July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
P. aeruginosa internalization into epithelial cells is associated with intracellular triggering of the type three secretion system
Author Affiliations & Notes
  • Abby Kroken
    School of Optometry, University of California, Berkeley, Berkeley, California, United States
  • Camille Chen
    School of Optometry, University of California, Berkeley, Berkeley, California, United States
  • David J Evans
    College of Pharmacy, Touro University California, Vallejo, California, United States
    School of Optometry, University of California, Berkeley, Berkeley, California, United States
  • Timothy Yahr
    Department of Microbiology, University of Iowa, Iowa City, Iowa, United States
  • Suzanne M J Fleiszig
    School of Optometry, University of California, Berkeley, Berkeley, California, United States
  • Footnotes
    Commercial Relationships   Abby Kroken, None; Camille Chen, None; David Evans, None; Timothy Yahr, None; Suzanne Fleiszig, None
  • Footnotes
    Support   NIH Grant F32 EY025969, NIH Grant R01 EY011221
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 1544. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Abby Kroken, Camille Chen, David J Evans, Timothy Yahr, Suzanne M J Fleiszig; P. aeruginosa internalization into epithelial cells is associated with intracellular triggering of the type three secretion system. Invest. Ophthalmol. Vis. Sci. 2018;59(9):1544.

      Download citation file:


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

      ×
  • Supplements
Abstract

Purpose : Pseudomonas aeruginosa is an ocular pathogen associated with contact lens wear and injury-related keratitis. While known to proliferate inside corneal epithelial cells, P. aeruginosa is often considered an extracellular pathogen and encodes type three secretion system (T3SS) effectors with anti-phagocytic activity. Previously we reported that one such effector ExoS is paradoxically needed for P. aeruginosa to survive and replicate intracellularly in corneal epithelial cells. Here, we sought to reconcile these apparently conflicting notions about P. aeruginosa because understanding bacterial localization during infection is key to development of effective management strategies.

Methods : T3SS expression versus bacterial location was examined using four P. aeruginosa isolates (PAO1, PAK, PA103, PA14) and their T3SS effector or fleQ mutants, with or without ExoS expression, using time lapse microscopy, a T3SS fluorescence reporter, and gentamicin protection assays.

Results : All isolates invaded cells when lacking T3SS effectors. When ExoS was exogenously expressed, the only isolate to lose capacity to enter cells was PA103, the strain used in most studies of ExoS antiphagocytic activity. For invading isolates, T3SS triggering occurred mostly after cell entry, and continued in daughter cells after intracellular replication. The impact of fleQ, a gene mutated in PA103, was then explored. Correcting fleQ in PA103, or mutating it in strain PAO1, modulated internalization ~10-fold, but that was irrespective of ExoS expression. Furthermore, PA103 remained ~10 fold less invasive than PAO1 when matched for fleQ status, and showed differences in excitability for T3SS triggering independently of fleQ (~80% T3SS-positive for PA103, ~40% T3SS-positive for PAO1).

Conclusions : By showing that only some bacteria in the population trigger their T3SS under inducing conditions, and that triggering can occur intracellularly, these findings explain how P. aeruginosa can thrive inside host cells whilst encoding antiphagocytic T3SS effectors. They also illustrate that strain PA103, used extensively for studying the P. aeruginosa T3SS, is hyper-excitable for T3SS triggering compared to other isolates, an abnormality independent of its known fleQ mutation. Finally, this study challenges current thinking that T3SS effectors are delivered into host cells across their plasma membrane.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×