{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T15:07:24Z","timestamp":1785424044012,"version":"3.56.0"},"reference-count":35,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2026,5,1]],"date-time":"2026-05-01T00:00:00Z","timestamp":1777593600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"crossref","award":["32573313"],"award-info":[{"award-number":["32573313"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"crossref"}]},{"award":["32573313"],"award-info":[{"award-number":["32573313"]}],"id":[{"id":"https:\/\/ror.org\/01h0zpd94","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Type II toxin\u2013antitoxin (TA) systems are significantly expanded in the Mycobacterium tuberculosis complex; however, the functional role of the VapBC40 system in Mycobacterium bovis (M. bovis) pathogenesis remains poorly characterized. This study aimed to investigate the role of VapBC40 in mycobacterial virulence and evaluate its potential as a target for rational vaccine attenuation. We performed evolutionary analysis and yeast two-hybrid assays to characterize VapBC40 system specificity, conducted in vitro macrophage infection models and in vivo murine studies to assess virulence contribution, and evaluated the immunoprotective efficacy of a VapC40 knockout strain. Evolutionary analysis revealed progressive sequence conservation and stringent homologous pairing specificity within the VapBC40 system. The VapC40 toxin correlates with enhanced intracellular bacterial survival, increased host cell death, and more severe pulmonary pathology with systemic dissemination. Based on these findings, we evaluated the vaccine potential of a vapC40 knockout strain. Immunization with this attenuated strain elicited a Th1 cellular immune response, characterized by enhanced IFN-\u03b3 production and increased frequency of CD4+IFN-\u03b3+ T cells. Upon challenge with virulent M. bovis, the knockout strain conferred superior protection compared to the conventional BCG vaccine, significantly reducing lung pathology and restricting extrapulmonary bacterial dissemination. Although the molecular mechanisms underlying VapC40-mediated effects remain to be fully elucidated, our findings suggest an important role of the VapBC40 system in mycobacterial-host interactions and support its potential as a target for next-generation tuberculosis vaccine development.<\/jats:p>","DOI":"10.3390\/ijms27094067","type":"journal-article","created":{"date-parts":[[2026,5,1]],"date-time":"2026-05-01T10:15:27Z","timestamp":1777630527000},"page":"4067","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["A Highly Protective Live-Attenuated Vaccine Generated by Targeted Deletion of the Mycobacterium bovis Virulence Factor VapC40"],"prefix":"10.3390","volume":"27","author":[{"given":"Xin","family":"Ge","sequence":"first","affiliation":[{"name":"State Key Laboratory of Veterinary Public Health and Safety, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Haoran","family":"Wang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Veterinary Public Health and Safety, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dingpu","family":"Liu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Veterinary Public Health and Safety, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2891-8449","authenticated-orcid":false,"given":"Yuhui","family":"Dong","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Veterinary Public Health and Safety, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lin","family":"Li","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Veterinary Public Health and Safety, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Puxiu","family":"Shen","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Veterinary Public Health and Safety, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yue","family":"Li","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Veterinary Public Health and Safety, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiaming","family":"Zhang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Veterinary Public Health and Safety, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1566-5745","authenticated-orcid":false,"given":"Xiangmei","family":"Zhou","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Veterinary Public Health and Safety, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ruichao","family":"Yue","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Veterinary Public Health and Safety, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,5,1]]},"reference":[{"key":"ref_1","unstructured":"World Health Organization (2025). Global Tuberculosis Report 2025, World Health Organization."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"788","DOI":"10.1038\/s41579-025-01201-x","article-title":"Mycobacterium tuberculosis biology, pathogenicity and interaction with the host","volume":"23","author":"Warner","year":"2025","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Ramage, H.R., Connolly, L.E., and Cox, J.S. (2009). Comprehensive functional analysis of Mycobacterium tuberculosis toxin-antitoxin systems: Implications for pathogenesis, stress responses, and evolution. PLoS Genet., 5.","DOI":"10.1371\/journal.pgen.1000767"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"5284","DOI":"10.1080\/21645515.2021.2007711","article-title":"BCG vaccination strategies against tuberculosis: Updates and perspectives","volume":"17","author":"Qu","year":"2021","journal-title":"Hum. Vaccin. Immunother."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"6","DOI":"10.1016\/j.medj.2021.11.006","article-title":"Protection against tuberculosis by Bacillus Calmette-Gu\u00e9rin (BCG) vaccination: A historical perspective","volume":"3","author":"Setiabudiawan","year":"2022","journal-title":"Med"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"453","DOI":"10.1016\/j.ijtb.2023.12.002","article-title":"Tuberculosis vaccine\u2014A timely analysis of the drawbacks for the development of novel vaccines","volume":"71","author":"Jeyachandran","year":"2024","journal-title":"Indian J. Tuberc."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"2630512","DOI":"10.1080\/21645515.2026.2630512","article-title":"Efficacy, immunogenicity, and safety of BCG revaccination against Mycobacterium tuberculosis: A systematic review and meta-analysis","volume":"22","author":"Ali","year":"2026","journal-title":"Hum. Vaccin. Immunother."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1","DOI":"10.3310\/hta17370","article-title":"Systematic review and meta-analysis of the current evidence on the duration of protection by bacillus Calmette-Gu\u00e9rin vaccination against tuberculosis","volume":"17","author":"Abubakar","year":"2013","journal-title":"Health Technol. Assess."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"ofaf611","DOI":"10.1093\/ofid\/ofaf611","article-title":"Safety of Bacillus Calmette-Gu\u00e9rin Vaccination and Impact on HIV-1 Latent Reservoir Size in People With Treated HIV-1 Infection","volume":"12","author":"West","year":"2025","journal-title":"Open Forum Infect. Dis."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"105703","DOI":"10.1016\/j.meegid.2024.105703","article-title":"BCG\u2019s role in strengthening immune responses: Implications for tuberculosis and comorbid diseases","volume":"127","author":"Naqvi","year":"2025","journal-title":"Infect. Genet. Evol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1809","DOI":"10.1093\/infdis\/jiy109","article-title":"System-Wide Analysis Unravels the Differential Regulation and In Vivo Essentiality of Virulence-Associated Proteins B and C Toxin-Antitoxin Systems of Mycobacterium tuberculosis","volume":"217","author":"Agarwal","year":"2018","journal-title":"J. Infect. Dis."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1587","DOI":"10.1080\/17460913.2024.2412447","article-title":"Deciphering the role of VapBC toxin-antitoxin systems in Mycobacterium tuberculosis stress adaptation","volume":"19","author":"Thakur","year":"2024","journal-title":"Future Microbiol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"9860","DOI":"10.1093\/nar\/gkw781","article-title":"VapCs of Mycobacterium tuberculosis cleave RNAs essential for translation","volume":"44","author":"Winther","year":"2016","journal-title":"Nucleic Acids Res."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Bordes, P., and Genevaux, P. (2021). Control of Toxin-Antitoxin Systems by Proteases in Mycobacterium Tuberculosis. Front. Mol. Biosci., 8.","DOI":"10.3389\/fmolb.2021.691399"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"e14220","DOI":"10.1016\/j.heliyon.2023.e14220","article-title":"Toxin-antitoxin systems in bacterial pathogenesis","volume":"9","author":"Sonika","year":"2023","journal-title":"Heliyon"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"eaba6944","DOI":"10.1126\/sciadv.aba6944","article-title":"VapBC22 toxin-antitoxin system from Mycobacterium tuberculosis is required for pathogenesis and modulation of host immune response","volume":"6","author":"Agarwal","year":"2020","journal-title":"Sci. Adv."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1417","DOI":"10.1038\/s42003-024-06998-6","article-title":"Deciphering the role of VapBC13 and VapBC26 toxin antitoxin systems in the pathophysiology of Mycobacterium tuberculosis","volume":"7","author":"Sharma","year":"2024","journal-title":"Commun. Biol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"3705","DOI":"10.1038\/s41467-023-38844-4","article-title":"Mycobacterium abscessus VapC5 toxin potentiates evasion of antibiotic killing by ribosome overproduction and activation of multiple resistance pathways","volume":"14","author":"Troian","year":"2023","journal-title":"Nat. Commun."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"9048","DOI":"10.1074\/jbc.RA118.006814","article-title":"Bioinformatic and mutational studies of related toxin-antitoxin pairs in Mycobacterium tuberculosis predict and identify key functional residues","volume":"294","author":"Tandon","year":"2019","journal-title":"J. Biol. Chem."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"e0189621","DOI":"10.1128\/aac.01896-21","article-title":"tRNA(fMet) Inactivating Mycobacterium tuberculosis VapBC Toxin-Antitoxin Systems as Therapeutic Targets","volume":"66","author":"Chauhan","year":"2022","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"136","DOI":"10.4014\/jmb.1709.09009","article-title":"Potentiation of Th1-Type Immune Responses to Mycobacterium tuberculosis Antigens in Mice by Cationic Liposomes Combined with De-O-Acylated Lipooligosaccharide","volume":"28","author":"Ko","year":"2018","journal-title":"J. Microbiol. Biotechnol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"202","DOI":"10.1038\/nrmicro.2018.8","article-title":"Ecology and evolution of Mycobacterium tuberculosis","volume":"16","author":"Gagneux","year":"2018","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"eaaw3307","DOI":"10.1126\/sciadv.aaw3307","article-title":"Genomic determinants of speciation and spread of the Mycobacterium tuberculosis complex","volume":"5","author":"Corander","year":"2019","journal-title":"Sci. Adv."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"6099","DOI":"10.1038\/s41467-021-26248-1","article-title":"Population structure, biogeography and transmissibility of Mycobacterium tuberculosis","volume":"12","author":"Freschi","year":"2021","journal-title":"Nat. Commun."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1401","DOI":"10.1080\/15548627.2021.1987671","article-title":"Mycobacterium bovis induces mitophagy to suppress host xenophagy for its intracellular survival","volume":"18","author":"Song","year":"2022","journal-title":"Autophagy"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"5467","DOI":"10.1038\/s41467-024-49246-5","article-title":"Mycobacterium tuberculosis strain with deletions in menT3 and menT4 is attenuated and confers protection in mice and guinea pigs","volume":"15","author":"Gosain","year":"2024","journal-title":"Nat. Commun."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"eads7377","DOI":"10.1126\/sciimmunol.ads7377","article-title":"Human LY9 governs CD4(+) T cell IFN-\u03b3 immunity to Mycobacterium tuberculosis","volume":"10","author":"Ogishi","year":"2025","journal-title":"Sci. Immunol."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"The UniProt Consortium (2018). UniProt: The universal protein knowledgebase. Nucleic Acids Res., 46, 2699.","DOI":"10.1093\/nar\/gky092"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1547","DOI":"10.1093\/molbev\/msy096","article-title":"MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms","volume":"35","author":"Kumar","year":"2018","journal-title":"Mol. Biol. Evol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"305","DOI":"10.1093\/bioinformatics\/15.4.305","article-title":"ESPript: Analysis of multiple sequence alignments in PostScript","volume":"15","author":"Gouet","year":"1999","journal-title":"Bioinformatics"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1335","DOI":"10.1080\/15548627.2025.2468139","article-title":"Mycobacterium bovis Mb3523c protein regulates host ferroptosis via chaperone-mediated autophagy","volume":"21","author":"Wang","year":"2025","journal-title":"Autophagy"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"e02364-19","DOI":"10.1128\/mBio.02364-19","article-title":"A CRISPR-Assisted Nonhomologous End-Joining Strategy for Efficient Genome Editing in Mycobacterium tuberculosis","volume":"11","author":"Yan","year":"2020","journal-title":"MBio"},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Ahidjo, B.A., Kuhnert, D., McKenzie, J.L., E Machowski, E., Gordhan, B.G., Arcus, V., Abrahams, G.L., and Mizrahi, V. (2011). VapC toxins from Mycobacterium tuberculosis are ribonucleases that differentially inhibit growth and are neutralized by cognate VapB antitoxins. PLoS ONE, 6.","DOI":"10.1371\/journal.pone.0021738"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"6059","DOI":"10.1038\/ncomms7059","article-title":"MazF ribonucleases promote Mycobacterium tuberculosis drug tolerance and virulence in guinea pigs","volume":"6","author":"Tiwari","year":"2015","journal-title":"Nat. Commun."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"116421","DOI":"10.1016\/j.celrep.2025.116421","article-title":"Mycobacterium bovis induces macrophage mitochondrial release of hexokinase 2 to enhance intracellular survival","volume":"44","author":"Li","year":"2025","journal-title":"Cell. Rep."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/9\/4067\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,5,12]],"date-time":"2026-05-12T04:18:30Z","timestamp":1778559510000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/9\/4067"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,5,1]]},"references-count":35,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2026,5]]}},"alternative-id":["ijms27094067"],"URL":"https:\/\/doi.org\/10.3390\/ijms27094067","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,5,1]]}}}