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Matteo Maria Emiliano Metruccio, Yvonne Wu, David J Evans, Suzanne M J Fleiszig; Pseudomonas aeruginosa adaptation to the ocular surface: transcriptional changes and virulence determinants. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4043.
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© ARVO (1962-2015); The Authors (2016-present)
Pseudomonas aeruginosa is a leading cause of contact lens-related corneal infection. Using a lens-wearing rodent model, we previously showed that bacteria could infect the cornea more efficiently if first pre-exposed to the ocular surface. This is consistent with the increased risk of infection with extended lens wear in humans. Here, we studied bacterial adaptions to the ocular surface that subsequently enable them to penetrate the corneal epithelium.
RNA-seq was used to compare the transcriptional profile of P. aeruginosa strain PAO1 exposed to human tear fluid for 5 h at 37 °C to PBS controls. Tn-seq was used to identify bacterial genes required for traversal of human corneal epithelial cell multilayers in vitro. For the latter, a pooled transposon mutant library of PAO1 was generated, and ~106 cfu of bacterial mutants incubated with Transwell filter-grown human telomerase-immortalized corneal epithelial cells for 4 h at 37 °C. Transposon insertion sites were deep sequenced for the input and traversed populations. HiSeq 2000 (Illumina) was used for sequencing and data analysis performed with Galaxy and IGB.
RNA-sequencing showed many P. aeruginosa genes were deregulated (up- or down-regulated) by exposure to tear fluid (~180 genes ≥ 8 fold). They included; the phoP/Q two-component regulatory system (down-regulated), oprH for antimicrobial resistance (down-regulated), and algF for biofilm formation and antiphagocytic activity (up-regulated). Two small non-coding RNAs, rsmZ and phrS, associated with virulence factor regulation, response to oxygen availability and quorum sensing were also down- and up-regulated respectively. Tn-seq identified ~200 gene insertions differentially represented in traversed populations as compared to the input (cut-off ≥ 18 fold), showing roles in epithelial traversal. Insertions mapped in or near 150 genes belonging to numerous important virulence categories, including pcrV (type three secretion), motC (motility and attachment) and mexF (multidrug efflux pump).
Use of an unbiased global genetic approach to study P. aeruginosa interaction with ocular surface components in vitro identified genes and genomic regions involved in bacterial adaptation to the host environment and ocular pathogenicity.
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