June 2013
Volume 54, Issue 15
ARVO Annual Meeting Abstract  |   June 2013
Pseudomonas aeruginosa uses type three secretion-dependent and -independent mechanisms for traversal of multilayered corneal epithelia
Author Affiliations & Notes
  • Aaron Sullivan
    School of Optometry, UC Berkeley, Berkeley, CA
  • Victoria Hritonenko
    School of Optometry, UC Berkeley, Berkeley, CA
  • Connie Tam
    School of Optometry, UC Berkeley, Berkeley, CA
  • David Evans
    College of Pharmacy, Turo University- California, Vellejo, CA
  • Suzanne Fleiszig
    School of Optometry, UC Berkeley, Berkeley, CA
  • Footnotes
    Commercial Relationships Aaron Sullivan, None; Victoria Hritonenko, None; Connie Tam, "Antimicrobial Peptides and Methods of Use Thereof" (P); David Evans, U.S. Provisional Patent Application No. 61/479,507. (P), U.S. Issued Patent 7,332,470 B2 (P); Suzanne Fleiszig, Allergan (C), Allergan (F), New methods for preventing infection (P)
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 5214. doi:
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      Aaron Sullivan, Victoria Hritonenko, Connie Tam, David Evans, Suzanne Fleiszig; Pseudomonas aeruginosa uses type three secretion-dependent and -independent mechanisms for traversal of multilayered corneal epithelia. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5214.

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

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Purpose: We previously reported that the type 3 secretion system (T3SS) of Pseudomonas aeruginosa can mediate corneal epithelial traversal by invasive strain PAO1 in susceptible mouse corneas. In vitro, we showed that cytotoxic strain PA14 uses its T3SS (specifically the toxin ExoU) to traverse human corneal epithelial cells. Here, we crossed over the models and examined invasive strain PAK, which hyper-expresses the T3SS in vitro.

Methods: In vitro traversal capacity was examined using air-lifted, filter-grown multilayered human corneal epithelial cells. Bacteria were added apically chamber and bacteria traversing to the basal chamber were collected and counted. For in vivo experiments, mouse corneas were PBS rinsed, blotted with tissue paper, and treated with 100 mM EGTA for 1 h at 37 °C to enable bacterial traversal. Corneas were inoculated with GFP-labeled P. aeruginosa (200 µl ~109 CFU/mL) strain PAK, PA14, or PA14 T3SS mutants including mutants lacking ExoU, all of its three known effectors, or the needle required for effector secretion) for 1.5, 3 or 6 h at 37°C. After rinsing with PBS, corneas were imaged using 2-photon (NADPH autofluorescence), and confocal reflection microscopy. Epithelial thickness and bacterial traversal were evaluated.

Results: In vitro assays with invasive strain PAO1 showed that the role of the T3SS decreased with incubation time: 4 h, 29 fold (p=2.32E-06): 6 h, 14 fold (p=1.58E-04); 8 h, 4 fold (p=6.53E-03). In vivo, cytotoxic strain PA14 required the T3SS (specifically ExoU) to traverse at 1.5. and 3 h, but not by 6 h. Surprisingly, a wild-type invasive strain that hyper-secretes T3SS effectors in vitro (PAK), did not traverse at all in vivo, contrasting with the efficient T3SS dependent traversal capacity we have reported for a different invasive strain (PA01) that secretes the same T3SS effectors much less well in vitro.

Conclusions: The data continue to support a role for T3SS effectors (e.g. ExoU) in P. aeruginosa traversal, but they also show that T3SS-independent mechanisms can mediate traversal at longer time points both in vivo (mouse) and in vitro (human cells) and for both cytotoxic and invasive strains. Why PAK does not traverse the corneal epithelium is unclear considering it is a T3SS hyper-secretor in vitro; possibilities include a different capacity for secretion in vivo or a lack of essential co-factors.

Keywords: 664 pseudomonas • 482 cornea: epithelium  

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