Listeria monocytogenes is a facultative intracellular pathogen that provides an extremely amenable model for basic studies on host-pathogen interactions. Importantly, L. monocytogenes is also a clinically relevant food-borne pathogen that causes a high rate of mortality in pregnant women and the immunocompromised. A primary determinant of L. monocytogenes pathogenesis and a target of the host's immune response is Listeriolysin O (LLO). LLO is a member of a large family of pore-forming cytolysins that is largely responsible for mediating escape of L. monocytogenes from a phagosome and for virulence;LLO-minus mutants are 5-logs less virulent in animal models of infection. LLO activity is a double-edged sword as its activity must be restricted to an acidic phagosome or the host cell will die due to LLO-mediated cell death (referred to as compartmentalization).
In Aim I of this proposal the molecular determinants mediating compartmentalization will be determined by using a combination of mutagenesis, cell biology and biochemistry. LLO mutants will be characterized with respect to phosphorylation, ubiquitylation, proteolysis, half-life, and aggregation.
In Aim II, the role played by autophagy will be examined with respect to escape from a phagosome and the fate of LLO secreted into the host cytosol. Macrophages that are defective for autophagy will provide an excellent system with which to examine these questions.
In Aim III, a newly developed, mariner-based, transposon mutagenesis system will be used to identify the role played by gene-products, other than LLO, on LLO expression, synthesis, secretion and toxicity. These studies should identify the hypothetical host protein(s) that interact with LLO and LLO mRNA to prevent toxicity, and open up new areas of investigations pertaining to LLO expression, synthesis, secretion and toxicity. Lastly, the role of post-translational modifications and autophagy will be examined with respect to the presentation of LLO epitopes in both the MHC Class I and Class II pathways of antigen processing and presentation. Novel in vivo assays will be used to select LLO mutants that are not recognized by the host's acquired immunity, thereby providing basic information on properties of foreign proteins that leads to immunogenicity.

Public Health Relevance

Diseases caused by intracellular pathogens, for example, tuberculosis, AIDS and Malaria, remain one of the largest challenges facing the international biomedical community. The proposed studies on Listeria monocytogenes will provide insight into the molecular biology, cell biology and immunology relevant to the treatment and prevention of diseases caused by intracellular pathogens.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
Project #
Application #
Study Section
Host Interactions with Bacterial Pathogens Study Section (HIBP)
Program Officer
Mills, Melody
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of California Berkeley
Schools of Arts and Sciences
United States
Zip Code
B├ęcavin, 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
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
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
Witte, Chelsea E; Whiteley, Aaron T; Burke, Thomas P et al. (2013) Cyclic di-AMP is critical for Listeria monocytogenes growth, cell wall homeostasis, and establishment of infection. MBio 4:e00282-13
Witte, Chelsea E; Archer, Kristina A; Rae, Chris S et al. (2012) Innate immune pathways triggered by Listeria monocytogenes and their role in the induction of cell-mediated immunity. Adv Immunol 113:135-56
Melton-Witt, Jody A; McKay, Susannah L; Portnoy, Daniel A (2012) Development of a single-gene, signature-tag-based approach in combination with alanine mutagenesis to identify listeriolysin O residues critical for the in vivo survival of Listeria monocytogenes. Infect Immun 80:2221-30
Portnoy, Daniel A (2012) Yogi Berra, Forrest Gump, and the discovery of Listeria actin comet tails. Mol Biol Cell 23:1141-5
Sauer, John-Demian; Witte, Chelsea E; Zemansky, Jason et al. (2010) Listeria monocytogenes triggers AIM2-mediated pyroptosis upon infrequent bacteriolysis in the macrophage cytosol. Cell Host Microbe 7:412-9
Vance, Russell E; Isberg, Ralph R; Portnoy, Daniel A (2009) Patterns of pathogenesis: discrimination of pathogenic and nonpathogenic microbes by the innate immune system. Cell Host Microbe 6:10-21
Auerbuch, Victoria; Brockstedt, Dirk G; Meyer-Morse, Nicole et al. (2004) Mice lacking the type I interferon receptor are resistant to Listeria monocytogenes. J Exp Med 200:527-33

Showing the most recent 10 out of 14 publications