Listeria monocytogenes is a gram-positive, opportunistic, intracellular bacterial pathogen that causes food borne illness. Given its well-characterized infection cycle and genetic amenability, L. monocytogenes provides a powerful tool to interrogate the fundamental aspects of intracellular bacterial pathogenesis and the host immune response to invasion by intracellular pathogens. L. monocytogenes that enter into the host cell cytosol secrete the second messenger signaling nucleotide, c-di-AMP, resulting in innate immune activation by the host cell. We recently identified the host protein RECON as a key mediator of this response. RECON is an enzyme whose activity is inhibited by c-di-AMP. C-di-AMP inhibition of RECON results in the accumulation of the ROS byproduct 4-hydroxy-2-nonenal (4-HNE), which augments NF-kB activation, resulting in elevated nitric oxide synthase expression and NO production during infection. Intriguingly, we have revealed that NO produced by the host cell promotes L. monocytogenes intracellular motility, rather than restricting bacterial growth, and that L. monocytogenes exhibits extreme NO resistance. Additionally, preliminary studies have revealed that 4-HNE is ubiquitously induced by eukaryotic cells following exposure to bacteria and that this reactive aldehyde exhibits antimicrobial effects on bacteria analogous to NO. Furthermore, L. monocytogenes exhibits extreme resistance to the antimicrobial effects of 4-HNE and induces a specific transcriptional response to 4-HNE exposure, which we hypothesize promotes survival within the infected host. We have begun in vitro biochemical studies, in vivo forward genetic studies, and the murine models of infection to interrogate the mechanisms by which L. monocytogenes counteracts the antimicrobial effects of NO and 4-HNE and utilizes NO to promote virulence.
In Aim I, we will interrogate the mechanisms used by L. monocytogenes to detoxify and counteract the antimicrobial effects of 4-HNE.
In Aim II, we propose to detail the molecular mechanisms of NO resistance and the impacts on bacterial virulence. Finally, in Aim III we will detail the mechanism of NO induced L. monocytogenes actin- based motility and explore the in vivo impacts of this process on bacterial invasion and dissemination. Together these studies will define the molecular mechanisms of exquisite antimicrobial innate immune responses employed by L. monocytogenes to promote survival within the eukaryotic host. .

Public Health Relevance

Intracellular pathogens remain on of the largest human health burdens in the 21st century. By characterizing the mechanism of host-pathogen interactions using the model intracellular bacterium L. monocytogenes we will define the fundamental mechanisms employed by intracellular pathogens to promote disease and the host response to these infections

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI116669-07
Application #
10084250
Study Section
Bacterial Pathogenesis Study Section (BACP)
Program Officer
Baqar, Shahida
Project Start
2015-02-01
Project End
2025-01-31
Budget Start
2021-02-01
Budget End
2022-01-31
Support Year
7
Fiscal Year
2021
Total Cost
Indirect Cost
Name
University of Washington
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
Pham, Huong Thi; Nhiep, Nguyen Thi Hanh; Vu, Thu Ngoc Minh et al. (2018) Enhanced uptake of potassium or glycine betaine or export of cyclic-di-AMP restores osmoresistance in a high cyclic-di-AMP Lactococcus lactis mutant. PLoS Genet 14:e1007574
McFarland, Adelle P; Luo, Shukun; Ahmed-Qadri, Fariha et al. (2017) Sensing of Bacterial Cyclic Dinucleotides by the Oxidoreductase RECON Promotes NF-?B Activation and Shapes a Proinflammatory Antibacterial State. Immunity 46:433-445
Whiteley, Aaron T; Garelis, Nicholas E; Peterson, Bret N et al. (2017) c-di-AMP modulates Listeria monocytogenes central metabolism to regulate growth, antibiotic resistance and osmoregulation. Mol Microbiol 104:212-233
Choi, Philip H; Vu, Thu Minh Ngoc; Pham, Huong Thi et al. (2017) Structural and functional studies of pyruvate carboxylase regulation by cyclic di-AMP in lactic acid bacteria. Proc Natl Acad Sci U S A 114:E7226-E7235
Lang, Kevin S; Hall, Ashley N; Merrikh, Christopher N et al. (2017) Replication-Transcription Conflicts Generate R-Loops that Orchestrate Bacterial Stress Survival and Pathogenesis. Cell 170:787-799.e18
Huynh, TuAnh Ngoc; Choi, Philip H; Sureka, Kamakshi et al. (2016) Cyclic di-AMP targets the cystathionine beta-synthase domain of the osmolyte transporter OpuC. Mol Microbiol 102:233-243
Huynh, TuAnh Ngoc; Woodward, Joshua J (2016) Too much of a good thing: regulated depletion of c-di-AMP in the bacterial cytoplasm. Curr Opin Microbiol 30:22-29
Choi, Philip H; Sureka, Kamakshi; Woodward, Joshua J et al. (2015) Molecular basis for the recognition of cyclic-di-AMP by PstA, a PII-like signal transduction protein. Microbiologyopen 4:361-74
Huynh, TuAnh Ngoc; Luo, Shukun; Pensinger, Daniel et al. (2015) An HD-domain phosphodiesterase mediates cooperative hydrolysis of c-di-AMP to affect bacterial growth and virulence. Proc Natl Acad Sci U S A 112:E747-56