Tuberculosis remains a major global health problem and vaccine development is a critical priority. T-cells play a pivotal role in protection against Mycobacterium tuberculosis. However, M. tuberculosis is known to inhibit human immunity, but the mechanisms are not well understood. Early secreted antigenic target of 6 kD (ESAT-6) is an immunogenic protein that is a potential vaccine candidate, but also contributes to virulence and cytolysis of mammalian cells. Our preliminary data indicate that ESAT-6 directly inhibits human T cell proliferation and IFN-g secretion, and these effects are not due to cytotoxicity or increased apoptosis. Our goal is to understand the molecular mechanisms by which ESAT-6 inhibits human T cell IFN-g production.
The specific aims are: 1. Identify the signaling pathways by which ESAT-6 inhibits T cell IFN-g production. We will determine the effect of ESAT-6 on transcription factors that bind to the IFN-g proximal promoter, using promoter pull-down assays and chromatin immunoprecipitation (aim 1.1). We will evaluate the effect of ESAT-6 on molecules that mediate signaling through the T-cell receptor (aim 1.2) and on the intermediary signaling molecules, mitogen-activated protein kinases (aim 1.3), using immunoprecipitation, Western blotting and functional kinase assays 2. Identify a mammalian ligand for ESAT-6 on human T-cells. We will use biotinylated ESAT-6 to pull down proteins from extracts of purified T-cells, elute the proteins, separate them by SDS-PAGE and identify them by mass spectrometry (aim 2.1). The putative ligand will be cloned and expressed, and interactions between ESAT-6 and its ligand will be confirmed by surface plasmon resonance and protein overlay assays (aim 2.2). To determine the functional significance of the interaction between ESAT-6 and its putative ligand, we will determine if neutralizing antibodies and/or siRNA to the ESAT-6 ligand abrogate the inhibitory effects of ESAT-6 on T-cells (aim 2.3).

Public Health Relevance

Tuberculosis remains a tremendous public health problem world-wide, and an effective vaccine would contribute greatly to disease control. Early secreted antigenic target of 6 kD (ESAT-6) is a Mycobacterium tuberculosis protein that is a promising vaccine candidate, but also contributes to virulence and tissue damage. This proposal seeks to understand the mechanisms by which ESAT-6 inhibits human T-cell immune function, information that is critical for development of improved vaccines and treatment strategies for tuberculosis.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Exploratory/Developmental Grants (R21)
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Immunity and Host Defense Study Section (IHD)
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Parker, Tina M
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University of Texas Health Center at Tyler
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United States
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