The epithelium covering the cornea usually resists traversal by Pseudomonas aeruginosa and almost all other bacteria. Yet, soft contact lens wear predisposes the cornea to sight threating-infections with this pathogen. Our published data show that while healthy rat corneas consistently succumb to infection if fitted with P. aeruginosa-contaminated soft contact lenses, there is a delay in disease onset (median ~8 days). Transferring lenses from infected to na?ve rat eyes, reduces the delay from ~8 to ~2 days, while superficially injuring the cornea before lens fitting has no impact on disease timing or its severity. These data suggest that the disease onset delay in rats occurs because bacteria need time to adapt to the ocular surface environment to become virulent, consistent with the increased risk of infection in people who wear contact lenses for extended time periods. Human (in vitro) data also support a role for bacterial adaptation, as there is a significant delay befor P. aeruginosa traverses cultured human corneal epithelium. The hypothesis is that exposure to host antimicrobials in tear fluid or corneal epithelial cells triggers expression of P. aeruginosa Type 3 Secretion System (T3SS) effectors, proteases and resistance genes that then mediate bacterial traversal of the corneal epithelium. Preliminary data supporting this hypothesis include: 1) Exposure to in vivo factors, not simply biofilm growth on a lens, enables bacterial adaptations promoting epithelial traversal in vivo. 2) After traversing multilayers of human corneal epithelial cells, P. aeruginosa demonstrates an enhanced (~100-1000-fold) capacity to traverse cells, correlating with an increased capacity to survive inside cells and compromise transepithelial resistance (TER). 3) Epithelial traversal alters P. aeruginosa gene expression. 4) A subset of the impacted genes modulate traversal, shown using bacterial mutants. 5) At least one of these genes is a novel regulator of the bacterial T3SS. 6) The T3SS mediates traversal in vitro (human cells) and in vivo (mouse). 7) Expression of the P. aeruginosa T3SS is upregulated on exposure to human tear fluid or human corneal epithelial cell lysates. 8) T3SS-independent factors can also mediate traversal given longer exposure, or if host innate defenses are compromised (e.g. MyD88 knockout).
Aim 1 will determine bacterial adaptations during host exposure that mediate traversal. We will use RNA-seq to study how the bacterial transcriptome changes with exposure to ocular surface factors, and Tn-seq to narrow down which of these changes modulate epithelial traversal.
Aim 2 will examine the mechanisms for their involvement in a human in vitro traversal assay and in the mouse cornea in situ using bacterial mutants.
Aim 3 will explore host triggers enabling the key bacterial adaptations, using qRT-PCR to monitor the impact of exposure to endogenous host antimicrobials. Since epithelial traversal is an early step in the pathogenesis of infection, studying bacterial adaptations and host triggers that enable bacteria to do it could lead to novel strategies for preventing, not simply treating, infection.

Public Health Relevance

Pseudomonas aeruginosa is a leading cause of sight-threatening corneal disease, most often associated with contact lens wear. Consistent with extended wear being a risk factor, preliminary data show that bacterial adaptation to the ocular surface is key to pathogenesis. Here, we will study the nature of these adaptations, the mechanisms for their contribution, and the host factors triggering them, with a view towards developing means to prevent infection.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
1R01EY024060-01A1
Application #
8774092
Study Section
(DPVS)
Program Officer
Mckie, George Ann
Project Start
2014-08-01
Project End
2019-07-31
Budget Start
2014-08-01
Budget End
2015-07-31
Support Year
1
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of California Berkeley
Department
Type
Schools of Optometry/Opht Tech
DUNS #
City
Berkeley
State
CA
Country
United States
Zip Code
94704