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Published on: March 17, 2014
Clearance of Pseudomonas aeruginosa from a healthy ocular surface involves surfactant protein D and is compromised by
James J Mun1, Connie Tam, David Kowbel
1School of Optometry, University of California, Berkeley, CA 94720-2020, USA.
Abstract:
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.
Insights
Surfactant protein D (SP-D) aids in clearing Pseudomonas aeruginosa from eyes. However, bacterial proteases can degrade SP-D, potentially increasing virulence and hindering clearance from the ocular surface.
Area of Science:
- Ophthalmology
- Immunology
- Microbiology
Background:
- Surfactant protein D (SP-D) is found in human tears and protects corneal cells from bacteria.
- The role of SP-D in clearing Pseudomonas aeruginosa from the ocular surface in vivo is not fully understood.
Purpose of the Study:
- To investigate the in vivo contribution of SP-D to the clearance of Pseudomonas aeruginosa from the healthy ocular surface.
- To determine if bacterial proteases affect SP-D-mediated clearance of P. aeruginosa.
Main Methods:
- A null-infection model was used in SP-D gene-targeted (SP-D(-/-)) knockout mice and wild-type mice.
- Mice corneas were inoculated with invasive (PAO1) or cytotoxic (6206) P. aeruginosa strains.
- Viable bacterial counts were assessed in tear fluid over 14 hours post-inoculation.
Main Results:
- Healthy ocular surfaces efficiently cleared both P. aeruginosa strains.
- Clearance of the low protease-producing strain (6206) was delayed in SP-D(-/-) mice.
- High protease-producing strain (PAO1) clearance was not significantly different in SP-D(-/-) mice compared to wild-type mice.
- Bacterial proteases, like elastase, were found to degrade tear fluid SP-D in vivo.
Conclusions:
- SP-D plays a role in clearing P. aeruginosa from the healthy ocular surface.
- Bacterial proteases can degrade SP-D, potentially compromising its clearance function and contributing to P. aeruginosa virulence.
- SP-D degradation is a potential mechanism by which P. aeruginosa proteases enhance ocular infection.
