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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI027655-24
Application #
8081849
Study Section
Host Interactions with Bacterial Pathogens Study Section (HIBP)
Program Officer
Mills, Melody
Project Start
1988-06-15
Project End
2013-05-31
Budget Start
2011-06-01
Budget End
2012-05-31
Support Year
24
Fiscal Year
2011
Total Cost
$427,490
Indirect Cost
Name
University of California Berkeley
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
124726725
City
Berkeley
State
CA
Country
United States
Zip Code
94704
Whiteley, Aaron T; Ruhland, Brittany R; Edrozo, Mauna B et al. (2017) A Redox-Responsive Transcription Factor Is Critical for Pathogenesis and Aerobic Growth of Listeria monocytogenes. Infect Immun 85:
Portman, Jonathan L; Huang, Qiongying; Reniere, Michelle L et al. (2017) Activity of the Pore-Forming Virulence Factor Listeriolysin O Is Reversibly Inhibited by Naturally Occurring S-Glutathionylation. Infect Immun 85:
Mitchell, Gabriel; Isberg, Ralph R (2017) Innate Immunity to Intracellular Pathogens: Balancing Microbial Elimination and Inflammation. Cell Host Microbe 22:166-175
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
Portman, Jonathan L; Dubensky, Samuel B; Peterson, Bret N et al. (2017) Activation of the Listeria monocytogenes Virulence Program by a Reducing Environment. MBio 8:
Xiong, Xiaolu; Jiao, Jun; Gregory, Anthony E et al. (2016) Identification of Coxiella burnetii CD8+ T-Cell Epitopes and Delivery by Attenuated Listeria monocytogenes as a Vaccine Vector in a C57BL/6 Mouse Model. J Infect Dis :
Burke, Thomas P; Portnoy, Daniel A (2016) SpoVG Is a Conserved RNA-Binding Protein That Regulates Listeria monocytogenes Lysozyme Resistance, Virulence, and Swarming Motility. MBio 7:e00240
Reniere, Michelle L; Whiteley, Aaron T; Portnoy, Daniel A (2016) An In Vivo Selection Identifies Listeria monocytogenes Genes Required to Sense the Intracellular Environment and Activate Virulence Factor Expression. PLoS Pathog 12:e1005741
Mitchell, Gabriel; Chen, Chen; Portnoy, Daniel A (2016) Strategies Used by Bacteria to Grow in Macrophages. Microbiol Spectr 4:
Durack, Juliana; Burke, Thomas P; Portnoy, Daniel A (2015) A prl mutation in SecY suppresses secretion and virulence defects of Listeria monocytogenes secA2 mutants. J Bacteriol 197:932-42

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