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Michael E Zegans, Kathryn Ray, Wesley Hebert, Amanda Naimie, John H Hammond, Lalitha Prajna, Deborah Hogan, Antonio DiGiandomenico, Thomas M Lietman, Muthiah Srinivasan, Steroids for Corneal Ulcers Trial Study Group; Pseudomonas aeruginosa isolates with lasR mutations were associated with worse visual outcomes in the Steroids For Corneal Ulcer Trial (SCUT). Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4847.
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© ARVO (1962-2015); The Authors (2016-present)
Pseudomonas aeruginosa (PA) uses quorum sensing to measure cell density and regulate production of virulence factors. In PA, quorum sensing is mediated through the activity of 3 hierarchically arranged signaling cascades, with the LasR transcription factor positioned as the master regulator. While lasR up regulates known virulence factors such as proteases, lasR mutants are frequently isolated from infections, suggesting that lasR is selected against in certain clinical contexts. We investigated 101 PA keratitis isolates from the SCUT study for evidence of lasR mutations and the effect of these mutations on visual outcomes. This is the first large scale study of lasR among PA keratitis strains.
101 PA SCUT isolates were screened for the lasR mutant sheen colony phenotype by streaking them on a lysogeny broth agar plate. Colonies with an iridescent sheen were considered possible lasR mutants. The lasR gene including the promoter was sequenced in all candidate isolates. Normal LasR function is associated with production of proteases, surfactant and homoserine lactones (HSL). Our candidate lasR mutants were assessed for these phenotypes. As a control, all of the above assays were performed with randomly selected SCUT PA isolates without the iridescent sheen phenotype. Finally, we compared vision at presentation and at 3 months in patients infected with las-intact vs. las- deficient Pseudomonas.
22 of 101 PA isolates from the SCUT study were noted to have colony morphology with a sheen phenotype characteristic of lasR mutations. Mutations in the lasR gene or promoter were identified among the candidate lasR mutants, but not in the other SCUT PA isolates. We verified the loss of LasR signaling by demonstrating decreased protease activity (p<0.01), decreased surfactant production (p<0.01), and a lack of production of HSL. Finally, SCUT patients infected with strains with lasR mutations had worse vision at presentation and at 3 months.
Mutations of lasR were common (22/101) among PA keratitis isolates from the SCUT study and associated with worse vision at presentation and at 3 months compared to patients infected with las-intact strains. These data suggest that conventional assumptions about the relationship between quorum sensing and virulence may need to be re-evaluated.
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