are well characterized, the microbial guilds contributing to methylmercury accumulation in rice paddies remain unclear. Here, we collected paddy soils across a mercury concentration gradient throughout the rice growing season to identify microbial and environmental factors influencing methylmercury dynamics. We show that hgcA gene abundance, the key gene required for methylation, was not a significant predictor of methylmercury concentration in paddy soils. We also show that merB gene abundance correlated with methylmercury in mercury-polluted rhizosphere samples. Methane cycling genes were actively expressed, and their beta-diversity was significantly associated with methylmercury levels. Methanogen abundance correlated with higher methylmercury under elevated total mercury concentrations. Analysis of the methanotroph-associated mbnT gene, implicated in demethylation, revealed an unexpected positive correlation with methylmercury. Multiple regression and machine learning models converged on mercury bioavailability and methanogen/methanotroph abundances as key predictors of methylmercury, with methanogen-associated hgcA gene abundance and methanogen-methanotroph interactions highlighted under flooded, low-redox conditions. These findings suggest that methane-cycling microbes play key roles in methylmercury cycling dynamics and point to management strategies that could simultaneously mitigate mercury pollution and greenhouse gas emissions. ImportanceMethylmercury is a microbially-derived neurotoxin that accumulates in rice, which is a global food staple. Predicting mercurys fate in rice paddies is challenging because of the interplay between microbes responsible for methylmercury cycling and variables that control mercury availability. Our study coupled genomic and geochemical measurements with machine learning to identify the key predictors of methylmercury accumulation in paddy soils. We demonstrate that methanogen and methanotroph abundance, and mercury bioavailability, are major predictors of methylmercury variability in paddies. We show that considering interactions between methane cycling guilds improves our capacity to predict methylmercury accumulation in soils compared to approaches that rely solely on mercury cycling genes. This work can inform remediation strategies for mercury in rice paddies but also wetlands and permafrost where methane and mercury cycling are tightly coupled. Such strategies could provide a solution to simultaneously mitigate methylmercury exposure and reduce greenhouse gas emissions amid global environmental change.

microbiology↗

bioRxiv2025

Chronic kidney disease exacerbates hyperlipidemia and atherosclerosis via miR-148a-3p targeting SIK1/AMPKα1 in liver

BackgroundCardiovascular complications are the leading cause of death in patients with chronic kidney disease (CKD). As a major risk factor for atherosclerotic cardiovascular disease (ASCVD), hyperlipidemia is a common sequela of CKD, which is linked to increased lipid synthesis, impaired lipoprotein clearance, and maladapted reverse cholesterol transport. Because exosomes can transport miRNAs to modulate intercellular and tissue communication, we hypothesized that exosomal miRNAs can act as endocrine-like molecules to modulate the CKD-associated hyperlipidemia and ASCVD. MethodsSmall RNA-seq and qPCR were used to determine the differential plasma level of exosomal miR-148a-3p in ASCVD patients with and without CKD. RNA-seq and in silico analysis were used to establish the regulation of lipid metabolism via miR-148a-3p/salt-inducible kinase 1 (SIK1)/AMP-activated protein kinase alpha 1 subunit (AMPK1) in the liver. Inhibition of miR-148a-3p or overexpression of SIK1/AMPK1 in ApoE-/- mice with 5/6 nephrectomy was used to elucidate the role of the miR-148a-3p/SIK1/AMPK1 axis in CKD-related hyperlipidemia and atherosclerosis. ResultsThe plasma level of exosomal miR-148a-3p was significantly elevated in patients with ASCVD and CKD (ASCVD/CKD) and ApoE-/- mice with 5/6 nephrectomy. Via circulating exosomes, kidney-generated miR-148a-3p was delivered to the liver, where it targeted SIK1/AMPK1 transcripts, thereby upregulating SREBP2/PCSK9 and reciprocally downregulating LDLR. Levels of SIK1 and AMPK1 were decreased in hepatocytes transfected with miR-148a-3p or treated with plasma exosomes from ASCVD/CKD patients, with attendant SREBP2 activation and attenuated LDL-C binding. The decreased LDL-C binding was rectified by SIK1/AMPK1 or LNA-miR-148a-3p overexpression. LNA-miR-148a-3p or AAV8-SIK1/AMPK administration in 5/6 nephrectomy/ApoE-/- mice significantly ameliorated hyperlipidemia and atherosclerosis. ConclusionIn ASCVD/CKD patients and nephrectomy/ApoE-/- mice, kidney-originating miR-148a-3p targeted hepatic SIK1/AMPK1 mRNA. This exosome-mediated endocrine effect upregulated SREBP2/PCSK9 and reciprocally reduced LDLR level, which elevated plasma LDL-C level and exacerbated ASCVD. These findings underscore a kidney-liver-artery axis involved in cardiovascular-kidney-metabolic syndrome.

pathology↗

bioRxiv2025

The DND1-NANOS3 complex shapes the primordial germ cell transcriptome via a heptanucleotide sequence in mRNA 3'UTRs

The RNA-binding proteins DND1 and NANOS3 are essential for primordial germ cell survival1-5. Their co-immunoprecipitation and overlapping loss-of-function phenotypes suggest joint function6-8, yet how they co-regulate target mRNAs remains unclear. Here, we developed Tandem PAR-CLIP and identified a DND1-NANOS3 ribonucleoprotein that specifically recognizes an AUGAAUU heptanucleotide on target mRNAs, termed the NANOS3-dependent DND1 Recognition Element (N3-DRE). mRNAs containing 3'-UTR N3-DREs are aberrantly upregulated in DND1- or NANOS3-deficient germ cells and encode key cell-cycle and epigenome regulators, such as CDK1. Genome editing showed that the N3-DRE is essential for Cdk1 repression in mouse PGCs in vivo. A 1.7-[A] crystal structure of the ternary complex of DND1, NANOS3, and CDK1- N3-DRE RNA revealed a continuous RNA-binding surface that confers high-affinity, sequence- specific recognition. Together, these findings define the molecular and functional basis of N3-DRE-mediated mRNA regulation in germ cell development. Moreover, we provide a paradigm of two RNA-binding proteins with low (DND1) or no (NANOS3) intrinsic sequence-specificity, jointly building a high-information-content RNA sequence motif that is different from the sum of their individual preferences. Because RNA-binding protein specificities are typically studied individually9-13, rather than in the context of ribonucleoproteins, this type of "two-factor authorization" may be an underappreciated mechanism to protect posttranscriptional gene regulatory networks from aberrant expression of an individual ribonucleoprotein component.

molecular biology↗

bioRxiv2025

A neural circuit for female-specific defensive homeostasis in risk assessment

HighlightsSC CB1R+ neurons encode visual survival threats and initiate risk assessment in both sexes CB1R+ SC-LHb GABAergic circuit maintains defensive homeostasis in risk assessment in females eCBs gate this circuit by disinhibiting presynaptic GABA release exclusively in females Disruption of this mechanism female-specifically impairs risk assessment and stress coping In BriefLiu et al. identify a female-specific subcortical circuit in which eCB signaling, via CB1R on GABAergic terminals in the SC, suppresses transmission to the LHb to maintain precise defensive homeostasis in risk assessment. These findings uncover the circuit and neuronal mechanisms that contribute to the sexually dimorphic maintenance of defensive homeostasis. Risk assessment in defensive behavior is an adaptive mechanism shaped by natural selection, enabling individuals to evaluate potential threats and thereby maintain defensive homeostasis. However, it remains unknown whether specific neural circuits maintain their behavioral homeostasis in a sex-specific manner. To address this, we investigated its neural basis by hierarchical behavior analysis framework with neural circuit dissection. In mice of both sexes, visual survival threats activated cannabinoid 1 receptor (CB1R)-expressing neurons in the superior colliculus (SC) to initiate consistent risk assessment. Deletion of CB1R in SC GABAergic neurons female-specifically impairs risk assessment by disinhibiting GABA release in SC-lateral habenula (LHb) projections, resulting in shortened risk assessment. This disruption furthermore increases the occurrence of abnormal spontaneous behavior following chronic stress exclusively in females. We identified a female-specific SC-LHb GABAergic circuit gated by CB1R maintains defensive homeostasis in risk assessment. Our findings reveal how a conserved neuromodulatory system sex-specifically gates a subcortical circuit to orchestrate distinct survival strategies.

neuroscience↗

bioRxiv2025

Single-nucleus transcriptome analyses uncover a dynamic transcriptional landscape of soybean roots in response to soybean cyst nematode infection

Soybean cyst nematode (SCN, Heterodera glycines) causes substantial yield losses by inducing the formation of specialized feeding structure called syncytia, which arise from extensive reprogramming of host root cells. Despite their pivotal role in SCN parasitism, the molecular mechanisms governing syncytium formation and function remain poorly understood, largely due to the technique challenges in precisely isolating these rare cell types. Here, we employed single-nucleus RNA sequencing (snRNA-seq) to generate a comprehensive, high-resolution transcriptomic atlas of SCN-infected soybean roots, comparing two resistant cultivars (PI 88788 and Forrest) with a susceptible cultivar (Williams 82) across multiple infection stages. By leveraging the well-characterized syncytium marker GmSNAP18, we successfully identified and transcriptionally profiled syncytial cells, and validate the spatial expression of known SCN-resistant genes. Our analyses revealed strong induction of resistance-associated genes specifically enriched in syncytia from resistant cultivars. Functional validation further demonstrated that overexpression of three novel candidate genes significantly suppressed SCN development. Pseudotime trajectory analyses traced syncytium development back to procambial cell origins, uncovering distinct, cultivar-specific developmental pathways. Collectively, this work provides a foundational single cell transcriptomic resource, advances our understanding of syncytial biology and SCN resistance mechanisms, and identifies promising molecular targets for engineering nematode-resistant soybean varieties.

plant biology↗

bioRxiv2025

CD133+ Vesicles Mediate Resistance to RAS-ERK Inhibition Regulated by YAP Activation in Liver Cancer Cells

CD133, a pentaspan plasma membrane protein, has been viewed as a biomarker of stem cells in normal and cancer tissues, although its function and mechanism are unclear. In previous studies, we identified a new type of CD133+ intracellular vesicles, named intercellsome, which is implicated in direct cell-cell communication under stress conditions. However, the regulatory mechanism and biological significance of these CD133+ vesicles are largely unknown, highlighting a gap in understanding of this cellular communication mechanism. We show here that CD133 acts as a stress response marker in cancer cells, with its expression and vesicle formation significantly induced under MEK inhibitor-mediated proliferative stress. The CD133+ vesicles are essential for maintaining cell proliferation under stress conditions in vitro. Mechanistically, the MEKi activates the Hippo-YAP signaling pathway, which promotes CD133 transcription, establishing a novel connection between YAP signaling and CD133+ vesicle biogenesis. Further, CD133 plays a critical role in YAP-driven liver cancer progression in mice. This study defines a critical role of CD133+ vesicles in stress response regulated by YAP, which advances the understanding of CD133 functions beyond its stem cell-associated roles and suggests new avenues for therapeutic intervention of liver cancer relapse.

cancer biology↗

bioRxiv2025

Sequence mismatch between gene-drive and target-site flanking regions significantly impairs homing efficiency in Culex quinquefasciatus

CRISPR/Cas9-based homing gene-drives (homing-drives) hold enormous potential as control tools for mosquito disease-vectors. These genomically-encoded technologies spread themselves through target populations by creating double-stranded DNA breaks on homologous chromosomes, into which the homing-drives are copied ( homed). Homing is dependent on sequence homology between the genomic regions flanking the transgene insertion and the break site. Homing efficiency (i.e. copying rate) substantially impacts the power of these systems: less efficient homing-drives spread slower, have fewer applications and are more resistance-prone. Understanding what influences homing-drive efficiency is therefore vital to the successful use of these technologies. Here we report a novel mechanism by which a homing-drives efficiency can be significantly impaired by natural sequence variation within a population into which it is spreading. Using a kmo-targeting split homing-drive in the West Nile virus mosquito Culex quinquefasciatus, we found that target-site heterology (sequence mismatch between the genomic regions flanking the target cut-site and the homing-drive transgene) of less than 10% reduced homing efficiency by up to 54%. While substantial research effort has been dedicated to increasing homing-drive efficiency through optimisation of within-construct components, our results highlight that the real-world efficacy of these systems may in part depend on variation beyond these controllable factors.

synthetic biology↗

bioRxiv2025

Integrative genomic and transcriptomic analyses uncover regulatory landscape of symbiotic nitrogen fixation in soybean natural population

Symbiotic nitrogen fixation (SNF) is a key trait in legume productivity, yet the genetic and regulatory basis underlying its natural variation remains poorly understood. Here, we integrated genome, transcriptome, and chromatin accessibility data from a soybean diversity panel comprising 380 accessions, including 108 wild and 272 cultivated lines. Genome-wide association studies (GWAS) detected multiple loci for SNF traits but with limited resolution due to polygenic architecture and environmental influences. Independent component analysis (ICA) identified 136 co-expression modules; ten ICs were strongly correlated with SNF phenotypes and enriched in circadian clock components (e.g., GmLHY1a/b), lipid metabolism, or defense signaling pathways. Transcriptome-wide association studies (TWAS) linked 1,453, 806, and 178 genes to NFP, NW, and NFE traits, respectively. Among TWAS hits, 185 transcription factors were identified, with 39.0% overlapping selective sweeps, suggesting regulatory evolution under domestication. To further dissect expression regulation, we performed eQTL mapping and detected 4,654 significant eQTLs, including 1,241 local (cis), 2,505 distal (trans), and 908 mixed. By integrating ATAC-seq data from sorted nodule nuclei, we found that eQTLs, particularly local eQTLs, are significantly enriched within open chromatin regions, indicating their regulatory potential. Notably, we validated the circadian clock gene GmLHY1b as a negative regulator of nodulation using CRISPR mutagenesis and CUT&Tag. Our integrative study provides comprehensive genomic and transcriptomic resources from a diverse soybean population, offering novel insights into SNF regulatory networks and a valuable foundation for future SNF research and soybean improvement.

plant biology↗

bioRxiv2025

Self-limiting population suppression gene drive in the West Nile vector mosquito, Culex quinquefasciatus

Culex mosquitoes transmit major pathogens including West Nile virus, encephalitis, filariasis, and avian malaria, threatening public health, poultry, and ecosystems. We engineered a CRISPR-based population suppression gene drive targeting a conserved exon of the doublesex (dsx) gene. The drive incorporates a recoded dsxM segment to preserve male function while converting genetic females into sterile intersexes, enabling male-biased propagation and removal of fertile females. It achieves super-Mendelian inheritance ([~]71%) and generates partially dominant sterile resistance alleles via end-joining, resulting in intersex phenotypes with reduced fertility and hatchability. Modeling predicts that this RIDD (Release of Insects carrying a Dominant-sterile Drive) system can suppress populations at low intrinsic growth rates and release ratios, outperforming SIT and fs-RIDL strategies in persistence and efficiency, with further gains achievable through improved cleavage rates. This study establishes a self-limiting gene drive framework for Culex suppression, highlightling the potential of targeting conserved sex-determination pathways for sustainable vector control.

genetics↗

bioRxiv2025

Isoliensinine Ameliorates Pathological Cardiac Hypertrophy After Aerobic Exercise by Activating Cytochrome c Oxidase Subunit 6A2

Pathological cardiac hypertrophy is an abnormal remodeling process of the heart under chronic pressure overload, characterized by myocardial fibrosis, apoptosis, and progressive decline in cardiac function, ultimately leading to heart failure or death. Although aerobic exercise, particularly swimming, has been shown to alleviate pathological cardiac hypertrophy, its molecular mechanisms remain incompletely understood. In this study, metabolomic and transcriptomic analyses revealed the molecular mechanisms underlying swimming-induced improvement of pathological cardiac hypertrophy. Results showed that swimming training significantly altered the level of isoliensinine in animals. Further studies demonstrated that isoliensinine exerts cardioprotective effects by regulating the expression of Cytochrome C Oxidase Subunit 6A2 (COX6A2), establishing for the first time the critical role of the Isoliensinine-COX6A2 signaling axis in cardiac function regulation. To validate this axis, a pathological cardiac hypertrophy model was constructed in mice through transverse aortic constriction (TAC). Both swimming training and isoliensinine intervention significantly improved cardiac structure and function. In vitro experiments using isoproterenol (ISO)-induced hypertrophy in H9C2 cells further confirmed the essential role of this signaling axis in suppressing pathological hypertrophy and maintaining mitochondrial homeostasis. Our findings demonstrate that swimming mitigates pathological cardiac hypertrophy by enhancing COX6A2 expression by isoliensinine, thereby improving mitochondrial function. This study not only expands the understanding of exercise-mediated mechanisms in pathological cardiac remodeling but also highlights the cardioprotective potential of isoliensinine and COX6A2 as therapeutic targets for preventing and treating heart failure.

molecular biology↗

bioRxiv2025

OmicsSankey: Crossing Reduction of Sankey Diagram on Omics Data

In bioinformatics, Sankey diagrams have been widely used to elucidate complex biological insights by visualizing gene expression patterns, microbial community dynamics, and cellular interactions. However, computational scalability remains a challenge for large-scale biological networks. In this work, we present OmicsSankey, a novel formulation of the layout optimization problem for Sankey Diagrams that employs eigen decomposition as a closed-form solution, addressing graph disconnection through a teleportation mechanism that enhances connectivity and stabilizes eigenvector solutions. Experimental results on synthetic datasets with varying layers and nodes validate the efficacy of OmicsSankey compared to state-of-the-art layout-optimizers. Improving the Sankey layouts for Cell Layers, BioSankey, and Sequence Flow further validates OmicsSankey in enhancing the interpretability of biological insights.

bioinformatics↗

bioRxiv2025

GPX4 Promotes Optic Nerve Regeneration and RGC Neuroprotection

Preventing retinal ganglion cells (RGCs)s soma and axon degeneration and promoting optic nerve (ON) regeneration holds great promise for effective glaucoma treatment. To explore potential neural repair strategies, we focused on glutathione peroxidase 4 (GPX4), a key regulator of lipid peroxidation. GPX4 is upregulated in surviving RGCs after acute ON crush or chronic ocular hypertension insult, and also in regenerating RGCs. AAV-mediated RGC-specific overexpression of GPX4 promotes significant ON regeneration and RGC survival, along with visual functional preservation, demonstrating the detrimental role of lipid peroxidation in glaucoma and the therapeutic potential of modulating lipid peroxidation through GPX4 in optic neuropathies.

neuroscience↗

bioRxiv2025

Amazon biodiversity is at risk from metal contamination due to mining activity

The Amazon basin hosts the most biodiverse and intact ecosystems on Earth, yet human activities are an increasing threat. Metal contamination due to mining constitutes one of these major threats, but its impacts remain poorly quantified. We provide the first quantitative assessment of biodiversity exposure to mining-associated metals--mercury [Hg], arsenic [As], copper [Cu], Zinc [Zn], and lead [Pb]--across the Amazon. Around 66% of the Amazons 38,890 species of birds, plants, mammals, reptiles, amphibians, and fishes are exposed to metal contamination, including biodiversity hotspots and Indigenous territories. Safeguarding the Amazons role as a global reservoir of biodiversity, ecosystem function, and cultural heritage requires addressing metal contamination not only as a localized issue, but as a pervasive threat to global biodiversity.

ecology↗

bioRxiv2025

How do bacterial extracellular Contractile Injection Systems bind target cells? A remarkable diversity of receptor binding domains

Extracellular contractile injection systems (eCISs) represent a diverse family of bacteriophage tail-derived toxin delivery complexes in prokaryotes. eCISs are vital for microbial interaction with hosts, and they use tail fiber proteins for target cell binding. However, the identity of the tail fiber proteins and their target cells are unknown. Here, we conducted a comprehensive exploration of eCIS tail fiber genes, providing insights into their remarkable diversity, target cells, functional adaptations, and intriguing evolutionary dynamics. We identified 3,445 eCIS tail fiber proteins found in 2,585 eCIS loci from 1,069 microbes. These fibers can be categorized by five new N-terminal domains responsible for tail fiber attachment to eCIS baseplates. Importantly, we leveraged structure prediction to divide the fibers into 276 structural clusters and dissected 1,177 domain fold families which likely mediate glycan and protein binding on the cell surface of eukaryotes or bacterial targets. These rapidly evolving domains are likely acquired from diverse genomes of eukaryotes, bacteria, and viruses. Finally, we experimentally showed that a candidate tail fiber from Paenibacillus eCIS can bind and direct effector injection into THP-1 human monocyte-like cells, and may bind D-mannose on the cell surface. This study reveals the exceptional diversity of eCIS target specificity determinants, suggests new eCIS target cells in Nature, and provides thousands of proteins that adhere to different cell types.

microbiology↗