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. .
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
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