Listeria monocytogenes is facultative intracellular food-borne pathogen that provides an extremely amenable model for basic studies on host-pathogen interactions. A primary determinant of L. monocytogenes pathogenesis is Listeriolysin O (LLO), 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 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 contrast, the ActA protein is only expressed by bacteria when they reach the host cytosol. Despite the fact that LLO and ActA act in different compartments and at different times during the infection cycle, the transcription of both is entirely dependent on the same Crp family transcription factor PrfA. PrfA is predicted to bind an activating ligand, but the nature of the ligand is not known. During the prior funding period, we developed innovative tools and performed extensive genetic screens to identify L. monocytogenes determinants that regulate compartment-specific expression of LLO and ActA. The results of the screens implicate a critical role of redox stress as a mediator of compartmentalization. L. monocytogenes mutants lacking glutathione synthase (GshF) expressed very low levels of ActA during cytosolic growth and were greater than 1000-fold less virulent in mice. These data suggest that cytosolic L. monocytogenes are experiencing redox stress, but of unknown origin. However, the defect in ActA expression was rescued by a mutation that locks PrfA in its activated state, strongly suggesting that a primary role for glutathione is to mediate PrfA activation and we hypothesize that glutathionylation is the activating ligand. We also found that LLO is glutathionylated. We hypothesize that glutathionylation protects the LLO cysteine from irreversible oxidation in a phagosome prevents its activity in the cytosol. We propose to test the role of glutathione on LLO activity and compartmentalization, evaluate the role played by glutathione in PrfA activation and test the hypothesis that PrfA is a sensor of phagosomal and cytosolic redox stress.

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 and cell biology 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 #
2R01AI027655-26A1
Application #
8698120
Study Section
Host Interactions with Bacterial Pathogens Study Section (HIBP)
Program Officer
Mills, Melody
Project Start
1988-06-15
Project End
2019-02-28
Budget Start
2014-03-01
Budget End
2015-02-28
Support Year
26
Fiscal Year
2014
Total Cost
$382,674
Indirect Cost
$132,674
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
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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
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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

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