{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T04:56:35Z","timestamp":1787028995675,"version":"build-2736575974"},"reference-count":51,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2026,4,11]],"date-time":"2026-04-11T00:00:00Z","timestamp":1775865600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Ministry of Science and Higher Education of the Republic of Kazakhstan","award":["AP26102743"],"award-info":[{"award-number":["AP26102743"]}]},{"award":["AP26102743"],"award-info":[{"award-number":["AP26102743"]}],"id":[{"id":"https:\/\/ror.org\/05tyne317","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Microorganisms"],"abstract":"<jats:p>The antigen 85 (Ag85) complex of Mycobacterium tuberculosis comprises secreted mycolyltransferases essential for cell wall biosynthesis and envelope integrity. Although extensively studied as immunodominant antigens and targets of small-molecule inhibitors, their potential for antibody-mediated functional modulation remains underexplored. In this review, we re-evaluate the Ag85 complex from a mechanistic and therapeutic perspective, focusing on its accessibility, structural organization, and functional vulnerabilities within the mycobacterial cell envelope. We highlight the distinction between antigen recognition and functional modulation, noting that conventional antibody responses often target immunodominant but functionally irrelevant epitopes. We propose that nanobodies represent a promising platform for targeting conformationally or spatially restricted regions of Ag85 enzymes that are not readily accessible to conventional antibodies. Based on structural and biochemical insights, we outline a framework in which Ag85-targeting nanobodies act as sensitizing agents that perturb cell wall biosynthesis and enhance susceptibility to antibiotics or cell wall-degrading enzymes. We further discuss key challenges, including antigen accessibility under physiological conditions, intracellular localization, and nanobody stability, which may influence the feasibility of this approach.<\/jats:p>","DOI":"10.3390\/microorganisms14040863","type":"journal-article","created":{"date-parts":[[2026,4,13]],"date-time":"2026-04-13T11:38:26Z","timestamp":1776080306000},"page":"863","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Revisiting the Antigen 85 Complex as a Target for Functional Antibody-Based Strategies Against Mycobacterium tuberculosis"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7739-7777","authenticated-orcid":false,"given":"Izat","family":"Smekenov","sequence":"first","affiliation":[{"name":"Department of Molecular Biology and Genetics, Faculty of Biology and Biotechnology, Al-Farabi Kazakh National University, Almaty 050040, Kazakhstan"},{"name":"Scientific Research Institute of Biology and Biotechnology Problems, Al-Farabi Kazakh National University, Almaty 050040, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9928-8021","authenticated-orcid":false,"given":"Nazym","family":"Tleumbetova","sequence":"additional","affiliation":[{"name":"Department of Molecular Biology and Genetics, Faculty of Biology and Biotechnology, Al-Farabi Kazakh National University, Almaty 050040, Kazakhstan"},{"name":"Scientific Research Institute of Biology and Biotechnology Problems, Al-Farabi Kazakh National University, Almaty 050040, Kazakhstan"},{"name":"Reference Laboratory for the Control of Especially Dangerous Infections, Almaty 050008, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sagit","family":"Bektassov","sequence":"additional","affiliation":[{"name":"National Scientific Center of Phthisiopulmonology of the Ministry of health of the Republic of Kazakhstan, Almaty 050010, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7837-8685","authenticated-orcid":false,"given":"Amangeldy","family":"Bissenbaev","sequence":"additional","affiliation":[{"name":"Department of Molecular Biology and Genetics, Faculty of Biology and Biotechnology, Al-Farabi Kazakh National University, Almaty 050040, Kazakhstan"},{"name":"Scientific Research Institute of Biology and Biotechnology Problems, Al-Farabi Kazakh National University, Almaty 050040, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,4,11]]},"reference":[{"key":"ref_1","unstructured":"World Health Organization (2023). Global Tuberculosis Report 2023, World Health Organization."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Allu\u00e9-Guardia, A., Garc\u00eda, J.I., and Torrelles, J.B. (2021). Evolution of Drug-Resistant Mycobacterium Tuberculosis Strains and Their Adaptation to the Human Lung Environment. Front. Microbiol., 12.","DOI":"10.3389\/fmicb.2021.612675"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"32244","DOI":"10.1021\/acsomega.3c02563","article-title":"Overcoming Mycobacterium Tuberculosis Drug Resistance: Novel Medications and Repositioning Strategies","volume":"8","author":"Sachan","year":"2023","journal-title":"ACS Omega"},{"key":"ref_4","first-page":"GPP3-0027-2018","article-title":"Unraveling the Structure of the Mycobacterial Envelope","volume":"7","author":"Marrakchi","year":"2019","journal-title":"Microbiol. Spectr."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"47","DOI":"10.1038\/s41579-019-0273-7","article-title":"The Mycobacterial Cell Envelope\u2014A Moving Target","volume":"18","author":"Dulberger","year":"2020","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"550","DOI":"10.1038\/nrmicro3299","article-title":"How Sisters Grow Apart: Mycobacterial Growth and Division","volume":"12","author":"Kieser","year":"2014","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1420","DOI":"10.1126\/science.276.5317.1420","article-title":"Role of the Major Antigen of Mycobacterium Tuberculosis in Cell Wall Biogenesis","volume":"276","author":"Belisle","year":"1997","journal-title":"Science"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"3651","DOI":"10.1074\/jbc.RA117.001681","article-title":"Mycolyltransferase from Mycobacterium Tuberculosis in Covalent Complex with Tetrahydrolipstatin Provides Insights into Antigen 85 Catalysis","volume":"293","author":"Goins","year":"2018","journal-title":"J. Biol. Chem."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1016\/S0065-2164(09)69002-X","article-title":"Biogenesis of the Cell Wall and Other Glycoconjugates of Mycobacterium Tuberculosis","volume":"69","author":"Kaur","year":"2009","journal-title":"Adv. Appl. Microbiol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"845","DOI":"10.1038\/nrmicro2236","article-title":"The Spectrum of Latent Tuberculosis: Rethinking the Biology and Intervention Strategies","volume":"7","author":"Barry","year":"2009","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"2748","DOI":"10.1038\/ncomms3748","article-title":"Mechanism of Inhibition of Mycobacterium Tuberculosis Antigen 85 by Ebselen","volume":"4","author":"Favrot","year":"2013","journal-title":"Nat. Commun."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1983","DOI":"10.1042\/BCJ20200194","article-title":"The Thick Waxy Coat of Mycobacteria, a Protective Layer against Antibiotics and the Host\u2019s Immune System","volume":"477","author":"Batt","year":"2020","journal-title":"Biochem. J."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"656","DOI":"10.1038\/nrmicro1211","article-title":"The Success and Failure of BCG-Implications for a Novel Tuberculosis Vaccine","volume":"3","author":"Andersen","year":"2005","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Huygen, K. (2014). The Immunodominant T-Cell Epitopes of the Mycolyl-Transferases of the Antigen 85 Complex of M. tuberculosis. Front. Immunol., 5.","DOI":"10.3389\/fimmu.2014.00321"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"3998","DOI":"10.1128\/IAI.67.8.3998-4007.1999","article-title":"Cloning, Expression, and Immunological Evaluation of Two Putative Secreted Serine Protease Antigens of Mycobacterium Tuberculosis","volume":"67","author":"Skeiky","year":"1999","journal-title":"Infect. Immun."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"189","DOI":"10.1038\/nm.2285","article-title":"A Multistage Tuberculosis Vaccine That Confers Efficient Protection before and after Exposure","volume":"17","author":"Aagaard","year":"2011","journal-title":"Nat. Med."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1147","DOI":"10.2217\/fmb.12.97","article-title":"Bacteriophage Endolysins as Novel Antimicrobials","volume":"7","author":"Schmelcher","year":"2012","journal-title":"Future Microbiol."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Allu\u00e9-Guardia, A., Saranathan, R., Chan, J., and Torrelles, J.B. (2021). Mycobacteriophages as Potential Therapeutic Agents against Drug-Resistant Tuberculosis. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms22020735"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"36771","DOI":"10.1074\/jbc.M400811200","article-title":"Mycobacterium Tuberculosis Antigen 85A and 85C Structures Confirm Binding Orientation and Conserved Substrate Specificity","volume":"279","author":"Ronning","year":"2004","journal-title":"J. Biol. Chem."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"217","DOI":"10.2217\/fmb.14.125","article-title":"The Future for Early-Stage Tuberculosis Drug Discovery","volume":"10","author":"Zuniga","year":"2015","journal-title":"Future Microbiol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1109","DOI":"10.1046\/j.1365-2958.2002.02953.x","article-title":"Evidence for a Partial Redundancy of the Fibronectin-Binding Proteins for the Transfer of Mycoloyl Residues onto the Cell Wall Arabinogalactan Termini of Mycobacterium Tuberculosis","volume":"44","author":"Puech","year":"2002","journal-title":"Mol. Microbiol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1702","DOI":"10.1002\/pmic.200600853","article-title":"Comprehensive Analysis of Exported Proteins from Mycobacterium Tuberculosis H37Rv","volume":"7","author":"Berven","year":"2007","journal-title":"Proteomics"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"3118","DOI":"10.1002\/pmic.201100043","article-title":"A Proteomic View of Mycobacteria","volume":"11","author":"Wiker","year":"2011","journal-title":"Proteomics"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"218","DOI":"10.1038\/nature12799","article-title":"Mycobacteria Manipulate Macrophage Recruitment through Coordinated Use of Membrane Lipids","volume":"505","author":"Cambier","year":"2014","journal-title":"Nature"},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Beghein, E., and Gettemans, J. (2017). Nanobody Technology: A Versatile Toolkit for Microscopic Imaging, Protein-Protein Interaction Analysis, and Protein Function Exploration. Front. Immunol., 8.","DOI":"10.3389\/fimmu.2017.00771"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"307","DOI":"10.1111\/j.1365-3083.1992.tb03104.x","article-title":"A Family of Cross-Reacting Proteins Secreted by Mycobacterium Tuberculosis","volume":"36","author":"Wiker","year":"1992","journal-title":"Scand. J. Immunol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1608","DOI":"10.1128\/jcm.30.6.1608-1610.1992","article-title":"Humoral Responses against the 85A and 85B Antigens of Mycobacterium Bovis BCG in Patients with Leprosy and Tuberculosis","volume":"30","author":"Drowart","year":"1992","journal-title":"J. Clin. Microbiol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"598","DOI":"10.1093\/infdis\/165.3.598","article-title":"Human Leukocyte Antigen, Tuberculosis, and Mycobacterium Tuberculosis-Specific Antibody","volume":"165","author":"Bothamley","year":"1992","journal-title":"J. Infect. Dis."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"14703","DOI":"10.1073\/pnas.1009080107","article-title":"Dynamic Antibody Responses to the Mycobacterium Tuberculosis Proteome","volume":"107","author":"Salamon","year":"2010","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_30","first-page":"491","article-title":"Prospective Evaluation of a Whole-Blood Test Using Mycobacterium Tuberculosis-Specific Antigens ESAT-6 and CFP-10 for Diagnosis of Active Tuberculosis","volume":"12","author":"Ravn","year":"2005","journal-title":"Clin. Diagn. Lab. Immunol."},{"key":"ref_31","first-page":"354","article-title":"Antigens of Mycobacterium Tuberculosis Recognized by Antibodies during Incipient, Subclinical Tuberculosis","volume":"12","author":"Singh","year":"2005","journal-title":"Clin. Diagn. Lab. Immunol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"648","DOI":"10.1128\/mr.56.4.648-661.1992","article-title":"The Antigen 85 Complex: A Major Secretion Product of Mycobacterium Tuberculosis","volume":"56","author":"Wiker","year":"1992","journal-title":"Microbiol. Rev."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"775","DOI":"10.1146\/annurev-biochem-063011-092449","article-title":"Nanobodies: Natural Single-Domain Antibodies","volume":"82","author":"Muyldermans","year":"2013","journal-title":"Annu. Rev. Biochem."},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Ortega, P.A., Silva-Miranda, M., Torres-Larios, A., Campos-Ch\u00e1vez, E., Franken, K.C.L.C.M., Ottenhoff, T.H.M., Ivanyi, J., and Espitia, C. (2020). Selection of a Single Domain Antibody, Specific for an HLA-Bound Epitope of the Mycobacterial Ag85B Antigen. Front. Immunol., 11.","DOI":"10.3389\/fimmu.2020.577815"},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Jin, B.K., Odongo, S., Radwanska, M., and Magez, S. (2023). NANOBODIES\u00ae: A Review of Diagnostic and Therapeutic Applications. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms24065994"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"236","DOI":"10.1002\/2211-5463.13850","article-title":"Nanobody Engineering: Computational Modelling and Design for Biomedical and Therapeutic Applications","volume":"15","author":"Cater","year":"2025","journal-title":"FEBS Open Bio"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"661","DOI":"10.1186\/s12951-024-02900-y","article-title":"Discovery of Nanobodies: A Comprehensive Review of Their Applications and Potential over the Past Five Years","volume":"22","author":"Alexander","year":"2024","journal-title":"J. Nanobiotechnol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"4586","DOI":"10.1073\/pnas.0505379103","article-title":"Molecular Basis for the Preferential Cleft Recognition by Dromedary Heavy-Chain Antibodies","volume":"103","author":"Silence","year":"2006","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Bannas, P., Hambach, J., and Koch-Nolte, F. (2017). Nanobodies and Nanobody-Based Human Heavy Chain Antibodies as Antitumor Therapeutics. Front. Immunol., 8.","DOI":"10.3389\/fimmu.2017.01603"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"3512","DOI":"10.1093\/emboj\/17.13.3512","article-title":"Potent Enzyme Inhibitors Derived from Dromedary Heavy-Chain Antibodies","volume":"17","author":"Lauwereys","year":"1998","journal-title":"EMBO J."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1707","DOI":"10.1038\/s41596-020-0304-x","article-title":"Generation of Synthetic Nanobodies against Delicate Proteins","volume":"15","author":"Zimmermann","year":"2020","journal-title":"Nat. Protoc."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1111\/j.1574-6968.1994.tb07194.x","article-title":"Mycobacterial Cell Wall: Structure and Role in Natural Resistance to Antibiotics","volume":"123","author":"Jarlier","year":"1994","journal-title":"FEMS Microbiol. Lett."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"367","DOI":"10.1111\/j.1365-2958.2009.06775.x","article-title":"Mycobacteriophage Lysin B Is a Novel Mycolylarabinogalactan Esterase","volume":"73","author":"Payne","year":"2009","journal-title":"Mol. Microbiol."},{"key":"ref_44","doi-asserted-by":"crossref","unstructured":"Singh, V. (2021). Editorial: Tuberculosis Drug Discovery & Development: Drug Targets, Chemical Matter, and Approaches. Front. Cell. Infect. Microbiol., 11.","DOI":"10.3389\/fcimb.2021.755459"},{"key":"ref_45","doi-asserted-by":"crossref","unstructured":"Shleider Carnero Canales, C., Marquez Cazorla, J., Furtado Torres, A.H., Monteiro Filardi, E.T., Di Filippo, L.D., Costa, P.I., Roque-Borda, C.A., and Pavan, F.R. (2023). Advances in Diagnostics and Drug Discovery against Resistant and Latent Tuberculosis Infection. Pharmaceutics, 15.","DOI":"10.3390\/pharmaceutics15102409"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"569","DOI":"10.1038\/35085034","article-title":"Mycobacterium Tuberculosis: Here Today, and Here Tomorrow","volume":"2","author":"Russell","year":"2001","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_47","doi-asserted-by":"crossref","unstructured":"Ivanyi, J. (2014). Function and Potentials of M. tuberc. Epitopes. Front. Immunol., 5.","DOI":"10.3389\/fimmu.2014.00107"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"67","DOI":"10.1038\/nrd2153","article-title":"Inhaling Medicines: Delivering Drugs to the Body through the Lungs","volume":"6","author":"Patton","year":"2007","journal-title":"Nat. Rev. Drug Discov."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"600","DOI":"10.1046\/j.1365-2125.2003.01893.x","article-title":"Pulmonary Drug Delivery. Part II: The Role of Inhalant Delivery Devices and Drug Formulations in Therapeutic Effectiveness of Aerosolized Medications","volume":"56","author":"Labiris","year":"2003","journal-title":"Br. J. Clin. Pharmacol."},{"key":"ref_50","first-page":"11","article-title":"The Therapeutic Potential of Nanobodies","volume":"34","author":"Muyldermans","year":"2019","journal-title":"Biodrugs"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"1076","DOI":"10.1016\/j.drudis.2016.04.003","article-title":"Nanobodies as Therapeutics: Big Opportunities for Small Antibodies","volume":"21","author":"Steeland","year":"2016","journal-title":"Drug Discov. Today"}],"container-title":["Microorganisms"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2607\/14\/4\/863\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,4,13]],"date-time":"2026-04-13T12:05:19Z","timestamp":1776081919000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2607\/14\/4\/863"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,4,11]]},"references-count":51,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2026,4]]}},"alternative-id":["microorganisms14040863"],"URL":"https:\/\/doi.org\/10.3390\/microorganisms14040863","relation":{},"ISSN":["2076-2607"],"issn-type":[{"value":"2076-2607","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,4,11]]}}}