Listeria monocytogenes (L.m.) is an intracellular bacterial pathogen that causes serious food-borne illness in pregnant women, the elderly and immunocompromised individuals. Listeriolysin O (LLO), a pore-forming cytolysin, and two bacterial phospholipases, PI-PLC and PC-PLC, are essential determinants of pathogenesis that mediate lysis of host cell vacuoles resulting from bacterial entry and intracellular spread. LLO is also expressed during intracytosolic growth and mediates numerous alterations in host cell physiology. During in vitro infection of cell lines, LLO is sufficient to facilitate lysis of all vacuolar membranes. Yet, PI-PLC and PC-PLC increase the efficiency of membrane lysis. It is hypothesized that these determinants play a specific role in the dissolution of each vacuolar membrane and the intracytosolic production of LLO is necessary for optimal intracellular growth. The focus of this proposal is to precisely define the temporal requirement of LLO for intracellular growth and cell-to-cell spread in primary host cells and for the maintenance of in vivo infection in a mouse infection model.
In Aim I, the precise requirement for LLO expression during intracellular growth and spread in primary host cells will be determined. This will be accomplished by using a novel genetic approach to allow regulated production of LLO during intracellular infection. Intracellular LLO levels will be varied during infection of primary host cells and bacterial replication and spread determined by microscopic analysis and enumeration of intracellular bacteria.
In Aim II, the precise roles of LLO, PI-PLC and PC-PLC in dissolution of vacuolar membranes during intracellular spread will be identified. L.m. strains allowing regulated expression of LLO in PI-PLC and PC-PLC mutants will be used in mixed host cell infections. Plaque formation in cell monolayers, differential time-lapse fluorescence microscopy and high-resolution electron microscopy will be used to evaluate progression of infection and dissolution of vacuolar membranes.
In Aim III, we will evaluate the requirement of LLO expression for maintenance of in vivo infection and the development of acquired immunity. BALB/c mice will be infected under varying times of in vivo LLO induction. Progression of infection will be evaluated by enumerating bacteria from organs and comparing to infection of wild-type and defined L.m. mutants. Immunological assessment will be determined by ELISPOT analysis and protection from wild-type bacterial challenge. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
1R01AI053669-01A1
Application #
6683675
Study Section
Special Emphasis Panel (ZRG1-BM-1 (05))
Program Officer
Hall, Robert H
Project Start
2003-07-01
Project End
2007-12-31
Budget Start
2003-07-01
Budget End
2003-12-31
Support Year
1
Fiscal Year
2003
Total Cost
$171,360
Indirect Cost
Name
Harvard University
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
02115
Grubaugh, Daniel; Regeimbal, James M; Ghosh, Pallab et al. (2018) The VirAB ABC Transporter Is Required for VirR Regulation of Listeria monocytogenes Virulence and Resistance to Nisin. Infect Immun 86:
Cemma, Marija; Lam, Grace Y; Stöckli, Martina et al. (2015) Strain-Specific Interactions of Listeria monocytogenes with the Autophagy System in Host Cells. PLoS One 10:e0125856
Fattouh, Ramzi; Kwon, Hyunwoo; Czuczman, Mark A et al. (2015) The diaphanous-related formins promote protrusion formation and cell-to-cell spread of Listeria monocytogenes. J Infect Dis 211:1185-95
Alonso, Almaris N; Perry, Kyle J; Regeimbal, James M et al. (2014) Identification of Listeria monocytogenes determinants required for biofilm formation. PLoS One 9:e113696
Czuczman, Mark A; Fattouh, Ramzi; van Rijn, Jorik M et al. (2014) Listeria monocytogenes exploits efferocytosis to promote cell-to-cell spread. Nature 509:230-4
Perry, Kyle J; Higgins, Darren E (2013) A differential fluorescence-based genetic screen identifies Listeria monocytogenes determinants required for intracellular replication. J Bacteriol 195:3331-40
Lam, Grace Y; Cemma, Marija; Muise, Aleixo M et al. (2013) Host and bacterial factors that regulate LC3 recruitment to Listeria monocytogenes during the early stages of macrophage infection. Autophagy 9:985-95
Lam, Grace Y; Fattouh, Ramzi; Muise, Aleixo M et al. (2011) Listeriolysin O suppresses phospholipase C-mediated activation of the microbicidal NADPH oxidase to promote Listeria monocytogenes infection. Cell Host Microbe 10:627-34
Kamp, Heather D; Higgins, Darren E (2011) A protein thermometer controls temperature-dependent transcription of flagellar motility genes in Listeria monocytogenes. PLoS Pathog 7:e1002153
Lieberman, Linda A; Higgins, Darren E (2010) Inhibition of Listeria monocytogenes infection by neurological drugs. Int J Antimicrob Agents 35:292-6

Showing the most recent 10 out of 26 publications