In this study, we developed a novel null-infection model and used it to show that P. aeruginosa is rapidly cleared from the healthy ocular surface of mice. We found that SP-D can contribute to this process and that it can be delayed by the expression of bacterial proteases. Suggesting a connection between those two findings are the results that SP-D within tear fluid can be degraded by P. aeruginosa elastase in vitro and in vivo, that SP-D−/− animals clear wild-type and protease-mutant bacteria similarly, and that protease-mutant bacteria (or strains producing less elastase) are cleared more effectively from wild-type animals than from SP-D−/− mice.
The ocular surface is constantly exposed to a diverse array of potentially pathogenic microbes. The efficient clearance of microorganisms entering the eye is likely to be important in the maintenance of ocular health. Factors that are likely to contribute include blinking and tear exchange for physical removal of bacteria in addition to tear biochemical factors that bind, aggregate, and/or inactivate microorganisms. Known activities of SP-D include binding and aggregation of P. aeruginosa
(and other microbial pathogens) (4
), direct antimicrobial activity (32
), and a role in limiting P. aeruginosa
-induced corneal pathology in an injury model of corneal infection (25
) and during infection of the respiratory tract (10
). SP-D is also known to facilitate phagocytosis by macrophages and to modulate activity of phagocytes (31
). It is known that there are resident dendritic cells within the cornea (13
). The upregulation of corneal epithelial cell SP-D in response to bacterial antigens, which we previously reported (28
), could be important in the mechanism by which SP-D contributes to clearance from the healthy ocular surface.
The data showing a relationship between protease expression and retention of P. aeruginosa
at the ocular surface could involve SP-D degradation in vivo by proteases. While P. aeruginosa
elastase and protease IV had already been shown to degrade purified SP-D into an inactive form (1
), those previous studies were done in vitro. In this study, we showed that elastase can also degrade SP-D when it is within tear fluid either in vitro or in vivo. The data also showed that differences in clearance between wild-type and protease-mutant bacteria seen in wild-type animals are no longer statistically significant in SP-D−/−
mice. Taken together, these data suggest that SP-D degradation provides a possible mechanism for the delayed clearance of protease-competent bacteria compared to protease mutants. However, the relationship between bacterial proteases, SP-D expression, and ocular clearance of bacteria will require further study to determine the contribution of elastase (and other P. aeruginosa
proteases) toward the in vivo degradation of SP-D and the biological significance of this finding given the continued renewal of this innate defense protein by the lacrimal apparatus and ocular surface epithelia (27
). In addition, proteases could also promote ocular colonization through other mechanisms. For example, P. aeruginosa
proteases are known to degrade tear immunoglobulins (19
), which could compromise their known ocular defense against infection (23
). Whether previously demonstrated roles for elastase and other proteases in increasing bacterial adherence to the mouse cornea (12
), or in invasion and penetration through epithelia (2
), relate to the role of proteases in colonization of the healthy cornea is yet to be determined.
Cytotoxic P. aeruginosa
(6206) was found to be cleared more rapidly than the invasive strain (PAO1). Interestingly, 6206 encodes and expresses a powerful cytotoxin, ExoU (absent in PAO1), which can repress phagocyte infiltration of infected corneas in vivo (33
), injure and kill corneal epithelial cells in vitro (8
), and also damage the intact corneal epithelium ex vivo (7
). Indeed, our data showed that at the inoculum used in this study, cytotoxic strain 6206 did damage corneal barrier function in vivo, as indicated by fluorescein staining. The rapid clearance of this cytotoxic strain (relative to PAO1), despite its capacity to cause superficial damage in vivo, may reflect its low level or lack of protease activity as previously reported (29
) and confirmed in the present study.
Traditional models for studying bacterial keratitis in which disease is induced do not allow normal resistance factors to be directly examined. In this study, we developed, and demonstrated, the usefulness of a new null-infection model for this purpose. While we have shown SP-D to be involved in clearance and have shown that P. aeruginosa proteases can compromise it, there are likely to be an array of host factors that protect the eye under normal circumstances and there are also likely to be bacterial factors with the potential to compromise clearance. Further studies using this model could facilitate our understanding of the circumstances surrounding resistance and susceptibility to infection and could eventually lead to new approaches for treatment or prevention of infection of the eye and of other sites.