Abstract
Purpose:
Pseudomonas aeruginosa, a leading cause of corneal infections, is able to enter and replicate within corneal epithelial cells. The T3SS of P. aeruginosa is necessary for intracellular bacterial survival within membrane blebs. CFTR is a receptor for P. aeruginosa; mutations in CFTR result in defective internalization and are highly correlated with chronic Pseudomonas lung infections in Cystic Fibrosis (CF). Here, we test the hypothesis that CFTR functions to promote intracellular survival.
Methods:
Telomerase-immortalized human corneal epithelial cells treated with DMSO or CFTR inhibitor-172 to block chloride channel function were infected with P. aeruginosa invasive strain PAO1. Telomerase-immortalized human bronchial epithelial cells from a healthy patient and a CF patient (ΔF508/ ΔF508 CFTR) were compared before and after infection with wild-type PAO1 and T3SS mutants. Infected cells were analyzed for bleb formation, and LysoTracker® dye used to selectively label acidic organelles. Cell death and apoptotic status were determined using propidium iodide staining and an annexin V-FITC conjugate, respectively. Gentamicin exclusion assays were used to determine bacterial internalization and survival.
Results:
CFTR-deficient infected bronchial epithelia displayed an enhanced blebbing phenotype (median bleb area is 440 µm2 in CFTR-deficient cells versus 138 µm2 in controls, p < 0.0001). Similar results were observed in corneal epithelial cells following treatment with CFTR inhibitor. CFTR-deficient cells also showed ~2 fold greater intracellular bacterial replication over 2-3 h compared to healthy epithelial cells (p < 0.02). Increased blebbing and bacterial survival in CFTR-deficient cells required the T3SS. CFTR-deficient cells displayed similar lysosome morphology and pH to control cells. However, 78% of blebbing cells were devoid of acidic vacuoles, independent of CFTR status, suggesting a relationship between bleb formation and bacterial modification of lysosomes. Blebbing cells lacked apoptotic markers and resisted infection-induced cell death.
Conclusions:
CFTR functions to inhibit P. aeruginosa intracellular survival, independent of a role for CFTR in lysosome acidification. Rather, enhanced intracellular survival in CFTR-defective cells appears to be related to the T3SS-induced blebbing ability in these cells.
Keywords: 664 pseudomonas •
482 cornea: epithelium •
569 ion channels