Comparative Proteomics Research Articles

Comparative Proteomics Highlights that GenX Exposure Leads to Metabolic Defects and Inflammation in Astrocytes.

Exposure to PFAS such as GenX (HFPO dimer acid) has become increasingly common due to the replacement of older generation PFAS in manufacturing processes. While neurodegenerative and developmental effects of legacy PFAS exposure have been studied in depth, there is a limited understanding specific to the effects of GenX exposure. To investigate the effects of GenX exposure, we exposed Drosophila melanogaster to GenX and assessed the motor behavior and performed quantitative proteomics of fly brains to identify molecular changes in the brain. Additionally, metabolic network-based analysis using the iDrosophila1 model unveiled a potential link between GenX exposure and neurodegeneration. Since legacy PFAS exposure has been linked to Parkinson's disease (PD), we compared the proteome data sets between GenX-exposed flies and a fly model of PD expressing human α-synuclein. Considering the proteomic data- and network-based analyses that revealed GenX may be regulating GABA-associated pathways and the immune system, we next explored the effects of GenX on astrocytes, as astrocytes in the brain can regulate GABA. An array of assays demonstrated GenX exposure may lead to mitochondrial dysfunction and neuroinflammatory response in astrocytes, possibly linking non-cell autonomous neurodegeneration to the motor deficits associated with GenX exposure.

Integrative Proteomic and Phosphoproteomic Profiling Reveals the Salt-Responsive Mechanisms in Two Rice Varieties (Oryza Sativa subsp. Japonica and Indica).

Salinity stress induces ionic and osmotic imbalances in rice plants that in turn negatively affect the photosynthesis rate, resulting in growth retardation and yield penalty. Efforts have, therefore, been carried out to understand the mechanism of salt tolerance, however, the complexity of biological processes at proteome levels remains a major challenge. Here, we performed a comparative proteome and phosphoproteome profiling of microsome enriched fractions of salt-tolerant (cv. IR73; indica) and salt-susceptible (cv. Dongjin/DJ; japonica) rice varieties. This approach led to the identification of 5856 proteins, of which 473 and 484 proteins showed differential modulation between DJ and IR73 sample sets, respectively. The phosphoproteome analysis led to the identification of a total of 10,873 phosphopeptides of which 2929 and 3049 phosphopeptides showed significant differences in DJ and IR73 sample sets, respectively. The integration of proteome and phosphoproteome data showed activation of ABA and Ca2+ signaling components exclusively in the salt-tolerant variety IR73 in response to salinity stress. Taken together, our results highlight the changes at proteome and phosphoproteome levels and provide a mechanistic understanding of salinity stress tolerance in rice.

Identification of candidate proteins related to oleic acid accumulation during sunflower (Helianthus annuus L.) seed development through comparative proteome analysis

Identification of candidate proteins related to oleic acid accumulation during sunflower (Helianthus annuus L.) seed development through comparative proteome analysis

Comparative Proteomics of ccRCC Cell Lines to Identify Kidney Cancer Progression Factors.

Clear cell renal cell carcinoma (ccRCC) is the most common type of kidney cancer, accounting for approximately 75% of kidney cancers. The objective of this study was to identify novel progression markers for ccRCC based on proteomics, with the goal of stage determination and early diagnosis of kidney cancer patients. We performed quantitative global proteomics coupled with Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and high-resolution tandem mass spectrometry on kidney-derived cells, including HEK-293, 786-O (primary ccRCC), and Caki-1 (metastatic ccRCC) cells, to investigate the novel progression factors of ccRCC. In this study, a total of 1,106 proteins were quantified. The Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were conducted for differentially expressed proteins (DEPs) that were increased in ccRCC cells compared to HEK-293 cells. Ultimately, 99 DEPs including 75 up-regulated and 24 down-regulated proteins, that were significantly altered in both ccRCC cells, were identified. Among DEPs, vimentin was identified as the most significantly changed protein. Its increased expression in ccRCC was verified through immunoblotting in ccRCC cell lines and immunohistochemistry in kidney tumors. From the global proteomics data detected in ccRCC, we propose 99 DEPs including vimentin as progression factors.

Mucin-driven ecological interactions in an in vitro synthetic community of human gut microbes.

Specific human gut microbes inhabit the outer mucus layer of the gastrointestinal tract. Certain residents of this niche can degrade the large and complex mucin glycoproteins that constitute this layer and utilise the degradation products for their metabolism. In turn, this microbial mucin degradation drives specific microbiological ecological interactions in the human gut mucus layer. However, the exact nature of these interactions remains unknown. In this study, we designed and studied an in vitro mucin-degrading synthetic community that included mucin O-glycan degraders and cross-feeding microorganisms by monitoring community composition and dynamics through a combination of 16S rRNA gene amplicon sequencing and qPCR, mucin glycan degradation with PGC-LC-MS/MS, production of mucin-degrading enzymes and other proteins through metaproteomics, and metabolite production with HPLC. We demonstrated that specialist and generalist mucin O-glycan degraders stably co-exist and found evidence for cross-feeding relationships. Cross-feeding on the products of mucin degradation by other gut microbes resulted in butyrate production, hydrogenotrophic acetogenesis, sulfate reduction and methanogenesis. Metaproteomics analysis revealed that mucin glycan degraders Akkermansia muciniphila, Bacteroides spp. and Ruminococcus torques together contributed 92% of the total mucin O-glycan degrading enzyme pool of this community. Furthermore, comparative proteomics showed that in response to cultivation in a community compared to monoculture, mucin glycan degraders increased carbohydrate-active enzymes whereas we also found indications for niche differentiation. These results confirm the complexity of mucin-driven microbiological ecological interactions and the intricate role of carbohydrate-active enzymes in the human gut mucus layer.

8182 New Mouse Model Mirrors Human MODY8: Opening Doors to Understanding Exocrine-Endocrine Crosstalk in the Diabetic Pancreas

Abstract Disclosure: K. El Jellas: None. V. Salerno: None. L.S. Katz: None. J. Hu: None. G. Fogarty: None. A. Mandal: None. A. DiStefano-Forti: None. D. Scott: None. M. Lowe: None. A. Molven: None. R.N. Kulkarni: None. Maturity-onset diabetes of the young, type 8 (MODY8) is a dominantly inherited form of monogenic diabetes, caused by heterozygous mutations in the carboxyl ester lipase (CEL) gene expressed in pancreatic acinar cells. MODY8 is characterized by chronic pancreatitis and exocrine dysfunction. Despite the intimate anatomical relationship between the endocrine and exocrine pancreas, little is known about the biological components mediating exocrine-endocrine communication in regulating glucose homeostasis. Here, we report a refined mouse model that bears the mutation present in MODY8 patients on one allele, and the humanized normal variable number of tandem repeat (VNTR) of CEL with 16 repeats (16R) on the other, mimicking the genetic make-up seen in MODY8 patients. Examination of glucose homeostasis revealed that inducing stress (e.g. by high-fat diet) leads to glucose intolerance in MODY8/16R mice and a blunted glucose-stimulated insulin-secretion compared to their control littermates. Histopathological analyses demonstrated features of chronic pancreatitis in some pancreatic lobes, including inflammation, acinar atrophy, acinar-to-ductal metaplasia, fibrosis and fat infiltration, mirroring the morphology of the human disease. Moreover, we applied immunolabeling-enabled three-dimensional imaging of solvent-cleared organs (iDISCO) to intact pancreata and found that beta-cell mass was significantly decreased in the MODY8/16R mice, with a large number of islets located within fatty lobes. These results are coupled with comparative proteomics and lipidomic profiling with the aim of understanding the molecular signatures of exocrine and endocrine cells during pathogenesis. In conclusion, we have generated a mouse that recapitulates several features of the exocrine and endocrine phenotype characteristic of human MODY8. This novel model provides a unique opportunity, not possible in humans, to characterize pancreatic endocrine-exocrine communication in a longitudinal manner as a response to diverse environmental stressors. Presentation: 6/2/2024

Analysis of Antibiotic Response in Clinical Wound Pseudomonas aeruginosa Isolates: Unveiling Proteome Dynamics of Tobramycin-Tolerant Phenotype

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic human pathogen, causing serious chronic infections. P. aeruginosa can adapt efficiently to antibiotic stressors via different genotypic or phenotypic strategies such as resistance and tolerance. The adaptation regulatory system is not always very well understood. In this study, we use shotgun proteomics to investigate the system-level response to tobramycin in two clinical wound P. aeruginosa isolates and PAO1. We profiled each strain for its antibiotic drug-tolerant phenotype using supra-minimum inhibitory concentrations (supra-MIC) of tobramycin and applied proteomics to investigate the protein expression profiles. The MIC revealed that all isolates were susceptible to tobramycin but at supra-MIC concentrations at stationary growth, a degree of tolerance was observed for the isolates. We identified around 40 % of the total proteins encoded by the P. aeruginosa genome and highlighted shared and unique protein signatures for all isolates. Comparative proteome profiling in the absence of antibiotic treatment showed divergent fingerprints, despite similarities in the growth behavior of the isolates. In the presence of tobramycin, the isolates shared a common response in the downregulation of proteins involved in the two-component system, whereas stress response proteins were present at higher levels. Our findings provide insight into the use of proteomic tools to dissect the system-level response in clinical isolates in the absence and presence of antibiotic stress.

Towards Characterization of Hass Avocado Peel and Pulp Proteome during Postharvest Shelf Life.

In recent years, avocados have gained worldwide popularity as a nutritive food. This trend is causing a rise in the production of this fruit, which is accompanied by several problems associated with monocultural practices. Despite massive economic gains, limited molecular and structural information has been generated about avocado ripening. In fact, limited studies have attempted to unravel the proteome complexity dynamics of avocado fruit. We therefore conducted a comparative proteomics study on avocado peel and pulp during the postharvest shelf life using tandem mass tag synchronous precursor selection triple-stage mass spectrometry. We identified 3161 and 1128 proteins in the peel and pulp, respectively. Peels exhibited major over-accumulation of proteins associated with water deprivation and oxidative stress, along with abscisic acid biosynthesis. Ethylene, jasmonic acid, phenylpropanoid, and flavonoid biosynthesis pathways were activated. Structurally, we observed the accumulation of lignin and a reduction in cuticular thickness, which coincides with the reduction in the levels of long-chain acyl-coenzyme A synthetase and a marginal increase in 10,16-dihydroxyhexadecanoic acid. Our study sheds light on the association of proteome modulation with the structural features of Hass avocado. Its detailed characterization will provide an alternative for better preservation during the postharvest period.

Network Medicine Approach Unravels Endophenotype Signature in Alzheimer's Disease through Large-Scale Comparative Proteomics Analysis: Vascular Dysfunction as a Prime Example.

Alzheimer's disease (AD) is the most common neurodegenerative disease burdening public health. We proposed a network-based infrastructure to identify protein signatures for five AD pathological endophenotypes: amyloidosis, tauopathy, vascular dysfunction, lysosomal dysfunction, and neuroinflammation. We analyzed 23 proteomic data sets from AD patients and transgenic mouse models, using network proximity to measure associations between endophenotype modules and differentially expressed proteins (DEPs) in the integrated AD proteome. We focused on the vascular dysfunction signature with 21 DEPs by integrating RNA-seq, single-cell transcriptomics, GWAS, and literature. Experiments on APP/PS1 and MCAO models highlighted three proteins (SEPT5, SNAP25, STXBP1) as novel AD biomarker candidates. This study demonstrates a network medicine framework for deciphering endophenotype signatures in AD.

Comparative Profiling of Milk Somatic Cells Proteomes Revealed Key Players in Mammary Immune Mechanisms During Mastitis in Tropical Sahiwal (Bos indicus) Cows.

Bovine mastitis poses a significant economic burden on the dairy industry worldwide. This pioneering proteomic study conducted a comparative profiling of milk somatic cell (SC) proteins contributing to mammary immune defense during subclinical and clinical mastitis (CM) in Sahiwal (Bos indicus) cows. Based on California mastitis test (CMT) scores, milk SC counts, differential leukocyte counts (DLCs), and bacteriological culture results, quarter milk SC samples were categorized into healthy (H), subclinical mastitis (SCM), and CM groups. Comparative proteome profiling of milk SCs was done using a label-free liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) proteomic approach. The identified upregulated proteins in mastitis groups such as Vanin 2, Thioredoxin reductase-like selenoprotein T, Ceramidase, Lymphocyte antigen 75, Misshapen-like kinase 1 (MINK1), Thrombospondin 1, Macrophage scavenger receptor 1, Leupaxin, and Lipoamide acyltransferase, involved in immune responses. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed immune functions and pathways like antigen processing, complement cascades, extracellular matrix receptor interaction, efferocytosis, leukocyte migration, chemokine, peroxisome proliferator-activated receptors (PPARs), and transforming growth factor (TGF)-beta signaling. These findings provide essential information on proteomic profiling in milk SCs and contribute valuable insights into immune-related proteins regulated during mastitis in dairy cows. Further, validated proteins (Vanin 2, MINK1, and Thrombospondin 1) offer potential inflammatory biomarkers for early mastitis detection in dairy cows.

Decoding recurrent pregnancy loss: insights from comparative proteomics studies.

Recurrent pregnancy loss (RPL) represents a common disorder that affects up to 2% of the women aiming at childbirth with long-term consequences on family and society. Factors contributing to it in more than half of the cases are still unknown. Comparative proteomic analysis can provide new insights into the biological pathways underlining the pathogenesis of RPL. Until now, chorionic villi, decidua, placenta, endometrium, and maternal blood from women with RPL have been analyzed by comparative proteomics studies. In this review, we aimed to provide a critical evaluation of the published comparative studies of RPL on human samples, gathered by systematic literature search using PubMed and Google Scholar databases. We provide a detailed overview of the analyzed materials, proteomics platforms, proposed candidate biomarkers and altered pathways and processes linked with RPL. The top, most identified and validated biomarker candidates from all studies are discussed, followed by bioinformatics analysis of the available high-throughput data and presentation of common altered processes and pathways in RPL. Finally, future directions aimed at developing new and efficient therapeutic strategies are discussed as well.

Restoration of LAMP2A expression in old mice leads to changes in the T cell compartment that support improved immune function

Chaperone-mediated autophagy (CMA) is a selective form of autophagy that contributes to the maintenance of cellular homeostasis. CMA activity declines with age in most tissues and systems, including the immune system, due to a reduction in levels of lysosome-associated membrane protein type 2A (LAMP2A), an essential CMA component. In this study, we show that overexpressing a copy of hLAMP2A within T cells since middle-age can prevent some of their age-associated loss of function. Our data support the idea that preserving LAMP2A expression with age through genetic means leads to enhanced proliferative responses, decreased number of regulatory T cell populations, and down-regulated expression of inhibitory receptors by T cells. During aging, elevated numbers of these immunosuppressive T cell populations significantly contribute to the age-associated downregulation of T cell responses. Using comparative proteomics, we confirm that preservation of CMA activity in old mice prevents age-related changes in both the resting and the activated T cell proteome. We also explore the effect of using first-in-class small molecule activators of CMA and demonstrate improved T cell response upon their administration to old mice. We conclude that sustaining CMA activity constitutes a potentially viable therapeutic approach to improving T cell function with age.

Comparative Proteomic Analysis Reveals the Effect Mechanisms of Glucose on the Biomass and Phenolic Glycoside Esters Synthesis Activity of Candida Parapsilosis ACCC 20221 Whole-Cell Catalyst.

A new Candida parapsilosis ACCC 20221 (C. parapsilosis ACCC 20221) whole-cell catalyst with a high phenolic glycoside esters synthesis activity and large biomass was obtained after culture with glucose. The possible mechanisms were revealed by using comparative proteomics. It found the expression of proteins involved in post-translational modification, protein turnover, and chaperone, and RNA processing and modification was upregulated, which ensured the metabolic balance and accurate translation, correct folding, and post-translational modification of proteins, thus enhancing the production of lipases in C. parapsilosis ACCC 20221 cultured with glucose. Moreover, the glycolysis pathway, tricarboxylic acid cycle, and fatty acids synthesis were enhanced, while the β-oxidation of fatty acids was weakened in C. parapsilosis ACCC 20221 cells cultured with glucose, which led to an increase in energy generation and cell membrane synthesis; thus, large biomass was obtained. In addition, CCE40476.1 and CAC86400.1, which were likely to exert arbutin esters synthesis activity in C. parapsilosis ACCC 20221, were screened, and it was found that vinyl propionate could easily enter the catalytic pocket of CCE40476.1 and form hydrogen bonding interactions with Leu191 and Ser266.

Multiomics Analysis of the Mechanism by Which Gibberellin Alleviates S-Metolachlor Toxicity in Rice Seedlings.

S-metolachlor is a selective pre-emergence herbicide used in dryland. However, it is challenging to employ in paddy fields due to its phytotoxic effects on rice. As a common phytohormone, Gibberellin-3 (GA3) is inferred to have the ability to alleviate herbicide phytotoxicity. This study first quantitatively verified the phytotoxicity of s-metolachlor to rice and then demonstrated the mitigative effect of GA3 on these adverse reactions. Furthermore, a transcriptome of rice seedlings subjected to different treatments was constructed to assemble the reference genes, followed by comparative metabolomics and proteomics analyses. Metabolomics revealed an enrichment of flavonoid metabolites in the group of adding GA3, and these flavonoids can eliminate ROS in plants. Proteomics analysis indicated that differential proteins were enriched in the phenylpropanoid biosynthesis pathway responsible for the synthesis of flavonoids and that the functions of most differential proteins are associated with peroxidase. The proteome, combined with the transcriptome, revealed that the expressions of proteins and genes was related to the POD activity in the group of adding GA3. It was speculated that the elimination of ROS is key to alleviating the stress of s-metolachlor on rice growth. It was inferred that the mechanism of GA3 in alleviating the phytotoxicity of the substance s-metolachlor is by increasing the activity of the POD and influencing the growth of rice seedlings through the restoration of flavonoid synthesis. In this study, we screened GA3 as a safener to alleviate the phytotoxicity of s-metolachlor on rice. On this basis, the mechanism of alleviating phytotoxicity was studied. The application range of s-metolachlor might be expanded, providing a new supplementary method for weed control and herbicide resistance management.

Lipoteichoic Acid from Heyndrickxia coagulans HOM5301 Modulates the Immune Response of RAW 264.7 Macrophages.

Heyndrickxia coagulans (formerly Bacillus coagulans) has been increasingly utilized as an immunomodulatory probiotics. Oral administration of H. coagulans HOM5301 significantly boosted both innate and adaptive immunity in mice, particularly by increasing the phagocytic capacity of monocytes/macrophages. Lipoteichoic acid (LTA), a major microbe-associated molecular pattern (MAMP) in Gram-positive bacteria, exhibits differential immunomodulatory effects due to its structural heterogeneity. We extracted, purified, and characterized LTA from H. coagulans HOM5301. The results showed that HOM5301 LTA consists of a glycerophosphate backbone. Its molecular weight is in the range of 10-16 kDa. HOM5301 LTA induced greater productions of nitric oxide, TNFα, and IL-6 in RAW 264.7 macrophages compared to Staphylococcus aureus LTA. Comparative transcriptome and proteome analyses identified the differentially expressed genes and proteins triggered by HOM5301 LTA. KEGG analyses revealed that HOM5301 LTA transcriptionally and translationally activated macrophages through two immune-related pathways: cytokine-cytokine receptor interaction and phagosome formation. Protein-protein interaction network analysis indicated that the pro-inflammatory response elicited by HOM5301 LTA was TLR2-dependent, possibly requiring the coreceptor CD14, and is mediated via the MAPK and NF-kappaB pathways. Our results demonstrate that LTA is an important MAMP of H. coagulans HOM5301 that boosts immune responses, suggesting that HOM5301 LTA may be a promising immunoadjuvant.

The coral pathogen Vibrio coralliilyticus uses a T6SS to secrete a group of novel anti-eukaryotic effectors that contribute to virulence.

Vibrio coralliilyticus is a pathogen of coral and shellfish, leading to devastating economic and ecological consequences worldwide. Although rising ocean temperatures correlate with increased V. coralliilyticus pathogenicity, the specific molecular mechanisms and determinants contributing to virulence remain poorly understood. Here, we systematically analyzed the type VI secretion system (T6SS), a contact-dependent toxin delivery apparatus, in V. coralliilyticus. We identified 2 omnipresent T6SSs that are activated at temperatures in which V. coralliilyticus becomes virulent; T6SS1 is an antibacterial system mediating interbacterial competition, whereas T6SS2 mediates anti-eukaryotic toxicity and contributes to mortality during infection of an aquatic model organism, Artemia salina. Using comparative proteomics, we identified the T6SS1 and T6SS2 toxin arsenals of 3 V. coralliilyticus strains with distinct disease etiologies. Remarkably, T6SS2 secretes at least 9 novel anti-eukaryotic toxins comprising core and accessory repertoires. We propose that T6SSs differently contribute to V. coralliilyticus's virulence: T6SS2 plays a direct role by targeting the host, while T6SS1 plays an indirect role by eliminating competitors.

Comparative proteomics analysis reveals the domesticated Lepista sordida primordium differentiation regulation mechanism and the subsequent different development patterns in the pileus and stipe

Comparative proteomics analysis reveals the domesticated Lepista sordida primordium differentiation regulation mechanism and the subsequent different development patterns in the pileus and stipe

Comparative proteomics analysis of the mouse mini-gut organoid: insights into markers of gluten challenge from celiac disease intestinal biopsies.

Organoid models enable three-dimensional representation of cellular systems, providing flexible and accessible research tools, and can highlight key biomolecules. Such models of the intestinal epithelium can provide significant knowledge for the study of celiac disease and provide an additional context for the nature of markers observed from patient biopsy data. Using LC-MS/MS, the proteomes of the crypt and enterocyte-like states of a mouse mini-gut organoid model were measured. The data were further compared with published biopsy data by comparing the changes induced by gluten challenge after a gluten-free diet. These analyses identified 4,850 protein groups and revealed how 400 putative biomarkers of dietary challenge were differentially expressed in the organoid model. In addition to the extensive changes within the differentiated cells, the data reiterated the disruption of the crypt-villus axis after gluten challenge. The mass spectrometry data are available via ProteomeXchange with the identifier PXD025690.

Functional characterization of a novel Chlamydomonas reinhardtii hydrolase involved in biotransformation of chloramphenicol

Microalgae-based biotechnology is one of the most promising alternatives to conventional methods for the removal of antibiotic contaminants from diverse water matrices. However, current knowledge regarding the biochemical mechanisms and catabolic enzymes involved in microalgal biodegradation of antibiotics is scant, which limits the development of enhancement strategies to increase their engineering feasibility. In this study, we investigated the removal dynamics of amphenicols (chloramphenicol, thiamphenicol, and florfenicol), which are widely used in aquaculture, by Chlamydomonas reinhardtii under different growth modes (autotrophy, heterotrophy, and mixotrophy). We found C. reinhardtii removed >92 % chloramphenicol (CLP) in mixotrophic conditions. Intriguingly, gamma-glutamyl hydrolase (GGH) in C. reinhardtii was most significantly upregulated according to the comparative proteomics, and we demonstrated that GGH can directly bind to CLP at the Pro77 site to induce acetylation of the hydroxyl group at C3 position, which generated CLP 3-acetate. This identified role of microalgal GGH is mechanistically distinct from that of animal counterparts. Our results provide a valuable enzyme toolbox for biocatalysis and reveal a new enzymatic function of microalgal GGH. As proof of concept, we also analyzed the occurrence of these three amphenicols and their degradation intermediate worldwide, which showed a frequent distribution of the investigated chemicals at a global scale. This study describes a novel catalytic enzyme to improve the engineering feasibility of microalgae-based biotechnologies. It also raises issues regarding the different microalgal enzymatic transformations of emerging contaminants because these enzymes might function differently from their counterparts in animals.

Cell-to-cell interactions revealed by cryo-tomography of a DPANN co-culture system

DPANN is a widespread and diverse group of archaea characterized by their small size, reduced genome, limited metabolic pathways, and symbiotic existence. Known DPANN species are predominantly obligate ectosymbionts that depend on their host for proliferation. The structural and molecular details of host recognition, host-DPANN intercellular communication, and host adaptation in response to DPANN attachment remain unknown. Here, we use electron cryotomography (cryo-ET) to show that the Microcaldus variisymbioticus ARM-1 may interact with its host, Metallosphaera javensis AS-7 through intercellular proteinaceous nanotubes. Combining cryo-ET and sub-tomogram averaging, we show the in situ architectures of host and DPANN S-layers and the structures of the nanotubes in their primed and extended states. In addition, comparative proteomics and genomic analyses identified host proteomic changes in response to DPANN attachment. These results provide insights into the structural basis of host-DPANN communication and deepen our understanding of the host ectosymbiotic relationships.