Clearance Of Pseudomonas Aeruginosa From A Healthy Ocular Surface Involves Surfactant Protein D And Is Compromised By Bacterial Elastase In A Murine Null-Infection Model

Our previous studies showed that surfactant protein D (SP-D) is present in human tear fluid and that it can protect corneal epithelial cells against bacterial invasion. Here we developed a novel null-infection model to test the hypothesis that SP-D contributes to the clearance of viable Pseudomonas aeruginosa from the healthy ocular surface in vivo. Healthy corneas of Black Swiss mice were inoculated with 10(7) or 10(9) CFU of invasive (PAO1) or cytotoxic (6206) P. aeruginosa. Viable counts were performed on tear fluid collected at time points ranging from 3 to 14 h postinoculation. Healthy ocular surfaces cleared both P. aeruginosa strains efficiently, even when 10(9) CFU was used: e. g., < 0.01% of the original inoculum was recoverable after 3 h. Preexposure of eyes to bacteria did not enhance clearance. Clearance of strain 6206 (low protease producer), but not strain PAO1 (high protease producer), was delayed in SP-D gene-targeted (SP-D-/-) knockout mice. A protease mutant of PAO1 (PAO1 lasA lasB aprA) was cleared more efficiently than wild-type PAO1, but this difference was negligible in SP-D-/- mice, which were less able to clear the protease mutant. Experiments to study mechanisms for these differences revealed that purified elastase could degrade tear fluid SP-D in vivo. Together, these data show that SP-D can contribute to the clearance of P. aeruginosa from the healthy ocular surface and that proteases can compromise that clearance. The data also suggest that SP-D degradation in vivo is a mechanism by which P. aeruginosa proteases could contribute to virulence.