The gram positive bacillus Listeria monocytogenes causes bacteremia and meningitis, while the gram negative bacillus Shigella flexneri causes severe diarrhea and dehydration. The pathogenesis of Listeriosis and Shigellosis absolutely requires these intracellular bacteria to usurp the host cell's contractile system to form membrane projections, called filopodia. Filopodia push into adjacent cells where they are ingested, allowing efficient cell-to-cell to spread and avoidance of the humoral immune system, as well as many antibiotics. The actin regulatory proteins and pathways that mediate bacteria-induced filopodia formation are poorly understood. We will:
Aim I - Explore the relative contribution of known actin-regulatory proteins to Listeria- and Shigella-induced filopodia formation. Immuno-fluorescence and immuno-gold electron microscopy, expression of GFP fusion proteins, RNAi, and knockout cell lines will be combined with freeze fracture electron microscopy, fluorescence recovery after photo-bleaching (FRAP), and laser nano-scissors to assess the roles of the actin-regulatory proteins Eps8, VASP , Arp2/3 complex, fascin, and myosin-X in filopodia formation.
Aim II - Dissect the signal transduction pathways that initiate Listeria-induced filopodia formation. The PI3K inhibitor LY294002 blocks Listeria-induced filopodia formation, and delivery of PIP3 results in a >6 x increase in filopodia number without a significant change in Listeria intracellular motility. Using targeted RNAi knockdown, the specific PI3K isotype responsible for initiating filopodia formation will be determined. The role of the downstream affecters Akt, PKC-gamma, Tapp-1 and Plekstrin-2, GAPs, GEFs, and small G-proteins will be examined. Filopodia formation in response to changes in [Ca2+]i is being assessed by Fura-2 and BAPTAM, and we find a >2x increase in filopodia length following Ca2+ chelation. The effects of polyamine calcium channel agonist and antagonists on Listeria cell-to-cell spread will be explored. These investigations promise to clarify the key regulatory pathways required for Listeria- and Shigella-induced filopodia formation and cell-to-cell spread, critical steps for virulence and new therapeutic approaches. These discoveries can be applied to all forms of cell motility, including tumor metastasis, neurite outgrowth, and platelet spreading.

Public Health Relevance

Listeria is responsible for deadly food poisoning and Shigella can cause severe bloody diarrhea. We are examining the ways these aggressive bacteria take over our bodies by secretly moving within our cells and avoiding antibiotics. Understanding how these microbes take over our cells'muscle systems may lead to new treatments for these dangerous infections, as well as provide a new understanding of how cancer cells spread in our body, nerve cells grow, and platelets control bleeding.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI034276-15
Application #
8279441
Study Section
Host Interactions with Bacterial Pathogens Study Section (HIBP)
Program Officer
Mills, Melody
Project Start
1993-07-01
Project End
2015-05-31
Budget Start
2012-06-01
Budget End
2013-05-31
Support Year
15
Fiscal Year
2012
Total Cost
$320,605
Indirect Cost
$97,855
Name
University of Florida
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
969663814
City
Gainesville
State
FL
Country
United States
Zip Code
32611
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Bubb, Michael R; Yarmola, Elena G; Gibson, Bruce G et al. (2003) Depolymerization of actin filaments by profilin. Effects of profilin on capping protein function. J Biol Chem 278:24629-35
Zhang, Fangliang; Southwick, Frederick S; Purich, Daniel L (2002) Actin-based phagosome motility. Cell Motil Cytoskeleton 53:81-8

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