Abstract
Purpose :
To examine the specific individual roles of bacterial virulence factors in the interactions between Pseudomonas aeruginosa (PA) bacteria and corneal fibroblasts in human microbial keratitis.
Methods :
Human corneal fibroblast (CF) were extracted from clinical samples from several patients and cultured to confluence in vitro. CFs were challenged with live wild type PAO1 bacteria and mutant strains deficient in type 4 pilus (TFP), flagella or both, and total bacterial association was quantified at 3h using the saponin-lysis and viable counting method and confirmed using laser scanning confocal microscopy (LSCM); and live wild type PA14 bacteria and mutant strains deficient in type III secretion system (TTSS) or flagella, and lactose dehydrogenase (LDH) release from CF was quantified and used as a measure of cytotoxicity.
Results :
Bacterial adherence to CF was significantly less (P=0.000, ANOVA) in mutant PAO1 strains deficient in TFP and flagella compared to wild type strain as demonstrated by viable counting assays and LSCM. Bacteria-induced cytotoxicity was significantly less (P=0.000, ANOVA) in mutant strains deficient in TTSS or flagella compared to wild type strain as demonstrated by reduced LDH release.
Conclusions :
We have established in vitro cell culture models of bacterial keratitis and demonstrated that PA utilises TFP and flagella to adhere to, and TTSS to induce cytotoxicity in, human CF.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.