Mycobacterium tuberculosis remains one of the most devastating human infectious diseases, causing two million deaths annually and latently infecting a third of the world's population. As an intracellular pathogen adapted to long-term survival, M. tuberculosis has evolved mechanisms to manipulate host events that rely on dynamic membrane processes such as phagosome maturation, phagolysome fusion and autophagy. Other pathogenic bacteria achieve similar effects by secreting protein virulence factors (called "effector proteins" or "effectors") that associate with host membranes to facilitat their activities. To date, few such effectors have been identified in M. tuberculosis and expanding this knowledge base may provide additional avenues for the development of new drugs. Because eukaryotic cellular membranes are the major organizational centers of the cell, we hypothesize that membranes are targeted by mycobacterial effector proteins. In preliminary experiments, we tested putative secreted mycobacterial effectors for their ability to bind host membranes using a high-throughput screening assay in the model eukaryotic organism, Saccharomyces cerevisiae (yeast). Of the 40 genes screened to date, 5 (12.5%) interact with membranes, and we have demonstrated that in a human cell line several of the genes associate with the major protein synthesis machinery center of the cell, the endoplasmic reticulum.
The aims of this proposal are to fundamentally understand how M. tuberculosis is able to use membrane targeting to manipulate the host. We propose to first identify a complete complement of membrane binding proteins secreted by M. tuberculosis by screening a total of 400 genes using our high- throughput cloning and expression system. Characterization will further include cell biologic assays to determine where each protein associates within eukaryotic cells and demonstration of direct secretion of mycobacterial effectors into host cells during infection. We will select several hits for further in-depth characterization including in vitro assays of membran association and identification of host protein targets. Lastly, we will use the knowledge gained to test the hypothesis that regulation of membrane and organelle dynamics by M. tuberculosis effectors is essential for mycobacterial survival within human macrophages. The proposed work will extend the current knowledge on M. tuberculosis's ability to manipulate host membrane dynamics and reveal novel microbial survival strategies.

Public Health Relevance

Tuberculosis is a major human pathogen, accounting for significant morbidity and mortality worldwide. Work outlined in this proposal will identify novel mechanisms that allow M. tuberculosis to survive and persist within humans. We expect that this work will help identify new potential drug targets for the treatment of tuberculosis. PUBLIC HEALTH RELEVANCE: The mechanisms used by Mycobacterium tuberculosis to manipulate host membrane dynamics to facilitate survival are incompletely understood. We propose to identify novel M. tuberculosis secreted virulence factors (effectors) that associate with host membranes using a unique screening approach in the model eukaryotic organism, Saccharomyces cerevisiae, and to further characterize putative effectors through cell biologic and biochemical studies. This approach is novel because it utilizes host membrane binding to identify M. tuberculosis effector activity.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21AI111023-01
Application #
8682011
Study Section
Special Emphasis Panel (ZRG1-IDM-B (80))
Program Officer
Jacobs, Gail G
Project Start
2014-02-01
Project End
2016-01-31
Budget Start
2014-02-01
Budget End
2015-01-31
Support Year
1
Fiscal Year
2014
Total Cost
$175,808
Indirect Cost
$63,308
Name
University of Texas Sw Medical Center Dallas
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
800771545
City
Dallas
State
TX
Country
United States
Zip Code
75390