Listeria monocytogenes is a facultative intracellular bacterial pathogen that causes serious illness in pregnant women, neonates, and immunocompromised individuals. Bacterial infections are generally food- borne and listeriosis is the leading cause of death from contaminated food products. L. monocytogenes serves as an excellent model system for exploring the interactions that take place between an intracellular parasite and its host. The overall goal of this proposal is to define the mechanisms by which L. monocytogenes senses its different host cell compartmental environments and responds with the regulated expression of gene products required for virulence.
In Aim I, the temporal expression of virulence genes of individual bacteria within different host cell compartmental environments will be measured. Bacterial gene fusions to the bioluminescent green fluorescent protein (GFP) gene of Aequorea Victoria will be constructed and introduced into the L. monocytogenes chromosome. Macrophage cells infected with the L. monocytogenes gfp fusion mutants will be monitored for intracellular bacterial gene expression using fluorescent microscopy. These experiments will determine which bacterial gene products are expressed in specific host cell environments. In addition, L. monocytogenes mutants with altered patterns of intracellular gene expression will be isolated and characterized. It has been previously demonstrated that the expression of L. monocytogenes virulence determinants is regulated by a key transcriptional activator protein, PrfA. A model has been proposed that suggests that the relative concentrations of PrfA within the bacterial cell govern which virulence genes are expressed.
In Aim II, the relative affinity of PrfA for its target promoters will be determined using gel mobility shift and foot printing assays, as well as a Bacillus subtilis-based expression system which enables characterization of promoter mutants. These experiments will assess if the DNA binding affinity of PrfA for a promoter determines the temporal expression and the level of transcriptional activation of that gene.
In Aim III, additional L. monocytogenes gene products subject to PrfA regulation will be identified and characterized. These gene, products may represent additional virulence determinants that contribute to L. monocytogenes pathogenesis.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29AI041816-05
Application #
6213321
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Project Start
1997-02-01
Project End
2002-01-31
Budget Start
2000-02-01
Budget End
2001-01-31
Support Year
5
Fiscal Year
2000
Total Cost
$124,502
Indirect Cost
Name
Seattle Biomedical Research Institute
Department
Type
DUNS #
City
Seattle
State
WA
Country
United States
Zip Code
98109
Xayarath, Bobbi; Alonzo 3rd, Francis; Freitag, Nancy E (2015) Identification of a peptide-pheromone that enhances Listeria monocytogenes escape from host cell vacuoles. PLoS Pathog 11:e1004707
McMullen, P David; Freitag, Nancy E (2015) Assessing bacterial invasion of cardiac cells in culture and heart colonization in infected mice using Listeria monocytogenes. J Vis Exp :e52497
Sheridan, Brian S; Romagnoli, Pablo A; Pham, Quynh-Mai et al. (2013) ?? T cells exhibit multifunctional and protective memory in intestinal tissues. Immunity 39:184-95
Xayarath, Bobbi; Freitag, Nancy E (2012) Optimizing the balance between host and environmental survival skills: lessons learned from Listeria monocytogenes. Future Microbiol 7:839-52
McMullen, P David; Gillaspy, Allison F; Gipson, Jenny et al. (2012) Genome sequence of Listeria monocytogenes 07PF0776, a cardiotropic serovar 4b strain. J Bacteriol 194:3552
Xayarath, Bobbi; Smart, Jennifer I; Mueller, Kimberly J et al. (2011) A novel C-terminal mutation resulting in constitutive activation of the Listeria monocytogenes central virulence regulatory factor PrfA. Microbiology 157:3138-49
Xayarath, Bobbi; Volz, Karl W; Smart, Jennifer I et al. (2011) Probing the role of protein surface charge in the activation of PrfA, the central regulator of Listeria monocytogenes pathogenesis. PLoS One 6:e23502
Alonzo 3rd, Francis; Xayarath, Bobbi; Whisstock, James C et al. (2011) Functional analysis of the Listeria monocytogenes secretion chaperone PrsA2 and its multiple contributions to bacterial virulence. Mol Microbiol 80:1530-48
Alonzo 3rd, Francis; McMullen, P David; Freitag, Nancy E (2011) Actin polymerization drives septation of Listeria monocytogenes namA hydrolase mutants, demonstrating host correction of a bacterial defect. Infect Immun 79:1458-70
Alonzo 3rd, Francis; Bobo, Linda D; Skiest, Daniel J et al. (2011) Evidence for subpopulations of Listeria monocytogenes with enhanced invasion of cardiac cells. J Med Microbiol 60:423-34

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