Abstract
Purpose: :
We have previously shown that Pseudomonas aeruginosa can use its Type 3 Secretion System (T3SS) to survive and replicate within corneal epithelial cells in vitro, and to mediate keratitis in vivo. Mutants lacking the T3SS traffic to perinuclear vacuoles after being internalized and do not thrive. In contrast, wild-type bacteria survive and replicate inside host plasma membrane bleb-niches. Whether or not wild-type bacteria also traffic to perinuclear vacuoles prior to escaping to membrane blebs, and whether these are acidic compartments (i.e. lysosomes) has not yet been established. Here, we tested the hypothesis that both wild-type and T3SS-mutant P. aeruginosa initially traffic to acidic perinuclear vacuoles.
Methods: :
Telomerase-immortalized corneal epithelial cells were grown on coverslips at sub-confluent levels and infected with GFP-labeled wild-type (PAO1-GFP) or T3SS-mutant (PAO1 exsA::omega-GFP) P. aeruginosa (~107 CFU/ml, 3 h). Cells were then washed and treated with amikacin to kill extracellular bacteria. Crystal violet was used to quench fluorescence of extracellular bacteria and/or cells were stained with the acidophilic LysoTracker (LT) dye prior to viewing by fluorescence microscopy. Quantification was based on > 10 fields/experiment, and bacterial viability was assessed in vitro in Tris-buffered solutions (pH 4.5-7.5) using quantitative plating.
Results: :
LT staining was mostly confined to perinuclear vacuoles in both healthy and infected cells. After 4.5 h, wild-type PAO1 were observed in the majority of cells (~80%) at ~7.5+/-0.6 bacteria/cell. About 14% of these bacteria colocalized with LT, in either perinuclear or newly-appearing peripheral vacuoles, with ~1.2+/-0.2 bacteria per LT+ vacuole. In contrast, the T3SS-mutant colocalized with LT more frequently with ~3.8+/-0.7 bacteria per LT+ vacuole (p = 0.01, t-Test). LT did not accumulate within bleb niches, or in vacuoles containing motile wild-type bacteria. In vitro, wild-type and T3SS-mutant bacteria showed a significant decrease in viability (~100-fold) after 5 h exposure to acidic pH (4.5).
Conclusions: :
The higher frequency of T3SS-mutant bacteria located within LT+ vacuoles, and their susceptibility to acidic pH in vitro, is consistent with their reduced capacity to thrive intracellularly. Colocalization of some wild-type bacteria with LT suggests that wild-type and T3SS mutants share a common intracellular trafficking pathway to lysosomes early in infection, prior to T3SS dependent escape to membrane bleb-niches.
Keywords: microbial pathogenesis: experimental studies • pseudomonas • keratitis