{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,29]],"date-time":"2026-08-29T09:53:08Z","timestamp":1787997188630,"version":"build-2784847793"},"reference-count":46,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2026,5,3]],"date-time":"2026-05-03T00:00:00Z","timestamp":1777766400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100010677","name":"INTA","doi-asserted-by":"publisher","award":["i114"],"award-info":[{"award-number":["i114"]}],"id":[{"id":"10.13039\/501100010677","id-type":"DOI","asserted-by":"publisher"}]},{"award":["i114"],"award-info":[{"award-number":["i114"]}],"id":[{"id":"https:\/\/ror.org\/04wm52x94","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Mycobacterium bovis, the causative agent of bovine tuberculosis, infects and persists within macrophages, triggering pro-inflammatory responses. While these mechanisms are well characterized for Mycobacterium tuberculosis, less is known about host responses to M. bovis. Inflammasome activation and IL-1\u03b2 production have been linked to ESAT-6, a substrate of the ESX-1 secretion system present in both species. Here, we examined inflammasome activation in bovine macrophages infected with the virulent M. bovis strain Mb04-303. M. bovis AF2122\/97 and NCTC10772 upregulated IL-1\u03b2 transcription, whereas Mb04-303 and BCG did not. Unexpectedly, deletion of the genes encoding ESAT-6 and CFP-10 from Mb04-303 enhanced inflammasome activation, as evidenced by increased NLRP3 and IL-1\u03b2 transcription. Complementation with either wild-type ESAT-6\/CFP-10 or the T63A ESAT-6 variant restored downregulation of the response, indicating that this substitution does not alter inflammasome modulation. In contrast, deletion of ESAT-6\/CFP-10 from an attenuated M. bovis vaccine candidate reduced IL-1\u03b2 transcription. No differences were observed between M. tuberculosis H37Rv and its ESAT-6-deficient mutant in bovine macrophages. Together, these findings demonstrate that ESAT-6\/CFP-10-mediated modulation of inflammasome activation in bovine macrophages is highly dependent on the mycobacterial genetic background.<\/jats:p>","DOI":"10.3390\/ijms27094099","type":"journal-article","created":{"date-parts":[[2026,5,4]],"date-time":"2026-05-04T01:12:09Z","timestamp":1777857129000},"page":"4099","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Mycobacterium bovis Strain-Dependent Effects of ESAT-6 and CFP-10 on Inflammasome Activation in Bovine Macrophages"],"prefix":"10.3390","volume":"27","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7820-2169","authenticated-orcid":false,"given":"Federico Carlos","family":"Blanco","sequence":"first","affiliation":[{"name":"Instituto de Agrobiotecnolog\u00eda y Biolog\u00eda Molecular (IABIMO), INTA-CONICET, Hurlingham 1686, Argentina"},{"name":"Instituto de Biotecnolog\u00eda, CICVyA, Instituto Nacional de Tecnolog\u00eda Agropecuaria, N. Repetto and De los Reseros, Hurlingham 1686, Argentina"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4831-9654","authenticated-orcid":false,"given":"Cristina Lourdes","family":"Vazquez","sequence":"additional","affiliation":[{"name":"Instituto de Agrobiotecnolog\u00eda y Biolog\u00eda Molecular (IABIMO), INTA-CONICET, Hurlingham 1686, Argentina"},{"name":"Instituto de Biotecnolog\u00eda, CICVyA, Instituto Nacional de Tecnolog\u00eda Agropecuaria, N. Repetto and De los Reseros, Hurlingham 1686, Argentina"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8711-1413","authenticated-orcid":false,"given":"Mar\u00eda Mercedes","family":"Bigi","sequence":"additional","affiliation":[{"name":"Instituto de Investigaciones Biom\u00e9dicas (INBIOMED), CONICET-Universidad de Buenos Aires, Buenos Aires C1121ABG, Argentina"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rosana Valeria","family":"Rocha","sequence":"additional","affiliation":[{"name":"Instituto de Agrobiotecnolog\u00eda y Biolog\u00eda Molecular (IABIMO), INTA-CONICET, Hurlingham 1686, Argentina"},{"name":"Instituto de Biotecnolog\u00eda, CICVyA, Instituto Nacional de Tecnolog\u00eda Agropecuaria, N. Repetto and De los Reseros, Hurlingham 1686, Argentina"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-9158-0078","authenticated-orcid":false,"given":"Elizabeth Andrea","family":"Garc\u00eda","sequence":"additional","affiliation":[{"name":"Instituto de Agrobiotecnolog\u00eda y Biolog\u00eda Molecular (IABIMO), INTA-CONICET, Hurlingham 1686, Argentina"},{"name":"Instituto de Biotecnolog\u00eda, CICVyA, Instituto Nacional de Tecnolog\u00eda Agropecuaria, N. Repetto and De los Reseros, Hurlingham 1686, Argentina"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8637-7180","authenticated-orcid":false,"given":"Fabiana","family":"Bigi","sequence":"additional","affiliation":[{"name":"Instituto de Agrobiotecnolog\u00eda y Biolog\u00eda Molecular (IABIMO), INTA-CONICET, Hurlingham 1686, Argentina"},{"name":"Instituto de Biotecnolog\u00eda, CICVyA, Instituto Nacional de Tecnolog\u00eda Agropecuaria, N. Repetto and De los Reseros, Hurlingham 1686, Argentina"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,5,3]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"471","DOI":"10.1016\/S1286-4579(02)01562-9","article-title":"Control of Mycobacterium bovis infections and the risk to human populations","volume":"4","author":"Wedlock","year":"2002","journal-title":"Microbes Infect."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"eadl3962","DOI":"10.1126\/science.adl3962","article-title":"BCG vaccination reduces bovine tuberculosis transmission, improving prospects for elimination","volume":"383","author":"Fromsa","year":"2024","journal-title":"Science"},{"key":"ref_3","first-page":"1","article-title":"Tuberculosis Vaccines for Cattle","volume":"18","author":"Blanco","year":"2023","journal-title":"CABI Rev."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"578","DOI":"10.1007\/s12026-022-09286-9","article-title":"Insights into inflammasome regulation: Cellular, molecular, and pathogenic control of inflammasome activation","volume":"70","author":"Challagundla","year":"2022","journal-title":"Immunol. Res."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"151","DOI":"10.1007\/s00018-010-0450-3","article-title":"Involvement of xanthine oxidase and hypoxia-inducible factor 1 in Toll-like receptor 7\/8-mediated activation of caspase 1 and interleukin-1\u03b2","volume":"68","author":"Nicholas","year":"2011","journal-title":"Cell. Mol. Life Sci."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"153","DOI":"10.1038\/nature18629","article-title":"Inflammasome-activated gasdermin D causes pyroptosis by forming membrane pores","volume":"535","author":"Liu","year":"2016","journal-title":"Nature"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"821","DOI":"10.1016\/j.cell.2010.01.040","article-title":"The Inflammasomes","volume":"140","author":"Schroder","year":"2010","journal-title":"Cell"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"36","DOI":"10.1038\/s41421-020-0167-x","article-title":"Inflammasome activation and regulation: Toward a better understanding of complex mechanisms","volume":"6","author":"Zheng","year":"2020","journal-title":"Cell Discov."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1046","DOI":"10.1111\/j.1462-5822.2010.01450.x","article-title":"Mycobacterium tuberculosis protein ESAT-6 is a potent activator of the NLRP3\/ASC inflammasome","volume":"12","author":"Mishra","year":"2010","journal-title":"Cell. Microbiol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"e2122161119","DOI":"10.1073\/pnas.2122161119","article-title":"The C terminus of the mycobacterium ESX-1 secretion system substrate ESAT-6 is required for phagosomal membrane damage and virulence","volume":"119","author":"Osman","year":"2022","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"104799","DOI":"10.1016\/j.isci.2022.104799","article-title":"Histone deacetylase-2 controls IL-1\u03b2 production through the regulation of NLRP3 expression and activation in tuberculosis infection","volume":"25","author":"Moreira","year":"2022","journal-title":"iScience"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Escobar-Chavarr\u00eda, O., Benitez-Guzman, A., Jim\u00e9nez-V\u00e1zquez, I., Carrisoza-Urbina, J., Arriaga-Pizano, L., Huerta-Y\u00e9pez, S., Baay-Guzm\u00e1n, G., and Guti\u00e9rrez-Pabello, J.A. (2023). Necrotic Cell Death and Inflammasome NLRP3 Activity in Mycobacterium bovis-Infected Bovine Macrophages. Cells, 12.","DOI":"10.3390\/cells12162079"},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Ramon-Luing, L.A., Olvera, Y., Flores-Gonzalez, J., Palacios, Y., Carranza, C., Aguilar-Duran, Y., Vargas, M.A., Gutierrez, N., Medina-Quero, K., and Chavez-Galan, L. (2022). Diverse Cell Death Mechanisms Are Simultaneously Activated in Macrophages Infected by Virulent Mycobacterium tuberculosis. Pathogens, 11.","DOI":"10.3390\/pathogens11050492"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1547","DOI":"10.1111\/j.1462-5822.2007.00892.x","article-title":"The ESAT6 protein of Mycobacterium tuberculosis induces apoptosis of macrophages by activating caspase expression","volume":"9","author":"Derrick","year":"2007","journal-title":"Cell. Microbiol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"195","DOI":"10.1016\/j.bbrc.2016.06.042","article-title":"ESAT6 inhibits autophagy flux and promotes BCG proliferation through MTOR","volume":"477","author":"Dong","year":"2016","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"60","DOI":"10.1016\/j.tube.2009.10.003","article-title":"Immune response to Mycobacterium tuberculosis specific antigen ESAT-6 among south Indians","volume":"90","author":"Kumar","year":"2010","journal-title":"Tuberculosis"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"2587","DOI":"10.1128\/iai.65.7.2587-2592.1997","article-title":"Predominant recognition of the ESAT-6 protein in the first phase of infection with Mycobacterium bovis in cattle","volume":"65","author":"Pollock","year":"1997","journal-title":"Infect. Immun."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1326","DOI":"10.1128\/IAI.01246-09","article-title":"Screening of predicted secreted antigens from Mycobacterium bovis reveals the immunodominance of the ESAT-6 protein family","volume":"78","author":"Jones","year":"2010","journal-title":"Infect. Immun."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1083","DOI":"10.1586\/erv.11.22","article-title":"DIVA reagents for bovine tuberculosis vaccines in cattle","volume":"10","author":"Vordermeier","year":"2011","journal-title":"Expert. Rev. Vaccines"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"795","DOI":"10.1016\/j.tube.2015.07.009","article-title":"Identification and evaluation of new Mycobacterium bovis antigens in the in vitro interferon gamma release assay for bovine tuberculosis diagnosis","volume":"95","author":"Eirin","year":"2015","journal-title":"Tuberculosis"},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Blanco, F.C., Gravisaco, M.J., Bigi, M.M., Garc\u00eda, E.A., Marquez, C., McNeil, M., Jackson, M., and Bigi, F. (2021). Identifying Bacterial and Host Factors Involved in the Interaction of Mycobacterium bovis with the Bovine Innate Immune Cells. Front. Immunol., 12.","DOI":"10.3389\/fimmu.2021.674643"},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Blanco, F.C., Bigi, M.M., Garc\u00eda, E.A., Elola, M.T., V\u00e1zquez, C.L., and Bigi, F. (2023). A Transcriptional Analysis of Cattle Immune Cells Reveals a Central Role of Type 1 Interferon in the In Vitro Innate Immune Response against Mycobacterium bovis. Pathogens, 12.","DOI":"10.3390\/pathogens12091159"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"108482","DOI":"10.1016\/j.vetmic.2019.108482","article-title":"Analysing nonsynonymous mutations between two Mycobacterium bovis strains with contrasting pathogenic profiles","volume":"239","author":"Bigi","year":"2019","journal-title":"Vet. Microbiol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"21598","DOI":"10.1074\/jbc.M201625200","article-title":"Conclusive evidence that the major T-cell antigens of the Mycobacterium tuberculosis complex ESAT-6 and CFP-10 form a tight, 1:1 complex and characterization of the structural properties of ESAT-6, CFP-10, and the ESAT-6\u00b7CFP-10 complex. Implications for pathogenesis and virulence","volume":"277","author":"Renshaw","year":"2002","journal-title":"J. Biol. Chem."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"139","DOI":"10.1111\/j.1365-2249.2009.03923.x","article-title":"Mycobacterium bovis with different genotypes and from different hosts induce dissimilar immunopathological lesions in a mouse model of tuberculosis","volume":"157","author":"Bernardelli","year":"2009","journal-title":"Clin. Exp. Immunol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1949","DOI":"10.1038\/s41467-020-15832-6","article-title":"Mycobacterium tuberculosis associated with severe tuberculosis evades cytosolic surveillance systems and modulates IL-1\u03b2 production","volume":"11","author":"Sousa","year":"2020","journal-title":"Nat. Commun."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"110","DOI":"10.1016\/j.it.2011.01.003","article-title":"Inflammasome activation and IL-1\u03b2 and IL-18 processing during infection","volume":"32","author":"Netea","year":"2011","journal-title":"Trends Immunol."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Barczak, A.K., Avraham, R., Singh, S., Luo, S.S., Zhang, W.R., Bray, M.A., Hinman, A.E., Thompson, M., Nietupski, R.M., and Golas, A. (2017). Systematic, multiparametric analysis of Mycobacterium tuberculosis intracellular infection offers insight into coordinated virulence. PLoS Pathog., 13.","DOI":"10.1371\/journal.ppat.1006363"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1866","DOI":"10.1111\/j.1462-5822.2008.01177.x","article-title":"ESX-1-dependent cytolysis in lysosome secretion and inflammasome activation during mycobacterial infection","volume":"10","author":"Koo","year":"2008","journal-title":"Cell. Microbiol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"417","DOI":"10.4103\/ijmy.ijmy_180_20","article-title":"Elimination of ESAT-6 and CFP-10 from a candidate vaccine against bovine tuberculosis impaired its protection efficacy in the BALBc mouse model","volume":"9","author":"Blanco","year":"2020","journal-title":"Int. J. Mycobacteriology"},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Liao, Y., Hussain, T., Liu, C., Cui, Y., Wang, J., Yao, J., Chen, H., Song, Y., Sabir, N., and Hussain, M. (2019). Endoplasmic reticulum stress induces marcrophages to produce IL-1\u03b2 during mycobacteria bovis infection via a positive feedback loop between mitochondrial damage and inflammasome activation. Front. Immunol., 10.","DOI":"10.3389\/fimmu.2019.00268"},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Zhou, Y., Shah, S.Z.A., Yang, L., Zhang, Z., Zhou, X., and Zhao, D. (2016). Virulent Mycobacterium bovis Beijing strain activates the NLRP7 inflammasome in THP-1 macrophages. PLoS ONE, 11.","DOI":"10.1371\/journal.pone.0152853"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1849","DOI":"10.1093\/infdis\/jit347","article-title":"The AIM2 inflammasome is involved in macrophage activation during infection with virulent mycobacterium bovis strain","volume":"208","author":"Yang","year":"2013","journal-title":"J. Infect. Dis."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"S35","DOI":"10.1016\/j.tube.2016.09.026","article-title":"Differential inflammasome activation signatures following intracellular infection of human macrophages with Mycobacterium bovis BCG or Trypanosoma cruzi","volume":"101","author":"Huante","year":"2016","journal-title":"Tuberculosis"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"5752","DOI":"10.1073\/pnas.95.10.5752","article-title":"A bacterial two-hybrid system based on a reconstituted signal transduction pathway","volume":"95","author":"Karimova","year":"1998","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"1371","DOI":"10.1111\/j.1462-5822.2011.01625.x","article-title":"Critical role for NLRP3 in necrotic death triggered by Mycobacterium tuberculosis","volume":"13","author":"Wong","year":"2011","journal-title":"Cell. Microbiol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"2270","DOI":"10.1038\/s41467-020-16143-6","article-title":"Plasma membrane damage causes NLRP3 activation and pyroptosis during Mycobacterium tuberculosis infection","volume":"11","author":"Beckwith","year":"2020","journal-title":"Nat. Commun."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"285","DOI":"10.1016\/j.molimm.2021.04.022","article-title":"Mycobacterium tuberculosis stimulates IL-1\u03b2 production by macrophages in an ESAT-6 dependent manner with the involvement of serum amyloid A3","volume":"135","author":"Jung","year":"2021","journal-title":"Mol. Immunol."},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Petrilli, J.D., M\u00fcller, I., Ara\u00fajo, L.E., Cardoso, T.M., Carvalho, L.P., Barros, B.C., Teixeira, M., Arruda, S., Riley, L.W., and Queiroz, A. (2020). Differential Host Pro-Inflammatory Response to Mycobacterial Cell Wall Lipids Regulated by the Mce1 Operon. Front. Immunol., 11.","DOI":"10.3389\/fimmu.2020.01848"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"185","DOI":"10.1016\/j.femsim.2004.08.013","article-title":"Comparison of mammalian cell entry operons of mycobacteria: In silico analysis and expression profiling","volume":"43","author":"Kumar","year":"2005","journal-title":"FEMS Immunol. Med. Microbiol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"106367","DOI":"10.1016\/j.micpath.2023.106367","article-title":"Mycobacterium tuberculosis Mce2D protein blocks M1 polarization in macrophages by inhibiting the ERK signaling pathway","volume":"184","author":"Weng","year":"2023","journal-title":"Microb. Pathog."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"110371","DOI":"10.1016\/j.vetmic.2025.110371","article-title":"Long-term evaluation in BALBc mice of a triple mutant of Mycobacterium bovis and the Bacillus Calmette-Gu\u00e9rin as potential vaccines against bovine tuberculosis","volume":"302","author":"Blanco","year":"2025","journal-title":"Vet. Microbiol."},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"Untergasser, A., Ruijter, J.M., Benes, V., and van den Hoff, M.J.B. (2021). Web-based LinRegPCR: Application for the visualization and analysis of (RT)-qPCR amplification and melting data. BMC Bioinform., 22.","DOI":"10.1186\/s12859-021-04306-1"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"100939","DOI":"10.1016\/j.isci.2020.100939","article-title":"MutaBind2: Predicting the Impacts of Single and Multiple Mutations on Protein-Protein Interactions","volume":"23","author":"Zhang","year":"2020","journal-title":"iScience"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"W306","DOI":"10.1093\/nar\/gki375","article-title":"I-Mutant2.0: Predicting stability changes upon mutation from the protein sequence or structure","volume":"33","author":"Capriotti","year":"2005","journal-title":"Nucleic Acids Res."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"e1008543","DOI":"10.1371\/journal.pcbi.1008543","article-title":"PremPS: Predicting the impact of missense mutations on protein stability","volume":"16","author":"Chen","year":"2020","journal-title":"PLoS Comput. Biol."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/9\/4099\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,5,4]],"date-time":"2026-05-04T01:15:49Z","timestamp":1777857349000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/9\/4099"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,5,3]]},"references-count":46,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2026,5]]}},"alternative-id":["ijms27094099"],"URL":"https:\/\/doi.org\/10.3390\/ijms27094099","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,5,3]]}}}