Purification and characterization of a low-molecular-mass T-cell antigen secreted by Mycobacterium tuberculosis
Open accessAbstract
A novel immunogenic antigen, the 6-kDa early secretory antigenic target (ESAT-6), from short-term culture filtrates of Mycobacterium tuberculosis was purified by hydrophobic interaction chromatography and anion-exchange chromatography by use of fast protein liquid chromatography. The antigen focused at two different pIs of 4.0 and 4.5 during isoelectric focusing, and each of these components separated into three spots ranging from 4 to 6 kDa during two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent differences in molecular masses or pIs of these isoforms were not due to posttranslational glycosylation. The molecular weight of the purified native protein was determined by applying gel filtration and nondenaturing polyacrylamide gel electrophoresis and found to be 24 kDa. ESAT-6 is recognized by the murine monoclonal antibody HYB 76-8, which was used to screen a recombinant lambda gt11 M. tuberculosis DNA library. A phage expressing a gene product recognized by HYB 76-8 was isolated, and a 1.7-kbp fragment of the mycobacterial DNA insert was sequenced. The structural gene of ESAT-6 was identified as the sequence encoding a polypeptide of 95 amino acids. The N terminus of the deduced sequence could be aligned with the 10 amino-terminal amino acids derived from sequence analyses of the native protein. N-terminal sequence analysis showed that the purified antigen was essentially free from contaminants, and the amino acid analysis of the antigen was in good agreement with the DNA sequence-deduced amino acid composition. Thus, the heterogeneities observed in the pI and molecular weight of the purified antigen do not derive from contaminating proteins but are most likely due to heterogeneity of the antigen itself. Native and recombinant ESAT-6 are immunologically active in that both elicited a high release of gamma interferon from T cells isolated from memory-immune mice challenged with M. tuberculosis. Analyses of subcellular fractions of M. tuberculosis showed the presence of ESAT-6 in cytosol- and cell wall-containing fractions. Interspecies analyses showed the presence of ESAT-6 in filtrates from M. tuberculosis complex species. Among filtrates from mycobacteria not belonging to the M. tuberculosis complex, reactivity was observed in Mycobacterium kansasii, Mycobacterium szulgai, and Mycobacterium marinum.
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The ESX-1 cluster is present in a range of mycobacteria, including Mycobacterium kansasii and Mycobacterium leprae.
“Apart from members of the M. tuberculosis complex, the ESX-1 cluster is also present in a range of mycobacteria, including Mycobacterium kansasii [23] and Mycobacterium leprae [42].”
From Systematic Genetic Nomenclature for Type VII Secretion Systems (2009) · cites this paper as [23]
Loss of ESX-1 is one of the main reasons for the attenuation of the BCG vaccine, while ESX-1 effectors are important antigens in immune responses.
“Loss of ESX-1 is one of the main reasons for the attenuation of the BCG vaccine (65), while ESX-1 effectors are important antigens in immune responses (6).”
From ESX/Type VII Secretion Systems—An Important Way Out for Mycobacterial Proteins (2019) · cites this paper as [6]
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- Authors
- Sørensen, A. L., Nagai, S., Houen, G., Andersen, P., & Andersen, A. B.
- Affiliations
- Statens Serum Institut (DK)
- Mycobacteria Department, Statens Seruminstitut, Copenhagen, Denmark.
- Venue
- Infection and Immunity
- Publisher
- American Society for Microbiology
- Published
- 1995-05
- Volume
- 63
- Issue
- 5
- Pages
- 1710-1717
- ISSN
- 0019-9567 (ISSN-L)
- 1098-5522
- PMC
- PMC173214
- OpenAlex
- W2100894095
- Cited by
- 586
- References
- 31
- Citations by year
- 2026: 5 · 2025: 12 · 2024: 6 · 2023: 15 · 2022: 8 · 2021: 16 · 2020: 8 · 2019: 10 · 2018: 12 · 2017: 7 · 2016: 15 · 2015: 20 · 2014: 22 · 2013: 30 · 2012: 25
- FWCI
- 13.06
- Type
- journal article
- Language
- en
- Open access
- green
- europepmc.org
- Locations
- Infection and Immunity (publishedVersion)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Health Sciences › Medicine › Infectious Diseases › Tuberculosis Research and Epidemiology
- Health Sciences › Medicine › Surgery › Infectious Diseases and Tuberculosis
- Health Sciences › Medicine › Epidemiology › Mycobacterium research and diagnosis
- Keywords
- ESAT-6, Mycobacterium tuberculosis, secreted antigens, T cell antigens, antigen purification, recombinant antigen, T cell memory, Mycobacterium tuberculosis complex, Mycobacterium kansasii, Mycobacterium szulgai, Mycobacterium marinum
- MeSH terms
- Amino Acid Sequence, Animals, Antigens, Bacterial, Bacterial Proteins, Base Sequence, Cell Compartmentation, Cell Fractionation, Cloning, Molecular, Genes, Bacterial, Mice, Inbred C57BL, Molecular Sequence Data, Molecular Weight, Mycobacterium tuberculosis, Recombinant Fusion Proteins, Species Specificity, T-Lymphocytes, Sequence Analysis, Mice
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