This proposal is Project 2 within a P01 renewal, entitled "Intracellular pathogens and innate immunity". M. tuberculosis is an important human pathogen that causes severe morbidity and mortality around the world. The goal of the previous proposal was to identify the mechanisms by which M. tuberculosis triggers and manipulates host responses of its primary host cell, the macrophage. We made the striking discovery that M. tuberculosis, a phagosomal pathogen, activates the host cytosolic surveillance pathway (CSP), an innate signaling pathway that senses bacterial molecules in the cytoplasm. We are interested in host and bacterial factors required for CSP activation, and in elucidating the functional role of the pathway in M. tuberculosis pathogenesis. We have shown that the ESX-1 secretion pathway and the cell wall lipid PDIM are critical for perturbing phagosomal membranes of macrophages, allowing activation of cytosolic DNA receptors. Unexpectedly, cytosolic access also targets bacteria to the autophagy pathway. We hypothesize that intracellular pathogens perturb intracellular membranes to promote virulence, but activation of the CSP allows host cells to discriminate and mount qualitatively different immune responses to pathogens versus non-pathogens.
In Aim 1, we propose to elucidate the mechanism by which M. tuberculosis gains access to the cytosol and activates the CSP, building upon our evidence that a single ESX-1 substrate, ESAT-6, functions to permeabilize the phagosomal membrane. We propose to identify the nature of the DNA that is recognized by the host, test the role of putative host receptors responsible for CSP activation, and probe the role of cytosolic signaling in M. tuberculosis infection.
In Aim 2, we will examine how cytosolic access leads to targeting of M. tuberculosis to the autophagy pathway. In particular, we will test the role of ubiquitination and ubiquitin-binding adapters in targeting of autophagic vesicles to M. tuberculosis during infection. In the final Aim, We will collaborate extensively with the Portnoy (Project 1) and Vance (Project 3) groups to screen for modulators of M. tuberculosis growth and host innate responses by carrying out a screen in macrophages isolated from ENU mutagenized mice, and by performing an RNAi screen in macrophages.
We recently discovered that M. tuberculosis utilizes its specialized secretion system to stimulate a robust host response leading to the production of interferon. The interferon response signature has recently been identified as a strong correlate of active disease in humans, thus these studies may impact the development of diagnostics and therapeutics that protect against tuberculosis.
|Kranzusch, Philip J; Lee, Amy S Y; Wilson, Stephen C et al. (2014) Structure-guided reprogramming of human cGAS dinucleotide linkage specificity. Cell 158:1011-21|
|Burke, Thomas P; Loukitcheva, Anastasia; Zemansky, Jason et al. (2014) Listeria monocytogenes is resistant to lysozyme through the regulation, not the acquisition, of cell wall-modifying enzymes. J Bacteriol 196:3756-67|
|Bécavin, Christophe; Bouchier, Christiane; Lechat, Pierre et al. (2014) Comparison of widely used Listeria monocytogenes strains EGD, 10403S, and EGD-e highlights genomic variations underlying differences in pathogenicity. MBio 5:e00969-14|
|Tenthorey, Jeannette L; Kofoed, Eric M; Daugherty, Matthew D et al. (2014) Molecular basis for specific recognition of bacterial ligands by NAIP/NLRC4 inflammasomes. Mol Cell 54:17-29|
|Lee, Bettina L; Barton, Gregory M (2014) Trafficking of endosomal Toll-like receptors. Trends Cell Biol 24:360-9|
|Sivick, Kelsey E; Arpaia, Nicholas; Reiner, Gabrielle L et al. (2014) Toll-like receptor-deficient mice reveal how innate immune signaling influences Salmonella virulence strategies. Cell Host Microbe 15:203-13|
|Archer, Kristina A; Durack, Juliana; Portnoy, Daniel A (2014) STING-dependent type I IFN production inhibits cell-mediated immunity to Listeria monocytogenes. PLoS Pathog 10:e1003861|
|Arpaia, Nicholas; Barton, Gregory M (2013) The impact of Toll-like receptors on bacterial virulence strategies. Curr Opin Microbiol 16:17-22|
|Isaac, Dervla T; Coady, Alison; Van Prooyen, Nancy et al. (2013) The 3-hydroxy-methylglutaryl coenzyme A lyase HCL1 is required for macrophage colonization by human fungal pathogen Histoplasma capsulatum. Infect Immun 81:411-20|
|Aachoui, Youssef; Leaf, Irina A; Hagar, Jon A et al. (2013) Caspase-11 protects against bacteria that escape the vacuole. Science 339:975-8|
Showing the most recent 10 out of 71 publications