Pseudomonas aeruginosa can cause severe sight- and life-threatening disease. Epithelial lined surface tissues such as the eye, the skin and the airways are the most commonly targeted sites. Susceptible populations include children with cystic fibrosis, immunocompromised individuals, burn victims, intubated patients, and contact lens wearers. The incidence of P. aeruginosa infection is rising;worrisome given that it is often highly destructive and associated with a poor prognosis. P. aeruginosa infection is notoriously difficult to treat using available therapies, in part because P. aeruginosa possesses a large number of genes devoted to survival and adaptation. Thus, new approaches to therapy are urgently needed. While it is known that P. aeruginosa can enter epithelial cells during infection, and that cell invasion can be a key component in pathogenesis, little is known about the intracellular lifestyle of P. aeruginosa within any cell type. The objective of this research is to understand intracellular survival strategies used by P. aeruginosa and to determine if they can be targeted to reduce virulence in vivo in an eye infection model. Preliminary data reveal that P. aeruginosa occupies a novel intracellular niche within epithelial cells;infection-induced plasma membrane blebs. In these blebs, bacteria replicate and demonstrate rapid (real-time visible) motility. The data show that the Type Three Secretion System (T3SS) is required for bleb-niche formation by P. aeruginosa. T3SS mutants fail to form blebs and instead localize to perinuclear vacuoles. In contrast to wild type bacteria, T3SS mutants (retain competency for invasion) lose viability after entering epithelial cells. The T3SS effectors and the translocon required for transporting T3SS effectors across host cell membranes both play roles in P. aeruginosa intracellular survival/trafficking. Effector mutants and translocon mutants each lack blebbing capacity and traffic to preinuclear vacuoles, however, only effector mutants lose capacity for intracellular replication. Thus, the data suggest at least two roles for the T3SS in intracellular survival;1) effector-dependent intracellular replication in perinuclear vacuoles, and 2) translocon-dependent bleb niche formation. The hypotheses to be tested are:
Aim 1 : That in the absence of T3SS effectors, P. aeruginosa is degraded within lysosomes, but specific T3SS effectors manipulate endocytic trafficking to enable survival in perinculear vacuoles.
Aim 2 : That the T3SS participates in bleb-niche formation by enabling bacterial escape from vacuolar compartments (translocon-dependent) and that there are also direct roles for the T3SS in bleb formation/trafficking to them.
Aim 3 : That intracellular survival in vivo is also T3SS-dependent, and can be targeted to manipulate virulence.
Aims 1 and 2 will involve in vitro cell culture infection methods, bacterial mutants, viability assays and imaging used with and without inhibitors/activators of molecular events.
Aim 3 will be done using a well-established in vivo corneal infection model, methods from aims 1 and 2, and quantification of bacterial colonization and of disease severity.

Public Health Relevance

Infections caused by Pseudomonas aeruginosa are often associated with a poor prognosis because this pathogen is inherently resistant to killing, can be highly destructive, and susceptible populations include people already debilitated by existing conditions such as cystic fibrosis, immunocompromise, burns or other injury. P. aeruginosa can become intracellular during infection, and this has been shown to contribute to pathogenesis in vivo. While cellular entry mechanisms have been studied, almost nothing is known about mechanisms used by P. aeruginosa for survival within cells after invasion. The focus of the research plan is to study strategies used by P. aeruginosa for surviving intracellularly and then to determine their potential as targets for new therapies.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI079192-05
Application #
8391254
Study Section
Special Emphasis Panel (ZRG1-BDCN-F (02))
Program Officer
Taylor, Christopher E,
Project Start
2008-12-05
Project End
2014-11-30
Budget Start
2012-12-01
Budget End
2014-11-30
Support Year
5
Fiscal Year
2013
Total Cost
$353,547
Indirect Cost
$123,223
Name
University of California Berkeley
Department
None
Type
Schools of Optometry/Ophthalmol
DUNS #
124726725
City
Berkeley
State
CA
Country
United States
Zip Code
94704
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