May 2007
Volume 48, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2007
Mutation of the Phospholipase Catalytic Domain of the Pseudomonas aeruginosa Cytotoxin ExoU Abolishes Colonization Promoting Activity and Reduces Corneal Disease Severity
Author Affiliations & Notes
  • D. J. Evans
    College of Pharmacy, Touro University-California, Vallejo, California
    School of Optometry, University of California-Berkeley, Berkeley, California
  • C. Tam
    School of Optometry, University of California-Berkeley, Berkeley, California
  • S. Lewis
    School of Optometry, University of California-Berkeley, Berkeley, California
  • W. Li
    School of Optometry, University of California-Berkeley, Berkeley, California
  • E. Lee
    School of Optometry, University of California-Berkeley, Berkeley, California
  • S. M. J. Fleiszig
    School of Optometry, University of California-Berkeley, Berkeley, California
Investigative Ophthalmology & Visual Science May 2007, Vol.48, 2659. doi:
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      D. J. Evans, C. Tam, S. Lewis, W. Li, E. Lee, S. M. J. Fleiszig; Mutation of the Phospholipase Catalytic Domain of the Pseudomonas aeruginosa Cytotoxin ExoU Abolishes Colonization Promoting Activity and Reduces Corneal Disease Severity. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2659.

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

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Abstract

Purpose:: We have shown that ExoU, a type III secreted cytotoxin of Pseudomonas aeruginosa, contributes to corneal disease pathology and ocular colonization in the murine scarification model of microbial keratitis. Subsequently, we showed that this is associated with ExoU-mediated repression of phagocyte infiltration of infected corneas in vivo. ExoU has patatin-like phospholipase activity which is required for its cytotoxic activity (injury and cell death) towards mammalian cells in vitro and disease in a murine model of acute pneumonia. We hypothesized that phospholipase activity was required for ExoU-mediated colonization and disease in the cornea.

Methods:: The murine scarification model was used to examine corneal disease pathology and bacterial colonization (viability) at 24 and 48 h after inoculation with ~106 cfu in 5 µl of a double effector mutant of P. aeruginosa strain PA103 (PA103ΔexoUexoT::Tc) complemented with exoU, phospholipase-inactive exoU (exoUD344A) or plasmid control (pUCP18). Eyes were photographed and disease severity scored. Colonization of the complemented mutants was also assessed at 6 h post-inoculation which is prior to significant phagocyte infiltration of the cornea. Complementation of the double effector mutant was used to avoid confounding effects of ExoT which also contributes to corneal disease and colonization.

Results:: Complementation with exoU caused significantly more disease pathology (increased disease severity scores) and ~1000-fold greater ocular colonization at 48 h post-infection than the phospholipase-inactive exoU which did not differ significantly from the effector mutant complemented with control plasmid. Surprisingly, neither exoU, nor its inactive phospholipase form, contributed significantly to early (6 h) ocular colonization.

Conclusions:: The phospholipase catalytic domain of ExoU is required for its contribution to ocular colonization in vivo and its contribution to corneal pathology at 48 h post-infection. The data suggest, however, that early cytotoxic effects on epithelial cells are not important for these in vivo effects at least in this model. The phospholipase-mediated colonization and disease promoting activity of ExoU at 48 h may be associated with repression of phagocyte infiltration of infected corneas associated with this toxin.

Keywords: Pseudomonas • keratitis • cornea: basic science 
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