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V. Hritonenko, J. Mun, C. Tam, D. J. Evans, S. M. J. Fleiszig; The in vivo Role of ExoY in P. aeruginosa-Induced Corneal Infection Involves Its Adenylate Cyclase Activity. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3896.
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© ARVO (1962-2015); The Authors (2016-present)
ExoY is an effector of the P. aeruginosa type three secretion system. Our in vitro studies with corneal epithelial cells have shown that ExoY inhibits bacterial internalization by corneal epithelial cells, and induces membrane bleb niche formation, and that both effects depend on its adenylate cyclase (AC) activity. Here, we examined the role of ExoY in virulence in vivo.
A triple effector mutant of P. aeruginosa PA01 (lacking ExoS, ExoT, ExoY) was complemented with plasmids carrying the exoY gene (pUCP_exoY), or exoY with an inactive AC (pUCP_exoY K81M) or a vector control (pUCP18). Black Swiss Surfactant Protein-D (SP-D) knockout mice (15-week-old female) were used to enhance corneal susceptibility. Mice were inoculated with ~106 cfu bacteria after corneal scarification and 6 h of epithelial healing. Disease severity was scored at 24 and 48 h using a 16 point scoring system. Colonization was quantified by viable count. Kruskal-Wallis (KW) and Mann-Whitney (MW) statistical tests were used.
There was no significant difference in disease scores between the groups at 24 h. Median [upper, lower quartile] scores were 6 [8, 5] for pUCP_exoY; 8 [8, 0] for pUCP_exoYK81M; 2 [2, 0] for pUCP18 (p = 0.3, KW). At 48 h, there was a significant difference in disease scores (p = 0.008, KW). Median scores of 10 [13, 10] were found for pUCP_exoY, 8 [8, 0] for pUCP_exoYK81M, and 2 [6, 0] for pUCP18. The difference between wild-type exoY and its AC mutant was significant (p = 0.015, MW), but that between the AC mutant and pUCP18 was not (p = 0.390, MW). Colonization differences at 48 h were not significant (p = 0.210, KW), but showed a trend towards increased colonization by bacteria containing pUCP_exoY.
ExoY contributes to P. aeruginosa keratitis in vivo in the absence of SP-D without a significant impact on colonization. The AC activity of ExoY is involved, but further studies are needed to determine the relationship between ExoY-mediated inhibition of invasion, bleb-niche formation, and disease pathogenesis in vivo.
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