Linking the behavior of individual host cells to their transcriptional signatures in tuberculosis Tuberculosis is an infectious disease of enormous proportions with no effective vaccine and the emergence of extensively drug resistant strains. Major holes in our understanding of its pathogenesis present roadblocks to new therapeutic and preventive interventions. My laboratory has developed zebrafish infection by Mycobacterium marinum as a powerful surrogate model for exploring tuberculosis pathogenesis. The transparency of zebrafish larvae allows real-time examination of cellular movements and behaviors in unprecedented detail. We have made surprising discoveries about central aspects of tuberculosis pathogenesis, in particular regarding the organized aggregate of immune cells called the granuloma. This hallmark structure was long thought to be a key host protective element. However, detailed kinetic monitoring of granuloma formation revealed that granulomas are converted by mycobacteria into tools for expansion and dissemination. Phagocytes are recruited to the nascent granuloma, where they phagocytose the contents of dying infected cells. A subset of newly infected cells leaves the granuloma to disseminate infection to new foci. Here I propose to systematically link the behaviors of immune cells to their expression profiles to gain insight into the molecular blueprint of the host processes that promote pathogenesis. I propose to use laser capture microdissection techniques combined with multiple fluorescent bacterial and host reporters to isolate cells engaged in distinct movements, i.e. migrating towards newly infecting bacteria, returning to tissues from peripheral infection sites, migrating to forming granulomas, and departing granulomas. The gene expression profiles of these cells will then be deciphered using single cell gene expression techniques. We will then perturb these molecular signatures with genetic techniques to determine their impact on infe

Agency
National Institute of Health (NIH)
Institute
National Institute of Mental Health (NIMH)
Type
NIH Director’s Pioneer Award (NDPA) (DP1)
Project #
5DP1MH099901-04
Application #
8542903
Study Section
Special Emphasis Panel (ZGM1-NDPA-B (01))
Program Officer
Beckel-Mitchener, Andrea C
Project Start
2010-09-30
Project End
2015-07-31
Budget Start
2013-08-01
Budget End
2014-07-31
Support Year
4
Fiscal Year
2013
Total Cost
$749,034
Indirect Cost
$268,884
Name
University of Washington
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
Cambier, C J; Takaki, Kevin K; Larson, Ryan P et al. (2014) Mycobacteria manipulate macrophage recruitment through coordinated use of membrane lipids. Nature 505:218-22
Ramakrishnan, Lalita (2013) Looking within the zebrafish to understand the tuberculous granuloma. Adv Exp Med Biol 783:251-66
Tobin, David M; Ramakrishnan, Lalita (2013) TB: the Yin and Yang of lipid mediators. Curr Opin Pharmacol 13:641-5
Tobin, David M; Roca, Francisco J; Oh, Sungwhan F et al. (2012) Host genotype-specific therapies can optimize the inflammatory response to mycobacterial infections. Cell 148:434-46