| 1.C.95 The Pore-forming ESAT-6 Protein (ESAT-6) Family
The ESX-1 secretion system (TC# 9.A.25) plays a critical role in virulence of M. tuberculosis and M. marinum. Virulent M. marinum is able to escape from the Mycobacterium-containing vacuole (MCV) into the host cell cytosol, polymerize actin, and spread from cell to cell. ESX-1 plays an essential role in M. marinum escape from the MCV.
Smith et al. (2008) provided evidence that M. marinum induces membrane pores approximately 4.5 nm in diameter, and this activity correlates with ESAT-6 secretion. Purified ESTAT-6, but not the other ESX-1-secreted proteins, is able to cause dose-dependent pore-formation in host cell membranes.
M. tuberculosis uses protein secretion systems, named 6 kDa early secretory
antigenic target (ESAT6) protein family secretion (ESX) systems to export a set of effector
proteins that help the pathogen resist or evade the host immune
response (Gröschel et al. 2016). Since the discovery of the esx loci during the M. tuberculosis H37Rv genome project, structural biology, cell biology and evolutionary
analyses have been conducted. Gröschel et al. 2016 reviewed roles that these studies have revealed for
ESX systems in bacterial survival and pathogenicity during infection
with M. tuberculosis. They discuss the diversity of ESX systems that have been
described among mycobacteria and selected non-mycobacterial species.
The reaction catalyzed for ESAT-6 is:
molecules (vacuoles) ⇌ molecules (cytoplasm)
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| References: |
Callahan, B., K. Nguyen, A. Collins, K. Valdes, M. Caplow, D.K. Crossman, A.J. Steyn, L. Eisele, and K.M. Derbyshire. (2010). Conservation of structure and protein-protein interactions mediated by the secreted mycobacterial proteins EsxA, EsxB, and EspA. J. Bacteriol. 192: 326-335.
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Gröschel, M.I., F. Sayes, R. Simeone, L. Majlessi, and R. Brosch. (2016). ESX secretion systems: mycobacterial evolution to counter host immunity. Nat. Rev. Microbiol. 14: 677-691.
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Nuñez-Garcia, J., S.H. Downs, J.E. Parry, D.A. Abernethy, J.M. Broughan, A.R. Cameron, A.J. Cook, R. de la Rua-Domenech, A.V. Goodchild, J. Gunn, S.J. More, S. Rhodes, S. Rolfe, M. Sharp, P.A. Upton, H.M. Vordermeier, E. Watson, M. Welsh, A.O. Whelan, J.A. Woolliams, R.S. Clifton-Hadley, and M. Greiner. (2018). Meta-analyses of the sensitivity and specificity of ante-mortem and post-mortem diagnostic tests for bovine tuberculosis in the UK and Ireland. Prev Vet Med 153: 94-107.
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Smith, J., J. Manoranjan, M. Pan, A. Bohsali, J. Xu, J. Liu, K.L. McDonald, A. Szyk, N. LaRonde-LeBlanc, and L.Y. Gao. (2008). Evidence for pore formation in host cell membranes by ESX-1-secreted ESAT-6 and its role in Mycobacterium marinum escape from the vacuole. Infect. Immun. 76: 5478-5487.
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Tak, U., T. Dokland, and M. Niederweis. (2021). Pore-forming Esx proteins mediate toxin secretion by Mycobacterium tuberculosis. Nat Commun 12: 394.
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| Examples: |
| TC# | Name | Organismal Type | Example |
| 1.C.95.1.1 | Pore-forming ESAT-6 (EsxA) (95 aas) (Nuñez-Garcia et al. 2018). Secreted from the bacterial cytoplasm via a ESX protein secretion system (Type VII; TC# 3.A.24.5.1). | Mycobacteria | ESAT-6 of Mycobacterium tuberculosis (bovis) (P0A564). |
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| 1.C.95.1.10 | Uncharacterized protein of 134 aas. | | UP of Paracoccus aminophilus |
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| 1.C.95.1.11 | Uncharacterized protein of 211 aas. | | UP of Nakamurella lactea |
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| 1.C.95.1.12 | Uncharacterized protein of 97 aas. | | UP of Holdemania filiformis |
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| 1.C.95.1.13 | Uncharacterized WXG100 family type VII secretion targetof 100 aas. | | UP of Salinispora arenicola |
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| 1.C.95.1.14 | EsxT/EsxY of 100 and 105 aas, respectively. Mycobacterium tuberculosis secretes proteins using five ESX
systems with distinctive functions essential for its growth and
virulence. Nair et al. 2025 showed that a non-canonical supercomplex of the
EsxU-EsxT proteins, encoded in the esx-4 locus, with the orphan EsxE-EsxF proteins, encoded in the cpnT operon, is required for toxin secretion by M. tuberculosis . Surprisingly, the outer membrane localization of all Esx proteins and
their secretion into the cytosol of infected macrophages also depend on
the EsxEF-EsxUT supercomplex and ESX-4. These results not only
demonstrate that the Esx proteins have dual functions as the long-sought
outer membrane components of ESX systems and as secreted effector
proteins, but also reveal a novel master control mechanism of protein
secretion in M. tuberculosis . The mutual dependency of EsxEF and EsxUT on each other synchronizes ESX effector protein secretion, enabling M. tuberculosis to block phagosomal maturation and to permeabilize the phagosomal
membrane only when it is capable of killing host cells by toxin
secretion (Nair et al. 2025). | | EsxT/EsxU of Mycobacterium tuberculosis |
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| 1.C.95.1.2 | ESAT-6-like protein, EsxA of 95 aas (Callahan et al. 2010). | | ESAT-6-like protein of Corynebacterium diphtheriae |
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| 1.C.95.1.3 | ESAT-6-like protein. a WXG100 (YukE) family type VII secretion target. | | ESAT-6 protein of Gordonia rhizosphera |
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| 1.C.95.1.4 | ESAT6-like protein of 95 aas. | | ESAT-6-like protein of Amycolatopsis nigrescens |
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| 1.C.95.1.5 | ESAT-6-like protein of 96 aas. | | ESAT-6 of Intrasporangium chromatireducens |
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| 1.C.95.1.6 | ESAT-6-like protein of 101 aas | | UP of Nesterenkonia sp. F |
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| 1.C.95.1.7 | ESAT-6-like protein of 103 aas. | | ESAT-6-like protein of Williamsia marianensis |
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| 1.C.95.1.8 | Uncharacterized protein of 96 aas | | UP of Paeniglutamicibacter antarcticus |
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| 1.C.95.1.9 | Uncharacterized WXG100 family type VII secretion target of 102 aas. | | UP of Herbivorax saccincola |
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| Examples: |
| TC# | Name | Organismal Type | Example |
| 1.C.95.2.1 | EsxE/EsxF pair of proteins of 90 and 103 aas, respectively (Tak et al. 2021). Mycobacterium tuberculosis secretes the tuberculosis necrotizing toxin
(TNT) to kill host cells. The WXG100 proteins EsxE
and EsxF are essential for TNT secretion. EsxE and EsxF form a
water-soluble heterodimer (EsxEF) that assembles into oligomers and long
filaments, binds to membranes, and forms stable membrane-spanning
channels. Electron microscopy of EsxEF reveals mainly pentameric
structures with a central pore (Tak et al. 2021). Mutations of both WXG motifs and of a
GXW motif do not affect dimerization, but abolish pore formation,
membrane deformation and TNT secretion. The WXG/GXW mutants are locked
in conformations with altered thermostability and solvent exposure,
indicating that the WXG/GXW motifs are molecular switches controlling
membrane interaction and pore formation. EsxF is accessible on the
bacterial cell surface, suggesting that EsxEF form an outer membrane
channel for toxin export. Thus, our study reveals a protein secretion
mechanism in bacteria that relies on pore formation by small WXG
proteins. | | EsxE/EsxF of Mycobacterium tuberculosis |
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