July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
High-resolution imaging and arabinose reporter induction to study Pseudomonas aeruginosa localization within the mouse cornea in situ
Author Affiliations & Notes
  • David J Evans
    College of Pharmacy, Touro University California, Vallejo, California, United States
    School of Optometry, University of California Berkeley, Berkeley, California, United States
  • Abby Kroken
    School of Optometry, University of California Berkeley, Berkeley, California, United States
  • Hart Horneman
    School of Optometry, University of California Berkeley, Berkeley, California, United States
  • Melinda Rose Grosser
    School of Optometry, University of California Berkeley, Berkeley, California, United States
  • Suzanne M J Fleiszig
    School of Optometry, University of California Berkeley, Berkeley, California, United States
  • Footnotes
    Commercial Relationships   David Evans, None; Abby Kroken, None; Hart Horneman, None; Melinda Grosser, None; Suzanne Fleiszig, None
  • Footnotes
    Support  NIH Grant EY011221, NIH Grant EY025969, American Heart Association
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 5186. doi:
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      David J Evans, Abby Kroken, Hart Horneman, Melinda Rose Grosser, Suzanne M J Fleiszig; High-resolution imaging and arabinose reporter induction to study Pseudomonas aeruginosa localization within the mouse cornea in situ. Invest. Ophthalmol. Vis. Sci. 2019;60(9):5186.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Many in vitro studies have shown that invasive P. aeruginosa strains can thrive inside eukaryotic cells, including corneal epithelial cells. After internalization, bacteria can escape endocytic trafficking, undergo rapid cytosolic replication and traffic to bacterial-induced membrane blebs. Yet P. aeruginosa is often regarded as a pathogen, exerting virulence by extracellular-mediated killing and extracellular disruption of tissue barriers. Here, we used high-resolution imaging and an arabinose-inducible reporter to quantitatively localize P. aeruginosa within the epithelium of the cornea in situ.

Methods : Eyes of C57BL/6 mice expressing membrane-localized tdTomato were challenged with P. aeruginosa PAO1 for 5 h after corneal blotting with tissue paper and EGTA treatment (100 mM, 1 h) that enables PAO1 to traverse normally resistant corneal epithelium. Other mice were infected in vivo for 4 h in a scarification model. Bacterial location relative to tdTomato was examined with high-resolution imaging over 24 h ex vivo. An arabinose-inducible fluorescent reporter plasmid was used to further distinguish and quantify intracellular bacteria. PAO1 was compared to an isogenic exsA mutant lacking the type 3 secretion system (T3SS), that does not thrive in cultured corneal epithelial cells. T3SS expression was monitored with a reporter plasmid (pJNE05).

Results : For both infection models, most bacteria within the corneal epithelium were located close to cell membranes, some appearing paracellular and others appearing intracellular. A proportion of intracellular bacteria were found actively swimming within epithelial cell membrane blebs, as described for cultured corneal epithelial cells. The arabinose reporter (blot/EGTA model) showed 48.7 +/- 6.9 % of bacteria in the central cornea expressed GFP above baseline controls lacking arabinose induction, similar to the proportion expressing the T3SS in this model. For the exsA mutant, only 12.5 +/- 2.1 % showed GFP levels above background in this model.

Conclusions : In the context of the cornea, a body surface with a limited phagocyte presence, P. aeruginosa internalization by corneal epithelial cells appears to be a relatively common phenomenon. This involves the T3SS and bleb-niche formation, similar to mechanisms involved in intracellular persistence within cultured corneal epithelial cells infected in vitro.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

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