Pseudomonas aeruginosa is among the most common causes of blinding corneal disease, while also being a major cause of life threating nosocomial infections such as pneumonia, bacteremia, urinary tract infections (UTIs), and cytstic fibrosis (CF), targeting immunocompromised and critically injured patients. Publications from the Fleiszig lab have shown that twitching motility, a type of surface associated movement, contributes to the ability of P. aeruginosa to penetrate human corneal epithelial cell multilayers in vitro and is critical to pathogenesis of P. aeruginosa corneal infection in a mouse model in vivo. Key to P. aeruginosa pathogenesis in the cornea is the capacity of the bacteria to invade corneal epithelial cells. While P. aeruginosa mutants that lack twitching motility can invade epithelial cells, and replicate inside them just as efficiently as wildtype bacteria, they have reduced capacity for exiting cells they have entered. During my postdoctoral fellowship in the Fleiszig lab, I used imaging and various other methods to study the mechanisms by which P. aeruginosa exits epithelial cells. Importantly, my preliminary data show that exit does not necessarily follow cell death, suggesting active/deliberate mechanisms contribute. My data further show that when twitching mutants invade and replicate in corneal epithelial cells, they differ from wildtype P. aeruginosa in being unable to distribute themselves in the cytoplasm and instead accumulate in aggregates. I have also screened a mutant library for exit capacity, and have found that mutants in either of two phospholipases, PlcB or PA2155, are exit defective. In contrast to twitching mutants, the phospholipase mutants spread normally throughout the host cell cytoplasm. Thus, my data mechanistically separate the exit process into two stages one dependent on twitching and the other dependent on phospholipases. My theoretical model for exit is that P. aeruginosa uses twitching motility to avoid forming a biofilm aggregate inside the cell and to access the host cell plasma membrane, where they use phospholipase activity (e.g. of PlcB and PA2155) to alter the plasma membrane to provide an exit route. Thus, in aim 1 I will the identify the genes transcripts that impact twitching mutant aggregation and exit compared to wildtype, and in aim 2 I will determine if phospholipases facilitate exit through their enzymatic activity. While contributing to our understanding of P. aeruginosa pathogenesis, this project could ultimately contribute to development of strategies for preventing and treating infections that act by preventing bacterial penetration through our protective surface epithelia.

Public Health Relevance

A key event in potentially blinding P. aeruginosa infections of the cornea is bacterial entry into corneal epithelial cells. Yet little is known about how the bacteria exit cells to further disseminate, except that Type IV mediated twitching motility, also required for trafficking through the corneal epithelium and causing disease in vivo, is a contributor. Here, I will follow up on my preliminary data showing two discrete steps in exit, one requiring twitching motility and the other involving two type 2 secreted phospholipases, to advance our understanding of the pathogenesis of infection and to potentially identify novel strategies for protecting our superficial epithelial against an important human pathogen.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
1F32EY029152-01
Application #
9541945
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Agarwal, Neeraj
Project Start
2018-05-01
Project End
2021-04-30
Budget Start
2018-05-01
Budget End
2019-04-30
Support Year
1
Fiscal Year
2018
Total Cost
Indirect Cost
Name
University of California Berkeley
Department
Type
Schools of Optometry/Opht Tech
DUNS #
124726725
City
Berkeley
State
CA
Country
United States
Zip Code
94704