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 viablePseudomonas 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.Pseudomonas aeruginosa, an opportunistic pathogen, is a leading cause of bacterial keratitis. While normal, healthy human corneas remain resistant to infection, contact lens wear or corneal injury/surgery can enable susceptibility (5,15,26). The mechanisms by which these factors predispose to infection are not yet well understood.A murine scarification model has been used exclusively to study the pathogenesis of P. aeruginosa corneal infection (3,9,30). That model involves scratching the cornea with a sterile needle prior to adding bacteria, which enables bacteria to directly access the exposed stroma. The resulting disease resembles P. aeruginosa infection in people. More recently, we used a healing model of murine corneal infection to show that 6 h after scratching, the mouse cornea remains susceptible to infection, but by 12 h, it regains resistance to infection despite loss of barrier function to fluorescein staining (18). These injury models that enable P. aeruginosa to infect the cornea have led to a wealth of information about how infection develops and resulting pathology. Yet, the mechanisms by which the normal ocular surface remains healthy under normal circumstances have not been explored in vivo. This cannot be studied using a scratch model. The corneas' ability to resist disease despite constant daily exposure to potential pathogens is remarkable, and learning about the mechanisms involved could help us to develop new therapies for disea...