INFECTION AND IMMUNITY, June 2009, p. 2392–2398
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 77, No. 6
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?
James J. Mun,1,2Connie Tam,1David Kowbel,1Samuel Hawgood,4Mitchell J. Barnett,5
David J. Evans,1,5and Suzanne M. J. Fleiszig1,2,3*
School of Optometry, University of California, Berkeley, California 947201; Vision Science Program, University of California, Berkeley,
California 947202; Graduate Groups in Microbiology and Infectious Disease and Immunity, University of California,
Berkeley, California 947203; Department of Pediatrics, Cardiovascular Research Institute, University of
California, San Francisco, California4; and College of Pharmacy, Touro University-California,
Vallejo, California 945925
Received 14 February 2009/Accepted 26 March 2009
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 107or 109CFU 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 109CFU 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 hu-
man 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 re-
sembles 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 de-
velops and resulting pathology. Yet, the mechanisms by which
the normal ocular surface remains healthy under normal cir-
cumstances 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 disease of the eye
and possibly other sites.
Results from the 12-hour healing situation suggested that
defense systems other than barrier function can protect the
ocular surface against infection. These defenses could involve
biochemical factors constitutively expressed or upregulated in
response to injury or bacterial exposure. Candidate factors
could include defensin or other antimicrobial peptides, secre-
tory immunoglobulin A, and mucin glycoproteins (11, 14, 23).
In this study we focused on surfactant protein D (SP-D),
which we have previously shown is present at the ocular sur-
face, is upregulated by P. aeruginosa or its antigens, and can
protect corneal epithelial cells against invasion (27, 28). Others
have shown that SP-D-deficient mice lose their capacity to
recover from P. aeruginosa keratitis when the eye is made
susceptible using an injury model (25). Here our aim was to
explore the role of SP-D in protecting the healthy eye against
bacterial colonization. Thus, we developed a novel null-infec-
tion model in which the cornea is not damaged prior to inoc-
ulation with P. aeruginosa. The objective was to allow bacteria
to interact with the healthy ocular surface (intact cornea) to
enable us to study host factors that normally protect the eye
from developing infection when it is not susceptible and the
potential role that bacterial factors might play in compro-
mising those defenses. This new model was then used to test
* Corresponding author. Mailing address: School of Optometry,
University of California, Berkeley, CA 94720-2020. Phone: (510) 643-
0990. Fax: (510) 643-5109. E-mail: email@example.com.
?Published ahead of print on 6 April 2009.
the hypothesis that SP-D contributes to the clearance of P.
aeruginosa from the healthy ocular surface and to explore
the role of P. aeruginosa proteases in promoting ocular
colonization in vivo.
MATERIALS AND METHODS
Bacterial strains. Cytotoxic strain 6206 and invasive strain PAO1 expressing
green fluorescent protein (GFP) on pSMC2 plasmid (PAO1-GFP) were used for
experiments comparing clearance of a cytotoxic strain and an invasive strain (see
Fig. 1 and 2). PAO1-GFP was provided by Gerald B. Pier (Harvard Medical
School, Boston, MA) and grown on Trypticase soy agar supplemented with
carbenicillin (300 ?g/ml). Wild-type PAO1 and its isogenic mutant PAO1 lasA
lasB aprA (6) without GFP were used in other experiments (see Fig. 3 and 4).
Inocula were prepared from overnight cultures grown on Trypticase soy agar
plates at 37°C for ?16 h before suspension in Dulbecco’s modified Eagle’s
medium (DMEM; Sigma-Aldrich, St. Louis, MO) to a concentration of 109or
1011CFU/ml. Bacterial concentrations were confirmed by viable count.
Murine null-infection model. Wild-type Black Swiss mice (6 to 8 weeks old)
and age-matched transgenic SP-D?/?mice were used. SP-D?/?Black Swiss mice
were obtained from Jeffrey Whitsett (Children’s Hospital Medical Center, Cin-
cinnati, OH) (16). Wild-type Black Swiss mice with a genetic background match-
ing that of the SP-D?/?mice were purchased from Taconic (Seattle, WA).
After induction of anesthesia (intraperitoneal injection with 21 mg/ml ketamine,
2.4 mg/ml xylazine, and 0.3 mg/ml acepromazine), 5 ?l of bacterial inoculum con-
taining ?107or 109CFU was applied to the healthy ocular surface. At 3, 6, 12, and
of viable bacteria within was determined. Tear fluid was collected by capillary action
using a 10-?l-volume glass capillary tube (Drummond Scientific Co., Broomall, PA)
from the lateral canthus after 4 ?l of phosphate-buffered saline was added to the
ocular surface. In some experiments, whole eyes were enucleated after collection of
tears and homogenized in 1 ml of phosphate-buffered saline with 0.25% Triton
X-100 for viable counts. In other experiments, eyes were preexposed to bacteria for
?16 h prior to inoculation in order to potentially stimulate innate defenses. Ocular
health was monitored throughout the experiment to ensure the absence of disease
pathology. All experiments involved between 3 and 10 animals per group and were
repeated at least twice. All procedures were carried out in accordance with the
protocol established by the Association for the Research in Vision and Ophthal-
mology and were approved by the Animal Care and Use Committee, University of
Bacterial elastolytic activity assay. Bacterial inocula were prepared in DMEM
at a concentration of 1011CFU/ml. Inocula were centrifuged for 5 min at 14,000 ?
g, and 50 ?l of the supernatant was added to Eppendorf tubes containing 10 mg
of elastin Congo red (Elastin Products Company, Owensville, MO) in 1 ml of
sodium phosphate buffer (Na2HPO4, 10 mM, pH 7.0). Known concentrations of
purified elastase (Elastin Products Company, Owensville, MO; Calbiochem,
Gibbstown, NJ) were included to form a standard curve. Samples were incubated
at 37°C for 2 h with constant shaking before centrifugation for 5 min at 5,000 ?
g to remove insoluble substrate. Absorbance of the supernatants was measured
as optical density at 495 nm, and elastase activity was determined by reference to
the standard curve.
Measurement of elastase-mediated SP-D degradation in vivo and in vitro. For
in vivo studies, both ocular surfaces of five anesthetized wild-type Black Swiss
mice were inoculated with 80 ?g/ml of purified elastase in a vehicle of DMEM
containing 4% (vol/vol) glycerol for 1 h. A control group of five anesthetized
mice were inoculated with vehicle only (DMEM with 4% [vol/vol] glycerol).
After 1 h, tear fluid was collected as described previously. The total protein
concentration of each sample was measured using the DC protein assay (Bio-
Rad, Hercules, CA), and equivalent amounts of each sample were resolved on a
10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis precast Tris-HCl
polyacrylamide gel (Bio-Rad, Hercules, CA) under reducing conditions (100 V,
1.5 h). SP-D was then detected by Western immunoblotting as previously de-
scribed using rabbit anti-mouse SP-D antiserum diluted 1:750. For in vitro
studies, recombinant SP-D or murine or human tear fluid was mixed with either
DMEM (vehicle) or 20 ?g/ml of purified elastase suspended in DMEM for 3 h
at 37°C and SP-D was detected as described above. Human tears were collected
from a healthy human volunteer using a 30-?l-volume capillary tube under a
protocol approved by the Committee for the Protection of Human Subjects,
University of California, Berkeley.
Statistical analysis. The statistical significances in numbers of viable bacteria
were compared between two groups nonparametrically using the Mann-Whitney
U test. Accordingly, the number of bacteria recovered is expressed as the median
and upper and lower quartiles. In the figures, asterisks show outliers within 3
interquartile ranges below the first quartile or above the third quartile and circles
show outliers beyond 3 interquartile ranges of the first and third quartiles. Each
panel of Fig. 2A to 4B is representative of independent experiments conducted
on different days. In each instance, data sets were analyzed using the Brown-
Forsythe test, which confirmed that there were no significant differences in the
variance between the two groups compared, thereby allowing use of the Mann-
Whitney U test. P ? 0.05 was considered statistically significant.
P. aeruginosa is efficiently cleared from the healthy ocular
surface. Healthy eyes of wild-type mice cleared both strains of
P. aeruginosa (6206 and PAO1-GFP) efficiently in a time-de-
pendent manner. After inoculation with 107CFU of invasive
strain PAO1-GFP, only 0.00012% of the original inoculum was
detected in the tear fluid after 6 h, and by 12 h no viable
bacteria were recovered from the tears (Fig. 1A). Similar re-
sults were obtained using a larger inoculum of this strain (109
CFU), with only 0.0018% and 0.000038% of the original inoc-
ulum recovered from the tears after 3 h and 14 h, respectively.
Even more efficient clearance was noted for the cytotoxic strain
6206, with only 0.000093% recovered after 3 h (?20-fold lower
than that of PAO1-GFP at the same time point), and no viable
bacteria were recovered after 14 h (Fig. 1A). Similar low per-
centages of bacteria were recovered from ocular homogenates
14 h after inoculation using 109CFU of either strain, showing
that differences in clearance from the tear fluid did not result
from bacterial relocation to other ocular sites. Fluorescein
staining 14 h after inoculation showed that 6206 caused more
superficial damage to the cornea than did PAO1-GFP (Fig.
1B). Nevertheless, 6206 bacteria were cleared more efficiently
from the ocular surface than were PAO1-GFP bacteria and
neither caused disease.
SP-D-deficient mice demonstrate delayed clearance of cyto-
toxic strain 6206. SP-D-deficient mice were used to study the
role of SP-D in clearance of P. aeruginosa from the healthy
ocular surface. Healthy eyes of wild-type and SP-D?/?Black
Swiss mice were inoculated with 109CFU of either 6206 or
PAO1-GFP for ?16 h and were then rechallenged for 3 h with
the same number of bacteria. Preexposure with bacteria was
used as a potential stimulant to upregulate potential innate
defense factors (17, 21, 28), although it is not known if this
occurs in this model. At 3 h after inoculation with 109CFU of
6206, significantly more bacteria (?12-fold) were isolated from
the tear fluid of the SP-D?/?mice than from that of the
wild-type mice (P ? 0.025, Mann-Whitney test) (Fig. 2A).
However, no significant differences were found for invasive
strain PAO1-GFP (Fig. 2B).
SP-D-deficient mice show delayed clearance of the protease
mutant of strain PAO1. Since the data indicated that 6206 was
cleared more efficiently from the ocular surface than was
PAO1-GFP and 6206 is known to express low protease activity
compared to PAO1 (1, 29), we hypothesized that proteases
may compromise clearance. Thus, clearance of PAO1 was
compared to that of an isogenic protease mutant in both wild-
type and SP-D?/?mice. The data showed that significantly
more protease-mutant bacteria remained in the tear fluid of
the SP-D?/?mice than in that of wild-type mice (P ? 0.009,
Mann-Whitney test) (Fig. 3A). No such differences were found
for PAO1 (P ? 0.4662, Mann-Whitney test) (Fig. 3B). In
VOL. 77, 2009 OCULAR SURFACE DEFENSES AGAINST BACTERIAL COLONIZATION2393
wild-type mice, significantly lower numbers of protease-mutant
bacteria than protease-competent PAO1 bacteria were recov-
ered from the tear fluid (?43-fold, P ? 0.016, Mann-Whitney
test) (Fig. 4A). Differences between PAO1 and protease-mu-
tant bacteria were not statistically significant in SP-D?/?mice
(P ? 0.4057, Mann-Whitney test) (Fig. 4B).
Purified P. aeruginosa elastase degrades tear fluid SP-D in
vitro and in vivo. SP-D is known to be cleaved by P. aeruginosa
proteases in vitro and in rat and human bronchoalveolar lavage
fluid into an inactive 35-kDa form (1, 20, 22). Since the data
showed that proteases delayed bacterial clearance and that
SP-D expedites this clearance process in the absence of pro-
teases, we next explored whether degradation of SP-D by P.
aeruginosa proteases could occur in tear fluid, which could
provide a potential mechanism for that effect. In vitro elasto-
lytic assays with typical inocula used in this study (1011CFU/
ml) revealed that PAO1 expressed ?70.4 ?g/ml of elastase
while 6206 expressed only ?5.6 ?g/ml. To avoid other inocu-
lum-related confounding factors (i.e., toxins that might inter-
fere with the assay), purified elastase, rather than bacteria, was
used in this study. Tear fluid collected from wild-type mice was
treated with 20 ?g/ml of purified elastase for 3 h at 37°C. The
same treatment was applied to tear fluid collected from healthy
human volunteers. Control samples were treated with vehicle
(DMEM). Western immunoblot analysis showed the presence
of monomeric SP-D (?43 kDa) in mouse tears and a slightly
larger (?50 kDa) form of SP-D in the human tears (Fig. 5A,
lanes 5 and 7, respectively), which may relate to a form of SP-D
of similar size reported previously by others in human respi-
ratory lavage and amniotic fluid (24). A degraded SP-D frag-
ment of ?35 kDa was observed only in elastase-treated mouse
and human tear fluid samples (Fig. 5A, lanes 6 and 8, respec-
tively) and in elastase-treated recombinant SP-D samples (Fig.
5A, lane 4). These data showed that in the context of tear fluid,
SP-D is susceptible to cleavage by P. aeruginosa elastase. To
determine if tear SP-D was also degraded in vivo, purified
elastase (80 ?g/ml), at levels similar to those present in our
experiments using bacteria, was added to the healthy ocular
FIG. 1. (A) Detection of invasive PAO1-GFP or cytotoxic 6206 P. aeruginosa in the tear fluid of Black Swiss mice after inoculation of healthy
eyes with 107or 109CFU of bacteria. Data were quantified as the means ? standard deviations of viable bacteria recovered from the tear fluid
at 3, 6, 12, or 14 h postinoculation and expressed as a percentage of the initial inoculum. (B) Photographs of representative eyes of Black Swiss
mice stained with fluorescein to observe any superficial disruption of the corneal epithelium 14 h after inoculation with 109CFU of 6206 or
PAO1-GFP. The 6206-treated (but not PAO1-GFP-treated) ocular surface shows superficial epithelial damage.
2394 MUN ET AL.INFECT. IMMUN.
surface of wild-type mice for 1 h. Control eyes were inoculated
with vehicle only. Western immunoblotting of collected tears
showed the presence of monomeric SP-D (?43 kDa) in control
eyes (Fig. 5B, lane 3). In contrast, elastase-treated eyes showed
both monomeric SP-D and a ?35-kDa band of degraded SP-D
(Fig. 5B, lane 4).
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 con-
tribute to this process and that it can be delayed by the expres-
sion 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 mi-
croorganisms entering the eye is likely to be important in the
maintenance of ocular health. Factors that are likely to con-
tribute 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 pa-
thology 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 activ-
ity 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 expres-
sion and retention of P. aeruginosa at the ocular surface could
involve SP-D degradation in vivo by proteases. While P. aerugi-
nosa elastase and protease IV had already been shown to
degrade purified SP-D into an inactive form (1, 20), 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 pro-
tease 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, 28). In addition, proteases could also promote ocular
colonization through other mechanisms. For example, P.
aeruginosa proteases are known to degrade tear immunoglobu-
lins (19), which could compromise their known ocular defense
against infection (23). Whether previously demonstrated roles
for elastase and other proteases in increasing bacterial adher-
ence to the mouse cornea (12), or in invasion and penetration
through epithelia (2, 6), 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
FIG. 2. Clearance of 6206 (A) or PAO1-GFP (B) from the healthy
ocular surface of wild-type versus SP-D?/?mice at 3 h postinoculation
with 109CFU bacteria. Eyes were preexposed to a similar inoculum for
16 h prior to the assay. Data are expressed as the median [lower
quartile:upper quartile] number of viable bacteria recovered from the
tear fluid. Data represent one of three independent experiments (n ?
3 to 5 mice per group).
VOL. 77, 2009 OCULAR SURFACE DEFENSES AGAINST BACTERIAL COLONIZATION 2395
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 func-
tion 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.
FIG. 3. Detection of the P. aeruginosa protease mutant PAO1 lasA lasB aprA (A) or its parent PAO1 (B) in the tear fluid of wild-type or
SP-D?/?mice at 3 h after inoculation with 109CFU of bacteria. Eyes were preexposed to the same inoculum for 16 h prior to the assay. Data are
expressed as the median [lower quartile:upper quartile] number of viable bacteria recovered from the tear fluid. Data represent one of three
independent experiments (n ? 7 to 8 mice per group).
FIG. 4. (A) Detection of viable P. aeruginosa strain PAO1 or its isogenic mutant PAO1 lasA lasB aprA in wild-type murine tear fluid 3 h after
inoculation with 109CFU of bacteria. Eyes were preexposed to the same inoculum for 16 h prior to the assay. (B) Clearance of PAO1 and that
of its isogenic mutant were also compared in SP-D?/?mice. Data are expressed as the median [lower quartile:upper quartile] number of viable
bacteria recovered in the tear fluid. Data represent one of three independent experiments (n ? 5 to 10 mice per group).
2396MUN ET AL.INFECT. IMMUN.
Traditional models for studying bacterial keratitis in which dis-
ease is induced do not allow normal resistance factors to be
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 stud-
ies using this model could facilitate our understanding of the
circumstances surrounding resistance and susceptibility to infec-
prevention of infection of the eye and of other sites.
This work was supported by NIH research grant R01-EY11221
(S.M.J.F.) and by unrestricted gift funds from Allergan Pharmaceuti-
cals (S.M.J.F.) and also by NIH HL24075 and HL58047 (S.H.).
S. M. J. Fleiszig and D. J. Evans are coinventors on a U.S. patent,
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FIG. 5. (A) Western immunoblot showing the effects of purified
elastase treatment (20 ?g/ml, 3 h) on murine or human tears and
recombinant SP-D in vitro. Under reducing conditions, the SP-D an-
tibody detected degraded 35-kDa product in each of the elastase-
treated samples but not in vehicle (DMEM) controls (lanes 4, 6, and
8). (B) Western immunoblot of murine tear fluid collected from
healthy eyes of wild-type Black Swiss mice 1 h after inoculation with
purified elastase (80 ?g/ml) in vivo (tears were pooled from five mice;
see Materials and Methods). Under reducing conditions, monomeric
SP-D (?43 kDa) was detected in the murine tear fluid (lanes 3 and 4).
The SP-D degradation product at 35 kDa was observed in eyes treated
with elastase (lane 4) but not in eyes treated with vehicle (DMEM with
4% glycerol) (lane 3). The rabbit anti-mouse SP-D antibody did not
react with vehicle (lane 1) or purified elastase (80 ?g/ml) (lane 2) when
used alone. Lanes 5 and 6 were loaded with recombinant murine SP-D
at different concentrations (positive control).
VOL. 77, 2009OCULAR SURFACE DEFENSES AGAINST BACTERIAL COLONIZATION2397
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2398 MUN ET AL.INFECT. IMMUN.