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Jianfang Li, Matteo Metruccio, David Evans, Suzanne M J Fleiszig; Human tear fluid suppresses Pseudomonas aeruginosa twitching motility. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5870. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
We previously reported that human tear fluid inhibits Pseudomonas aeruginosa virulence in corneal epithelial cells in vitro independently of antimicrobial activity, and that it protects injured murine corneas from P. aeruginosa keratitis in vivo. We also used RNA-seq to reveal that P. aeruginosa exposure to human tear fluid decreases transcription of multiple motility-related genes within 5 h. Here, we explored impact of human tears on twitching motility, a surface-associated movement mediated by type IV pili, because we have shown it allows internalized P. aeruginosa to exit corneal epithelial cells, and contributes to the pathogenesis of bacterial keratitis.
Human tears collected from healthy volunteers as approved by the UC Berkeley CPHS, contrived (artificial) tears containing lysozyme, albumin, and γ-globulins (Ursa BioScience), purified lactoferrin and lysozyme (2 mg/mL) and PBS as control were tested. Solutions, pipetted (10 μl) onto semi-solid twitching media, were inoculated with P. aeruginosa PAO1 or PAO1ΔpilA (twitching negative). Samples were imaged through a coverslip using time-lapse wide-field microscopy after 4 or 24 h at 37 °C. Bacterial velocity was quantified with IMARIS. ANOVA was used for statistical analysis.
Human tears suppressed PAO1 twitching compared to PBS at 4 h. Mean velocity dropped from 700 (+/- 610) nm/min in PBS to 170 (+/- 100) nm/min in 25 % tear fluid (p < 0.0001), similar to that of twitching mutants of 110 (+/- 40) nm/min. Inhibition continued to the 24 h time point, and was retained through to 25% dilution in PBS. Tear fluid also inhibited twitching in two other P. aeruginosa strains tested, PAK and PA103. Neither lactoferrin, lysozyme, nor a cocktail of both, had any impact compared to controls [820 (+/- 560), 680 (+/- 470), and 790 (+/- 950) nm/min respectively (p > 0.4)]. Surprisingly, twitching was unaffected by contrived tears. Removal of proteins < 30 kDa had no impact on tear inhibition of twitching, which was abrogated by boiling or proteinase K digestion (100 μg/mL, 42 °C, 2 h).
Tear suppression of P. aeruginosa twitching motility may contribute to tear protection of corneal epithelial cells. This inhibition likely involves one or more proteins larger than 30 kDa, does not involve known tear components identified in contrived tear fluid, and is a host defense to which P. aeruginosa does not readily adapt.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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