Abstract
Purpose:
Pseudomonas aeruginosa is a leading cause of corneal infection. We have previously shown that P. aeruginosa can invade and survive inside human corneal epithelial cells, and that intracellular bacteria can traffic to membrane blebs. Our previous work has shown that this unique intracellular niche formed by P. aeruginosa requires its type III secretion system (T3SS), and that deficiency of the host cell CFTR chloride channel enhances the quantity and size of the blebs. Here, we examined mechanisms for bleb biogenesis and impact on fate of intracellular bacteria.
Methods:
Telomerase-immortalized epithelial cells were infected with P. aeruginosa PAO1 (MOI 100:1), adding gentamicin 3 h post inoculation to kill extracellular bacteria. The effect of cell medium osmolality on bleb-niche formation and bacterial survival was examined with and without a CFTR inhibitor (20 uM CFTR(Inh)-172). Gentamicin exclusion assays were performed to quantify the impact of these treatments on bacterial intracellular survival.
Results:
Bacterial-induced blebbing was completely eliminated by increasing the medium osmolality to 600 mOsM at 3 h after bacteria had entered cells, using either NaCl or mannitol (p < 0.0001). Increasing osmolality to only 400 mOsM eliminated enhanced bleb formation observed in CFTR inhibitor treated cells. Bleb inhibition with 600 mOsM mannitol also resulted in a 75% reduction in total intracellular bacteria by the end of the assay (p < 0.0001). This corresponded to a decreased intracellular replication rate (p < 0.05) over 3 h similar to T3SS mutant bacteria that do not form blebs. Controls showed that 600 mOsM medium had no impact on epithelial cell viability or replication by T3SS mutants. Imaging experiments using osmolality to manipulate bleb formation also revealed that bacteria-occupied vacuoles can fuse with and deposit bacteria into newly formed blebs.
Conclusions:
The data suggest that; 1) P. aeruginosa-induced bleb formation involves dysregulated ion channel conductance of the CFTR causing increased fluid influx due to loss of osmoregulatory function, 2) blebbing contributes to bacterial intracellular survival, 3) P. aeruginosa does not require the T3SS for intracellular survival when the cell is CFTR deficient, 4) bleb formation can occur before bacteria traffic to them, and 5) vacuoles can be used to deliver bacteria into this niche.
Keywords: 482 cornea: epithelium •
448 cell membrane/membrane specializations •
664 pseudomonas