Corneal epithelial barrier function against microbes is generally thought to involve surface mucins, junctional barriers, and extracellular antimicrobials. Additionally, we have found that corneal epithelial cells can behave as non-professional phagocytes, i.e. they can internalize bacteria and then suppress their viability within acidified lysosomes. The importance of this potential innate defense against microbes remains to be determined and is a goal of this project. The other goal is to determine how bacteria evade this defense. Pseudomonas aeruginosa is among the most common and the most devastating of corneal pathogens. The statistics are even worse for other epithelial lined body sites, where P. aeruginosa infection very often results in mortality. While often referred to as an extracellular pathogen, we have shown P. aeruginosa can thrive in corneal (and airway) epithelial cells, by evading acidified lysosomes and establishing replicative niches inside plasma membrane blebs. While lysosome evasion and bleb-niche formation both require the bacterial type III secretion system (T3SS), these capacities are separable. Lysosome evasion depends on the ADP-ribosylation (ADPr) activity of the T3SS effector ExoS, but not PopB, one of two T3SS translocators. In stark contrast, both ExoS and PopB are needed for P. aeruginosa to establish bleb-niches. The three aims of this project are:
Aim 1. To study mechanisms by which corneal epithelial cells kill intracellular bacteria; specifically exploring the role of MyD88, which we hae found is involved.
Aim 2 : To determine how P. aeruginosa uses ExoS to evade acidified lysosomes in corneal epithelial cells.
Aim 3. To explore how PopB and ExoS then allow intracellular P. aeruginosa to establish membrane bleb niches. Phagocytic activity of corneal epithelial cells and bacterial evasion of this defense are both novel concepts. Understanding the host-pathogen interactions that result in these very different outcomes, could lead to new strategies for managing infection.

Public Health Relevance

The epithelium of the cornea is a formidable barrier against infection, yet Pseudomonas aeruginosa can penetrate it to cause vision-threatening eye disease in some situations. We have made two discoveries that help explain this dichotomy; 1) that corneal epithelial cells can kill bacteria intracellularly, and 2) that P. aeruginosa can subvert tis defense to form an intracellular replicative niche within plasma membrane blebs. Here, we will study the details of the host-pathogen interactions that determine the fate of intracellular bacteria, the long-term goal being to develop therapies that tip the balance in favor of host defense.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY011221-20
Application #
9388348
Study Section
Diseases and Pathophysiology of the Visual System Study Section (DPVS)
Program Officer
Mckie, George Ann
Project Start
1995-07-01
Project End
2019-11-30
Budget Start
2017-12-01
Budget End
2018-11-30
Support Year
20
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
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Jolly, Amber L; Agarwal, Paresh; Metruccio, Matteo M E et al. (2017) Corneal surface glycosylation is modulated by IL-1R and Pseudomonas aeruginosa challenge but is insufficient for inhibiting bacterial binding. FASEB J 31:2393-2404
Metruccio, Matteo M E; Evans, David J; Gabriel, Manal M et al. (2016) Pseudomonas aeruginosa Outer Membrane Vesicles Triggered by Human Mucosal Fluid and Lysozyme Can Prime Host Tissue Surfaces for Bacterial Adhesion. Front Microbiol 7:871
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Robertson, Danielle M; Alexander, Larry J; Bonanno, Joseph A et al. (2014) Cornea and ocular surface disease: application of cutting-edge optometric research. Optom Vis Sci 91:S3-16
Borkar, Durga S; Acharya, Nisha R; Leong, Chelsia et al. (2014) Cytotoxic clinical isolates of Pseudomonas aeruginosa identified during the Steroids for Corneal Ulcers Trial show elevated resistance to fluoroquinolones. BMC Ophthalmol 14:54
Heimer, Susan R; Evans, David J; Mun, James J et al. (2013) Surfactant protein D contributes to ocular defense against Pseudomonas aeruginosa in a murine model of dry eye disease. PLoS One 8:e65797
Mun, James; Tam, Connie; Chan, Gary et al. (2013) MicroRNA-762 is upregulated in human corneal epithelial cells in response to tear fluid and Pseudomonas aeruginosa antigens and negatively regulates the expression of host defense genes encoding RNase7 and ST2. PLoS One 8:e57850

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