{"doi":"10.1128/spectrum.01131-25","title":"Assembly of the <i>Mycobacterium tuberculosis</i> type VII ESX-1 secretion system in <i>Mycobacterium smegmatis</i> identifies a new transcriptional activator of <i>esx-1</i> genes and a novel TB vaccine","abstract":"ABSTRACT Mycobacterium tuberculosis ( M. tb ) uses its type VII secretion system (T7SS) ESX-1 to export immunogenic, virulence-mediating protein effectors. In this study, the fast-growing, non-pathogenic model mycobacteria Mycobacterium smegmatis mc 2 -155 was engineered to express the M. tb T7SS ESX-1 system. We found that M. smegmatis transformed with M. tb esx-1 locus genes only, as well as M. smegmatis transformed with M. tb esx-1 and espACD operon genes (designated MSX-1), produces and secretes the M. tb ESX-1 protein effectors EsxA, EsxB, and EspB. However, the abundance of these proteins was higher inside the cell and culture filtrate of the MSX-1 strain. Although ESX-1 is critical for M. tb pathogenesis, expression of M. tb ESX-1 did not make the recombinant M. smegmatis strains virulent in macrophages. Serendipitously, transformation of M. smegmatis with a modified esx-1 locus in this study revealed rv3860 , a gene of previously unknown function, to be required for the transcription of pe35 , ppe68 , esxB, and esxA genes. Finally, mice vaccinated with MSX-1 were found to be as protected as mice vaccinated with Mycobacterium bovis BCG against M. tb infection, without becoming sensitized to tuberculin. These results show that a functional M. tb ESX-1 system can be assembled in M. smegmatis to uncover novel facets of the secretion machinery and that the modified M. smegmatis strain can function as a tuberculosis (TB) vaccine. Unlike BCG, however, its deployment may be compatible with tests currently used to diagnose TB. IMPORTANCE In this study, we modified Mycobacterium smegmatis , which is often used as a surrogate model organism in mycobacterial research, to produce and assemble a functional Mycobacterium tuberculosis ( M. tb ) ESX-1 protein secretion system. One such M. smegmatis strain named MSX-1 was found to make a functional M. tb ESX-1 system without becoming virulent. And in using M. smegmatis as a chassis to study the ESX-1 system, we found that rv3860 , an M. tb gene of previously unknown function, is needed for the production of key ESX-1 proteins. Finally, mice vaccinated with MSX-1 were as protected from tuberculosis (TB) as mice given BCG, the only approved TB vaccine. Notably, we found that unlike BCG, MSX-1 does not sensitize mice to the antigens used in existing TB diagnostic tests. These observations, taken together, highlight the utility of M. smegmatis as a chassis to study the M. tb ESX-1 secretion machinery.","journal":"Microbiology Spectrum","year":2025,"id":546846,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":1,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9637,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1439340,"name":"Ze Long Lim","orcid":null,"position":1,"is_corresponding":false},{"id":1439341,"name":"Marlene Snider","orcid":null,"position":2,"is_corresponding":false},{"id":1438909,"name":"Nirajan Niroula","orcid":"0000-0002-3243-0131","position":3,"is_corresponding":false},{"id":1439342,"name":"Marie Hardouin","orcid":null,"position":4,"is_corresponding":false},{"id":1438910,"name":"Jeffrey M. Chen","orcid":"0000-0001-8431-3802","position":5,"is_corresponding":false},{"id":1438908,"name":"Slim Zriba","orcid":"0000-0001-8553-8317","position":0,"is_corresponding":true}],"reference_count":48,"raw_metadata":null,"created_at":"2026-07-19T02:53:36.567932Z","pmid":"40938095","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}