Purpose: The authors have shown that twitching motility, a pilus-mediated form of bacterial surface movement, is required for Pseudomonas aeruginosa virulence in a murine model of keratitis. To study the role of twitching motility in virulence, Pseudomonas traversal of multilayered corneal epithelia in vitro was investigated.
Methods: Translocation of multilayered corneal epithelia was investigated with the invasive strain PAK and isogenic twitching motility mutants. Rabbit corneal epithelial cells were grown to multilayers with filters and inoculated on their apical surfaces with 10(6) colony-forming unit bacteria, and translocating bacteria were quantified by viable counts of the basal chamber. Transepithelial resistance (TER) was recorded. Cellular exit of P. aeruginosa after invasion was quantified with modified gentamicin survival assays, and the role of apoptosis in exit was explored.
Results: PAK translocated the epithelia as early as 1 hour after infection, and by 8 hours apical and basal numbers of bacteria were similar. Bacterial translocation did not reduce TER. Each twitching motility mutant (pilU, pilT with pili, pilA lacking pili) was defective in translocation (>2 log reduction vs. PAK; P < 0.005). All twitching mutants were competent for cell invasion but defective in cellular exit, accumulating intracellularly to numbers exceeding those of PAK. Inhibiting apoptosis reduced the cellular exit of PAK.
Conclusions: These results show that twitching motility enables P. aeruginosa to translocate corneal epithelial layers and suggest that it contributes to epithelial cell exit by a mechanism involving apoptosis. The relationship between these in vitro findings and the role of twitching motility in P. aeruginosa virulence in vivo remains to be determined.