This proposal is Project 1 within a P01 renewal, entitled Intracellular pathogens and innate immunity. Listeria monocytogenes is a highly amenable model intracellular pathogen that induces robust and long-lived cell mediated immunity. The goal of the previous proposal was to identify L. monocytogenes determinants that activate a host cytosolic surveillance pathway (CSP) of innate immunity that leads to the expression of IFN? and co-regulated genes. We hypothesize that this pathway contributes significantly to the potent and long-lived cell-mediated immunity induced by L. monocytogenes. Using a genetic screen and subsequent biochemistry, we identified c-di-AMP as the listerial ligand that is secreted through a multidrug efflux pump and activates the CSP. Thus, c-di-AMP is a newly discovered PAMP. C-di-AMP is also a novel and conserved small bacterial signaling molecule that appears essential for bacterial growth. We have identified the di-adenylate cyclase (DacA) and c-di-AMP phosphodiesterase (Pde).
In Aim 1, we propose to further characterize the molecular determinants that lead to expression of c-di-AMP and generate a panel of well-characterized strains that express either enhanced or diminished levels of c-di-AMP. We will also address the following questions: What are the bacterial components that regulate c-di-AMP production? Does c-di- AMP act from the inside and/or the outside of bacteria? What is the physiologic role of c-di-AMP? In Aim 2, we will use the strains constructed above to determine the role of c-di-AMP during infection of macrophages and dendritic cells in vitro. We will also evaluate the effects of purified c-di-AMP on dendritic cell activation. In collaboration with the Vance Lab (Project 3), we will use ENU mutagenesis to identify novel host factors that control the CSP. In preliminary data, we identified Sting (Stimulator of interferon genes) as a critical host component necessary for response to L. monocytogenes, M. tuberculosis, and c-di-AMP. In the final Aim, we will test the hypothesis that c-di-AMP production is essential for the full expression of L. monocytogenes pathogenesis, and importantly, contributes to the induction of cell-mediated immunity. We will examine the role of c-di-AMP as an adjuvant.

Public Health Relevance

We recently discovered that the intracellular bacterial pathogen, Listeria monocytogenes, secretes a novel small di-cyclic molecule that is essential for bacterial growth and stimulates a robust host response leading to the production of interferon. These studies may impact the development of vaccines and therapeutics that protect against infectious disease agents.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Program Projects (P01)
Project #
5P01AI063302-12
Application #
8890064
Study Section
Special Emphasis Panel (ZAI1)
Project Start
Project End
Budget Start
2015-07-01
Budget End
2016-06-30
Support Year
12
Fiscal Year
2015
Total Cost
Indirect Cost
Name
University of California Berkeley
Department
Type
DUNS #
124726725
City
Berkeley
State
CA
Country
United States
Zip Code
94704
Cheng, Mandy I; Chen, Chen; Engström, Patrik et al. (2018) Actin-based motility allows Listeria monocytogenes to avoid autophagy in the macrophage cytosol. Cell Microbiol 20:e12854
Mitchell, Gabriel; Cheng, Mandy I; Chen, Chen et al. (2018) Listeria monocytogenes triggers noncanonical autophagy upon phagocytosis, but avoids subsequent growth-restricting xenophagy. Proc Natl Acad Sci U S A 115:E210-E217
Penn, Bennett H; Netter, Zoe; Johnson, Jeffrey R et al. (2018) An Mtb-Human Protein-Protein Interaction Map Identifies a Switch between Host Antiviral and Antibacterial Responses. Mol Cell 71:637-648.e5
Chen, Chen; Nguyen, Brittney N; Mitchell, Gabriel et al. (2018) The Listeriolysin O PEST-like Sequence Co-opts AP-2-Mediated Endocytosis to Prevent Plasma Membrane Damage during Listeria Infection. Cell Host Microbe 23:786-795.e5
Nguyen, Brittney N; Peterson, Bret N; Portnoy, Daniel A (2018) Listeriolysin O: a phagosome-specific cytolysin revisited. Cell Microbiol :e12988
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Deng, Weiwen; Lira, Victor; Hudson, Thomas E et al. (2018) Recombinant Listeria promotes tumor rejection by CD8+ T cell-dependent remodeling of the tumor microenvironment. Proc Natl Acad Sci U S A 115:8179-8184
Price, April E; Shamardani, Kiarash; Lugo, Kyler A et al. (2018) A Map of Toll-like Receptor Expression in the Intestinal Epithelium Reveals Distinct Spatial, Cell Type-Specific, and Temporal Patterns. Immunity 49:560-575.e6
Mitchell, Gabriel; Isberg, Ralph R (2017) Innate Immunity to Intracellular Pathogens: Balancing Microbial Elimination and Inflammation. Cell Host Microbe 22:166-175

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