Abstract
Purpose :
Invasion of corneal epithelial cells contributes to the pathogenesis of Pseudomonas aeruginosa keratitis. Within these cells, P. aeruginosa escapes trafficking to lysosomes to form plasma membrane blebs that they subsequently occupy. The role of bleb-niche formation in pathogenesis is not well understood. Previously, we showed that pili gene mutants (pilA, pilU) of P. aeruginosa strain PAK were defective in the capacity to exit rabbit corneal epithelial cells. Here, we explored the relationship between bleb-niche formation and cellular exit using pili mutants and human cells.
Methods :
Exit of intracellular wild-type PAO1 or pili mutants (pilA, pilT, pilU) from hTCEpi cells was quantified 3 h post-infection by killing bacteria not yet internalized with gentamicin, washing with PBS, then supplying antibiotic-free media to allow intracellular bacteria to exit. Bacteria released into the medium were collected each hour for 6 h and viable counts were performed. Images of hTCEpi cell morphology after infection by WT or pili mutants for 8 h were captured using phase-contrast microscopy.
Results :
All three mutants were competent for cellular invasion, but defective for cellular exit (@6 h x105 CFU/mL; wild-type 7.7 ± 1.2, pilA 1.3 ± 0.3, pilT 2.1 ± 0.5, pilU 1.5 ± 0.4, p < 0.005, ANOVA). Differences among mutants were not significant. While the pilA and pilT mutants both caused membrane blebbing in corneal epithelial cells, there were fewer blebs, and only wild-type infected cells had blebs occupied by bacteria. Surprisingly, cells infected with the pilU mutant lacked blebs altogether and instead exhibited significant cell rounding.
Conclusions :
Pili gene mutations impede PAO1 exit from human corneal epithelial cells, and block membrane bleb-niche formation, suggesting that these events might be related. Empty blebs in cells infected with mutants possessing pili, but defective in pili retraction (pilT mutants), suggest twitching motility (not pili) is required for bacterial trafficking to blebs. The absence of any blebs in cells infected with another twitching mutant (pilU), suggests that these two twitching genes play distinct roles in bleb-niche formation in corneal epithelial cells.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.