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PubMed2025

Assembly of the Mycobacterium tuberculosis type VII ESX-1 secretion system in Mycobacterium smegmatis identifies a new transcriptional activator of esx-1 genes and a novel TB vaccine.

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 mc2-155 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.IMPORTANCEIn 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.

Mycobacterium smegmatis↗

PubMed2025

WhiB6 Transduces Contact-Dependent Signaling in Mycobacterium smegmatis and Coordinately Induces Both ESX-1 and ESX-4.

Bacteria have evolved complex conditional pathways that respond to environmental stresses and signals. We use conjugation in Mycobacterium smegmatis to identify contact-recognition and response pathways that mediate interactions between donor and recipient cells. Contact with a compatible donor cell initiates a response in the recipient that requires the ESX-1 secretion system and subsequently activates the dormant ESX-4 secretion system. The links of this signal transduction pathway, the mechanism of coordination and dependency between ESX-1 and ESX-4 secretion systems, are unknown. Previous studies identified SigM as a cell-contact responsive sigma factor dedicated to activating ESX-4. WhiB proteins are iron-sulfur-binding stress-response transcription factors exclusively found in Actinobacteria. WhiB6 has been shown to regulate ESX-1 associated gene expression in other mycobacteria. Here, we show that WhiB6 is required both for conjugation and for transducing cell-contact dependent signaling in the recipient cell. Our RNA-seq, ChIP-seq, and proteomic profiling data define a WhiB6 regulon that supports conjugative cell-cell interaction. The WhiB6 regulon includes genes encoding ESX-1, ESX-4, SigM, as well as dispersed operons that likely support ESX secretion. Our data demonstrate that WhiB6 is epistatic to SigM and ESX-4 in this signal transduction pathway. This work shows that WhiB6 functions as a signal transduction node in recipient cells: it coordinates the expression of two ESX systems and it also induces uncharacterized proteins that collectively constitute a complete secretion response to recipient contact with a donor cell.

Mycobacterium smegmatis↗

PubMed2023

Proteomics from compartment-specific APEX2 labeling in Mycobacterium tuberculosis reveals Type VII secretion substrates in the cell wall.

The cell wall of mycobacteria plays a key role in interactions with the environment. Its ability to act as a selective filter is crucial to bacterial survival. Proteins in the cell wall enable this function by mediating the import and export of diverse metabolites, from ions to lipids to proteins. Identifying cell wall proteins is an important step in assigning function, especially as many mycobacterial proteins lack functionally characterized homologues. Current methods for protein localization have inherent limitations that reduce accuracy. Here we showed that although chemical labeling of live cells did not exclusively label surface proteins, protein tagging by the engineered peroxidase APEX2 within live Mycobacterium tuberculosis accurately identified the cytosolic and cell wall proteomes. Our data indicate that substrates of the virulence-associated Type VII ESX secretion system are exposed to the periplasm, providing insight into the currently unknown mechanism by which these proteins cross the mycobacterial cell envelope.

Mycobacterium tuberculosis↗

PubMed2026

Phylogenomic and Clinical Perspectives of an East Asia-Specific Cluster of Mycobacterium massiliense.

BACKGROUND: Mycobacterium abscessus subspecies massiliense (MAM) can form genetically related clusters through continuous within-host adaptations. RESEARCH QUESTION: What is the epidemiologic and clinical significance of the sequence type 120 (ST120) strain of MAM, an East Asia-specific cluster? STUDY DESIGN AND METHODS: Isolates were obtained from patients with MAM pulmonary disease at Seoul National University Hospital between October 1, 2019, and December 31, 2023. These isolates were analyzed using multilocus sequence typing and colony morphotyping and were evaluated for glycopeptidolipid biosynthesis-related gene deletions. Whole-genome sequencing was performed for phylogenomic and pangenome analyses, incorporating MAM genome data from public databases. Finally, the clinical course and treatment outcomes of patients infected with ST120 were evaluated. RESULTS: Among the isolates obtained from 136 patients, 50 isolates (36.8%) were classified as ST120. All ST120 isolates exhibited a rough colony morphotype and harbored deletions in glycopeptidolipid biosynthesis-related genes. From 796 global strains, ST120 was identified exclusively in the isolates obtained from South Korea (51/137 isolates [37.1%]), Japan (3/53 [5.7%]), and Taiwan (2/52 [3.8%]). Genomic analysis revealed the stepwise genetic evolution of ST120, suggesting transmission from Taiwan to Japan and from Japan to South Korea. The ST120 strains exhibited genetic mutations associated with virulence and structural alterations within the ESX system. Consequently, patients with ST120 strain infections had a higher prevalence of cavitary disease (23/50 [46%]) than those infected with non-ST120 strains (19/86 [22.1%]; P = .004). Among those who initiated treatment, the proportion of microbiological cure was lower in patients with ST120 infection (6/15 [40.0%]) than in those with non-ST120 infection (24/34 [70.6%]; P = .043). INTERPRETATION: Our results show that ST120, a strain predominantly found in South Korea, is characterized by the extensive loss of glycopeptidolipid biosynthesis-related genes and is associated with increased disease severity and worse treatment outcomes.

Humans↗

PubMed2026

Sec and Tat Mediated Secretion Safeguards Mycobacterium tuberculosis Membrane Homeostasis.

Protein secretion is essential for the growth and virulence of Mycobacterium tuberculosis, yet the organization and function of its secretion pathways remain poorly understood. We reviewed the existing literature, combined it with systematic queries, and finalized annotations based on experimental data and computational predictions to compile a curated list of 92 secretory components and 198 reactions involved in Sec, twin-arginine translocation (Tat), and ESX pathways. Using CRISPRi, targeted depletion of SecA1 or TatAC impaired both in vitro growth and ex vivo survival. Label-free quantitative secretome analysis revealed decreased export of substrates dependent on SecA1 and TatAC, with enrichment of cytosolic proteins in culture filtrates, indicating increased membrane dysbiosis. Membrane proteomics showed elevated levels of proteins engaged in intermediary and lipid metabolism, while proteins associated with the cell wall and cell processes decreased, suggesting weakened membrane integrity. Loss of SecA1 or TatAC increased membrane permeability, with the effect being more pronounced in the case of TatAC, and caused structural abnormalities seen under electron microscopy. Overall, our integrated multi-omics and functional genetics studies demonstrate that the SecA1 and Tat pathways are essential for maintaining membrane homeostasis in Mycobacterium tuberculosis. These results suggest that essential secretory proteins may be promising targets for therapeutic intervention.

Mycobacterium tuberculosis↗

PubMed2025

Evaluating selection at intermediate scales within genes provides robust identification of genes under positive selection in M. tuberculosis clinical isolates.

Multiple studies have reported genes in the M. tuberculosis (Mtb) genome that are under diversifying selection, based on genetic variants among Mtb clinical isolates. These might reflect adaptions to selection pressures associated with modern clinical treatment of TB. Many, but not all, of these genes under selection are related to drug resistance. Most of these studies have evaluated selection at the gene-level. However, positive selection can be evaluated on different scales, including individual sites (codons) and local regions within an ORF. In this paper, we use GenomegaMap, a Bayesian method for calculating selection, to evaluate selection of genes in the Mtb genome at all three levels. We present evidence that the intermediate analysis (windows of codons) yields the most credible list of candidate genes under selection (excluding PPE and PE_PGRS genes, which are predicted less reliably due to frequent sequencing errors). A further advantage of this approach is that it identifies specific regions within proteins that are under selective pressure, which is useful for structural and functional interpretation. In an analysis of two separate collections of Mtb clinical isolates (from Moldova; and a globally-representative set), we observed 53 and 173 significant genes under selection, with 36% overlap. The lists of genes under selection include many drug-resistance genes, as well as other genes that have previously been reported to be under selection (resR, phoR). The specific regions under selection identified within drug-resistance genes are shown to correspond to protein structural features known to be involved in resistance, supporting accuracy of the method. Positive selection in several ESX-1-related genes was also observed, suggesting adaptation to immune pressure.

adaptation↗