Pseudomonas aeruginosa (PA) is one of the most virulent opportunistic pathogens of man. The morbidity of PA infections results from the ability of the bacterium to colonize previously injured or disrupted epithelial cell layers leading to further epithelial cell damage, inhibition of wound healing, and access to other tissues or the blood stream. Our initial work utilized a novel genetic screen to identify new virulence factors of PA required for epithelial cell injury. These studies identified new virulence factors (the type III secretion system and the secreted effector protein ExoU) and also suggested new functions in virulence for previously identified virulence factors (type IV pill). The role of these genes in pathogenesis was validated using assays testing for virulence in the tissue culture system and in a mouse model of acute pneumonia. We discovered that PA can damage epithelial cells and macrophages by at least two type Ill-secretion dependent pathways. The first involves ExoU-mediated necrosis and the second pathway has features of apoptosis. In this competitive renewal we will continue these studies with the long term goals of (i) understanding the complex interplay between the bacterial type III secretion system, its secreted effectors, and the host eukaryotic cell and (ii) elucidating the role of type IV fimbriae as virulence factors in acute infections caused by PA. Our short term goals will focus on (i) the pathways by which ExoT alters the host cell cytoskeleton, (ii) the mechanism of type III secretion-dependent apoptosis, (iii) and the role of type IV pill in type III secreetion.
Specific aim 1. We will test the hypothesis that multiple domains of ExoT contribute to its role in inhibiting bacterial internalization, inducing cytoskeletal changes and cell rounding, and inhibiting wound healing of eukaryotic cells.
Specific Aim 2. We will dissect the mechanism by which type III secretion induces apoptotic like death in host cells.
Specific Aim 3. We will explore the biological roles of the polarly located type IV pill in virulence. We will test the hypothesis that specific components of type IV pill are required for discrete steps in type Ill-mediated secretion and translocation.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
3R01AI042806-10S1
Application #
7493894
Study Section
Special Emphasis Panel (ZRG1-EVR (02))
Program Officer
Taylor, Christopher E,
Project Start
1998-06-01
Project End
2008-05-31
Budget Start
2007-06-01
Budget End
2008-05-31
Support Year
10
Fiscal Year
2007
Total Cost
$45,767
Indirect Cost
Name
University of California San Francisco
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
094878337
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Inclan, Yuki F; Persat, Alexandre; Greninger, Alexander et al. (2016) A scaffold protein connects type IV pili with the Chp chemosensory system to mediate activation of virulence signaling in Pseudomonas aeruginosa. Mol Microbiol 101:590-605
Bucior, Iwona; Tran, Cindy; Engel, Joanne (2014) Assessing Pseudomonas virulence using host cells. Methods Mol Biol 1149:741-55
Tran, Cindy S; Eran, Yoni; Ruch, Travis R et al. (2014) Host cell polarity proteins participate in innate immunity to Pseudomonas aeruginosa infection. Cell Host Microbe 15:636-43
Wood, Stephen; Sivaramakrishnan, Gayathri; Engel, Joanne et al. (2011) Cell migration regulates the kinetics of cytokinesis. Cell Cycle 10:648-54
Inclan, Yuki F; Huseby, Medora J; Engel, Joanne N (2011) FimL regulates cAMP synthesis in Pseudomonas aeruginosa. PLoS One 6:e15867
Bertrand, Jacob J; West, Joyce T; Engel, Joanne N (2010) Genetic analysis of the regulation of type IV pilus function by the Chp chemosensory system of Pseudomonas aeruginosa. J Bacteriol 192:994-1010
Endoh, Takayuki; Engel, Joanne N (2009) CbpA: a polarly localized novel cyclic AMP-binding protein in Pseudomonas aeruginosa. J Bacteriol 191:7193-205
Engel, Joanne; Balachandran, Priya (2009) Role of Pseudomonas aeruginosa type III effectors in disease. Curr Opin Microbiol 12:61-6
Shafikhani, Sasha H; Mostov, Keith; Engel, Joanne (2008) Focal adhesion components are essential for mammalian cell cytokinesis. Cell Cycle 7:2868-76
Pielage, Julia F; Powell, Kimberly R; Kalman, Daniel et al. (2008) RNAi screen reveals an Abl kinase-dependent host cell pathway involved in Pseudomonas aeruginosa internalization. PLoS Pathog 4:e1000031

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