Visualization of the Biogenesis, Dynamics, and Host Interactions of Bacterial Extracellular Vesicles

Chemical & Biomedical Imaging, Год журнала: 2025, Номер 3(3), С. 132 - 136

Опубликована: Янв. 30, 2025

InfoMetricsFiguresRef. Chemical & Biomedical ImagingASAPArticle This publication is Open Access under the license indicated. Learn More CiteCitationCitation and abstractCitation referencesMore citation options ShareShare onFacebookX (Twitter)WeChatLinkedInRedditEmailJump toExpandCollapse ViewpointJanuary 30, 2025Visualization of Biogenesis, Dynamics, Host Interactions Bacterial Extracellular VesiclesClick to copy article linkArticle link copied!Sandrine TruchetSandrine TruchetUniversité Paris-Saclay, INRAE, AgroParisTech, Micalis Institute, 78350 Jouy-en-Josas, FranceMore by Sandrine TruchetJeanne Malet-VillemagneJeanne Malet-VillemagneUniversité Jeanne Malet-VillemagneGilles TessierGilles TessierSorbonne Université, CNRS UMR7210, INSERM UMRS968, Institut de la Vision, 75012 Paris, Gilles TessierJasmina Vidic*Jasmina VidicUniversité France*Email: [email protected]More Jasmina Vidichttps://orcid.org/0000-0002-8549-8199Open PDFChemical ImagingCite this: Chem. Biomed. Imaging 2025, XXXX, XXX, XXX-XXXClick citationCitation copied!https://pubs.acs.org/doi/10.1021/cbmi.5c00002https://doi.org/10.1021/cbmi.5c00002Published January 2025 Publication History Received 10 2025Accepted 22 2025Published online 30 2025article-commentary© The Authors. Co-published Nanjing University American Society. licensed CC-BY-NC-ND 4.0 . License Summary*You are free share (copy redistribute) this in any medium or format within parameters below:Creative Commons (CC): a Creative license.Attribution (BY): Credit must be given creator.Non-Commercial (NC): Only non-commercial uses work permitted. No Derivatives (ND): Derivative works may created for purposes, but sharing prohibited. View full license*DisclaimerThis summary highlights only some key features terms actual license. It not has no legal value. Carefully review before using these materials. underCC-BY-NC-ND share(copy below: Attribution *DisclaimerThis creator. Non-Commercial ACS Publications© SocietySubjectswhat subjectsArticle subjects automatically applied from Subject Taxonomy describe scientific concepts themes article.BacteriaDyes pigmentsGeneticsMembranesVesiclesThe first discovery bacterial Vesicles (bEVs) was made Gram-negative bacterium Escherichia coli 1960s. (1) secretion vesicles Gram-positive bacteria uncovered until 1990s because thick cell wall seen as physical barrier their release. (2) now evident that all domains life, secrete spherical membrane ranging diameter 20 400 nm (Figure 1A). bEVs found associated with numerous cellular factors, suggesting pivotal role homeostasis, survival, stress response adaptation environment. (3,4) involved communication other microorganisms host cells. They can transport proteins, lipids, peptidoglycan fragments, DNA, RNA, phages small signaling molecules. In addition, pathogenic bacteria, carry deliver toxins virulence factors. (5,6) To date, researchers able purify isolate them, very few protocols allow tracking biogenesis fusion cells.Figure 1Figure 1. Monitoring secretion. (A) Ultrathin sections (red arrows) S. aureus examined TEM. Bar: 100 nm. (B) Production increased when cross-linking reduced. 200 Reprinted ref (7). Copyright 2018 Springer Nature Ltd. (C) Confocal time-lapse formation Bacteroides coexpressing Inulinase-GFP OmpF-mCherry. 2 μm. (11). 2023 NAS. (D) Representative multicolor STORM image secreting colabeled (Nile red, green) Enterotoxin B (EntB, red). 1 (12). 2022 (E) Time-lapse sequence (3D-SIM) blebbing E. phage infection stained dye FM1–43X (green). Red arrows indicate blebs. μm (14). 2021 Microbiological Society.High Resolution ImageDownload MS PowerPoint SlideDue nanometric sizes, reference method characterizing remains transmission electron microscopy (TEM). (7) However, TEM suitable visualizing native state since it requires fairly drastic sample pretreatment 1B). contrast, super-resolution techniques including structured illumination (SIM), STED, PALM/STORM microscopies have become tools choice them. While spatial resolution unmatched, label-free methods, such ISCAT interferometry, emerged recently, arguably much simpler setups. (8) Optical phase also evolved way beyond simple contrast imaging provide quantitative imaging. (9,10) Here, we present state-of-the-art methods labeling strategies bEVs.Biogenesis bEVsClick section linkSection copied!In recent report, Sartorio et al. employed an elegant dual marker system through outer vesicle markers fluorescent proteins visualize prominent member human intestinal microbiota thetaiotaomicron. (11) Using (widefield) they showed production resulted orchestrated process assisted cells gain fitness 1C).Previously unresolved bEV addressed at nanoscale STORM. (12) By staining Staphylococcus (or epidermidis) Nile red label plasma membrane, WGA walls, antiprotein A antienterotoxin antibodies were observed surface correlative analysis scanning images suggested explosive lysis main pathways 1D). Notably, degradation plays significant regulating release bacteria. another study, live cryo-tomography revealed mechanism Bacillus subtilis. (13) Through vesicles' membranes FM4-64 nucleic acid cargo SYTOX green dye, shown death induces neighboring Moreover, induced genotoxic activation endolysin system. Indeed, expression prophage-encoded subpopulation B. subtilis holes wall. These acted open doors facilitating bEVs. involvement coli. Mandal used phase-contrast (3D-SIM, Structured Illumination Microscopy) study bacteriophage (14) sequences labeled indicated generated 1E).bEVs Modulating Microbial EcosystemsClick copied!bEVs facilitate interactions between same species play essential different species, influencing competition, cooperation, overall community dynamics microbial ecosystems. (15) Recently, Meyer Nodwell fluorescence transfer lipid soluble dyes confocal assess delivery Streptomyces content yeast fusion. (16) demonstrated antimicrobial containing achieve direct target microbes. For instance, FM4–64-, Vancomycin-Bodipy-Fl-, CFSE (5-(and-6)-Carboxyfluorescein Diacetate Succinimidyl Ester)-stained stationary actively growing subtilis, less than 15 min. delivered multiple specifically cytoplasm surrounding 2A). Elsewhere, self-quenching octadecyl rhodamine chloride (R18)-labeled released mixed Pseudomonas aeruginosa cells, signal over time microscope. (17) P. assessed, revealing promote pathogenicity.Figure 2Figure 2. targets. extracellular DNA (SYTOX, (labeled FM4–64, red) Numbers (3, merge 120 min) progression death. 5 (13). 2017 costained (PKH2, RNA (SYTO RNASelect, green). (20). Joshi, Singh, Nadeem, Askarian, Wai, Johannessen Hegstad. Single plane showing internalization (Dil, H. pylori A549 (LC3-GFP, phalloidin, magenta). Arrowheads colocalized LC3-GFP puncta DiI-labeled (merge, yellow) actin. (22). Wiley-VCH. bEVs, (DiD, magenta SYTO into macrophages DAPI (blue). ref. (23) Intravital tail-vein-injected A. actinomycetemcomitans cortex CX3CR1-GFP mice. point colocalization blood brain barrier-crossed DiD-stained (red) CX3CR1-positive microglial (green) localized exterior vessels (Alexa Fluor 555-conjugated anti-CD31 antibody, blue). Scale bar: 50 μm; magnified scale: (24). 2019 Wiley-VCH.High SlidebEV-based interbacterial particular interest case hydrophobic molecules transport, notably living aqueous environments. N-hexadecanoyl-L-homoserine lactone solubilized Paracoccus sp. aid (18) epifluorescence FM4–64-labeled Paracoccus-delivered preferentially targeted strain rather suggests recognize types. carriage indispensable requirement cargo's function cell–cell bacteriocin micrococcin P1 (MP1) important interaction microbiome. (19) MP1 poorly active due its low solubility, both activity depend on incorporation PKH26, R18.bEVs Host–Microbe InteractionClick copied!By acting efficient protective carriers signals genetic material, coordinate mutual interaction, exchanges delivering factors modulating immune responses promoting host. Most studies lipid-specific DiD PKH2 lipophilic Vybrant DiI DiO monitor Sahr Legionella pneumophila translocate RNAs (6) Automated conformal purified DiD-labeled which then defense pathways. proposed general L. interact eukaryotic Similarly, RNASelect dye-labeled influence microbe–microbe host–pathogen (20) 2B).Besides Bitto visualized packaging chromosomal (21) membrane-permeant SYTO-61 epithelial lung (A549) microscopy. transported encoded virulence, response, antibiotic resistance metabolism. aureus, immunostimulatory fragments 2C D) activate innate receptors induce autophagy (22,23)Codemo stimulated monocyte-derived dendritic pneumococcal characterize immunomodulatory effect. (25) high-resolution immunofluorescence upon incubation antipneumolysin intracellular localization vesicles. Internalized triggered proinflammatory cytokine regardless presence cytosolic pore-forming toxin pneumolysin indicating transferred. Interestingly, heterogeneity Aggregatibacter actinomycetemcomitans, implicated periodontal disease. (26) modulation functions during monitoring internalized dynamin-dependent endocytosis. (27)Finally, distribution after uptake monitored whole animals. Han administrated RNA-Select mice cardiac injection. (28) Cleared mouse brains imaged two-dimensional light-sheet microscopy, providing evidence cross blood-brain promoted TNFα (24) 2E). group laser intravital show went earlier meningeal intravenous injection transgenic vivo biodistribution coli-delivered SpyCatcher protein anchored flowed PET/MRI via synthetic SpyTag-NODAGA conjugates combined radiolabeling (64Cu). (29) approach liver spleen had highest following administration.Concluding RemarksClick copied!BEVs crucial bacterial–host serving various biomolecules. presents still several technical challenges size, structural complexity, dynamic nature. Electron detailed structures does functional insights while conventional light limit closes diameters (20–400 nm). Advanced techniques, specific provided deeper biogenesis, mechanisms support survival pathogenicity.Author InformationClick copied!Corresponding AuthorJasmina Vidic - Université France; https://orcid.org/0000-0002-8549-8199; Email: protected]AuthorsSandrine Truchet FranceJeanne Malet-Villemagne FranceGilles Tessier Sorbonne FranceAuthor ContributionsJ.V. defined scope S.T. structure Viewpoint article. All authors prepared manuscript writing initial draft, reviewing editing.NotesThe declare competing financial interest.AcknowledgmentsClick copied!This supported Agence Nationale Recherche (ELISE-ANR-21-CE42), European Union (grant agreement no. 101135402, Mobiles project), Department MICA INRAE (Vélib project).ReferencesClick copied! references 29 publications. 1Bishop, D.; Work, An glycolipid produced grown lysine-limiting conditions. Biochem. J. 1965, 96 (2), 567, DOI: 10.1042/bj0960567 Google ScholarThere corresponding record reference.2Dorward, D. W.; Garon, C. F. packaged membrane-derived Applied environmental microbiology 1990, 56 (6), 1960– 1962, 10.1128/aem.56.6.1960-1962.1990 reference.3Lee, Y.; Choi, Kim, K.; Park, O.; S.; H.; Desiderio, M.; Y. K. Gram positive produce vesicles: proteomics based characterization derived Proteomics 2009, 9 (24), 5425– 5436, 10.1002/pmic.200900338 reference.4Gill, Catchpole, R.; Forterre, three life beyond. FEMS reviews 2019, 43 (3), 273– 303, 10.1093/femsre/fuy042 reference.5Malet-Villemagne, J.; Vidic, pathogenesis Campylobacter jejuni. Microbes Infection 2024, 26, 105377, 10.1016/j.micinf.2024.105377 reference.6Sahr, T.; Escoll, P.; Rusniok, C.; Bui, Pehau-Arnaudet, G.; Lavieu, Buchrieser, Translocated mimic microRNAs targeting response. Nat. Commun. 2022, 13 (1), 762, 10.1038/s41467-022-28454-x reference.7Wang, X.; Thompson, Weidenmaier, Lee, Release application vaccine platform. 2018, 1379, 10.1038/s41467-018-03847-z reference.8Priest, L.; Peters, Kukura, Scattering-based microscopy: metal nanoparticles single proteins. Rev. 2021, 121 (19), 11937– 11970, 10.1021/acs.chemrev.1c00271 reference.9Gentner, Rogez, B.; Robert, Aggoun, A.; Tessier, Bon, Berto, Enhanced Quantitative Wavefront Nano-Object Characterization. Nano 18 (29), 19247– 19256, 10.1021/acsnano.4c05152 reference.10Chaumet, Maire, Sentenac, Baffou, G. microscopies: accuracy comparison. Light: Science Applications 288, 10.1038/s41377-024-01619-7 reference.11Sartorio, M. Pardue, Scott, N. E.; Feldman, Human gut tailor breakdown diet-and host-derived glycans. Proc. Natl. Acad. Sci. U. 2023, (27), e2306314120, 10.1073/pnas.2306314120 reference.12Jeong, Chung, Kweon, H.-S.; Kang, N.-G.; Hwang, Youn, Visualizing gram-positive BMC biology 270, 10.1186/s12915-022-01472-3 reference.13Toyofuku, Cárcamo-Oyarce, Yamamoto, Eisenstein, F.; Hsiao, C.-C.; Kurosawa, Gademann, Pilhofer, Nomura, N.; Eberl, Prophage-triggered damage 2017, 8 481, 10.1038/s41467-017-00492-w reference.14Mandal, Ballerin, Nolan, Petty, Whitchurch, Bacteriophage leads blebbing. Microbiology 167 (4), 001021, 10.1099/mic.0.001021 reference.15Toyofuku, Schild, Kaparakis-Liaskos, Composition Reviews 21 (7), 415– 430, 10.1038/s41579-023-00875-5 reference.16Meyer, Nodwell, R. broad permissive Bacteriol. 206 e00325– 00323, 10.1128/jb.00325-23 reference.17Subsomwong, Teng, Ishiai, Narita, Sukchawalit, Nakane, Asano, pathogenicity aeruginosa. Research 281, 127612, 10.1016/j.micres.2024.127612 reference.18Toyofuku, Morinaga, Hashimoto, Uhl, Shimamura, Inaba, Schmitt-Kopplin, Membrane vesicle-mediated communication. ISME journal 11 1504– 1509, 10.1038/ismej.2017.13 reference.19Liu, Liu, Q.; Zhao, Dickey, Wang, Xu, Chen, Jian, Lv, Essential biological P1. Journal e12212, 10.1002/jev2.12212 reference.20Joshi, Johannessen, Hegstad, Transcriptome profiling reveals RNA-cargo. Frontiers molecular biosciences 7, 566207, 10.3389/fmolb.2020.566207 reference.21Bitto, Chapman, Pidot, Costin, Lo, D'Cruze, Reynolds, Dashper, Turnbull, Rep. 7 1– 11, 10.1038/s41598-017-07288-4 reference.22Bitto, Cheng, Johnston, Pathirana, Phan, T. Poon, I. O'Brien Simpson, Hill, Stinear, Kaparakis Liaskos, contain autophagy. e12080, 10.1002/jev2.12080 reference.23Rodriguez, V.; Kuehn, secretes 2020, 18293, 10.1038/s41598-020-75108-3 reference.24Ha, S.-Y.; Hong, S.-H.; H.-J. Delivery periodontopathogenic monocytes IL-6 promotion cargo. 596366, 10.3389/fmolb.2020.596366 reference.25Codemo, Muschiol, Iovino, Nannapaneni, Plant, Henriques-Normark, Immunomodulatory effects humoral defenses. MBio e00559-18, 10.1128/mBio.00559-18 reference.26Singh, Nice, Wu, Brown, Wittenberg, Multivariate Analysis Individual Outer Fluorescence Microscopy. (5), 352– 361, 10.1021/cbmi.4c00014 reference.27Wang, Eagen, W. Orchestration macrophage NLRP3 inflammasome 117 3174– 3184, 10.1073/pnas.1915829117 reference.28Han, E.-C.; J.-W.; TNF-α FASEB 33 (12), 13412, 10.1096/fj.201901575R reference.29Szöllősi, Hajdrik, Tordai, Horváth, I.; Veres, Gillich, Shailaja, Smeller, Bergmann, Bachmann, Molecular display. 18752, 10.1038/s41598-023-45628-9 reference.Cited Click yet been cited publications.Download PDFFiguresReferences Get e-AlertsGet e-AlertsChemical copied!https://doi.org/10.1021/cbmi.5c00002Published 2025© Article Views-Altmetric-Citations-Learn about metrics closeArticle Views COUNTER-compliant sum text downloads November 2008 (both PDF HTML) across institutions individuals. regularly updated reflect usage leading up last days.Citations number articles citing article, calculated Crossref daily. Find more information counts.The Altmetric Attention Score measure attention research received online. 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Язык: Английский

Engineering Planar Gram-Negative Outer Membrane Mimics Using Bacterial Outer Membrane Vesicles

Langmuir, Год журнала: 2024, Номер 40(44), С. 23289 - 23300

Опубликована: Окт. 25, 2024

Antibiotic resistance is a major challenge in modern medicine. The unique double membrane structure of Gram-negative bacteria limits the efficacy many existing antibiotics and adds complexity to antibiotic development by limiting transport bacterial cytosol. New methods mimic this barrier would enable high-throughput studies for development. In study, we introduce an innovative approach modify outer vesicles (OMVs) from Aggregatibacter actinomycetemcomitans, generate planar supported lipid bilayer membranes. Our method first involves incorporation synthetic lipids into OMVs using rapid freeze–thaw technique form hybrid (OM-Hybrids). Subsequently, these OM-Hybrids can spontaneously rupture when contact with SiO2 surfaces (OM-SB). We assessed formation dynamic light scattering fluorescence quenching assay. To analyze OM-SBs used quartz crystal microbalance dissipation monitoring (QCM-D) recovery after photobleaching (FRAP). Additionally, conducted assays detect surface-associated DNA proteins on OM-SBs. interaction antimicrobial peptide, polymyxin B, was also assessed. These findings emphasize capability our platform produce membranes, which turn, could function as valuable tool streamlining antibiotics.

Язык: Английский

Engineering Planar Gram-Negative Outer Membrane Mimics Using Bacterial Outer Membrane Vesicles

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2023, Номер unknown

Опубликована: Дек. 12, 2023

Abstract Antibiotic resistance is a major challenge in modern medicine. The unique double membrane structure of gram-negative bacteria limits the efficacy many existing antibiotics and adds complexity to antibiotic development by limiting transport bacterial cytosol. New methods mimic this barrier would enable high-throughput studies for development. In study, we introduce an innovative approach modify outer vesicles (OMVs) from Aggregatibacter actinomycetemcomitans, generate planar supported lipid bilayer membranes. Our method first involves incorporation synthetic lipids into OMVs using rapid freeze-thaw technique form hybrid (OM-Hybrids). Subsequently, these OM-Hybrids can spontaneously rupture when contact with SiO 2 surfaces (OM-SB). We assessed formation dynamic light scattering fluorescence quenching assay. To analyze OM-SBs used quartz crystal microbalance dissipation monitoring (QCM-D) recovery after photobleaching (FRAP). Additionally, conducted assays detect surface-associated DNA proteins on OM-SBs. interaction antimicrobial peptide, polymyxin B, was also assessed. These findings emphasize capability our platform produce membranes, which turn, could function as valuable tool streamlining antibiotics.

Язык: Английский