Surfactant Protein D Is Present in Human Tear Fluid and the Cornea and Inhibits Epithelial Cell Invasion by Pseudomonas aeruginosa

ABSTRACT We have previously shown that human tear fluid protects corneal epithelial cells against Pseudomonas aeruginosa in vitro and in vivo and that protection does not depend upon tear bacteriostatic activity. We sought to identify the responsible tear component(s). The hypothesis tested was that collectins (collagenous calcium-dependent lectins) were involved. Reflex tear fluid was collected from healthy human subjects and examined for collectin content by enzyme-linked immunosorbent assay (ELISA) and Western blot with antibody against surfactant protein D (SP-D), SP-A, or mannose-binding lectin (MBL). SP-D, but not SP-A or MBL, was detected by ELISA of human reflex tear fluid. Western blot analysis of whole tears and of high-performance liquid chromatography tear fractions confirmed the presence of SP-D, most of which eluted in the same fraction as immunoglobulin A. SP-D tear concentrations were calculated at ∼2 to 5 μg/ml. Depletion of SP-D with mannan-conjugated Sepharose or anti-SP-D antibody reduced the protective effect of tears against P. aeruginosa invasion. Recombinant human or mouse SP-D used alone reduced P. aeruginosa invasion of epithelial cells without detectable bacteriostatic activity or bacterial aggregation. Immunofluorescence microscopy revealed SP-D antibody labeling throughout the corneal epithelium of normal, but not gene-targeted SP-D knockout mice. SP-D was also detected in vitro in cultured human and mouse corneal epithelial cells. In conclusion, SP-D is present in human tear fluid and in human and mouse corneal epithelia. SP-D is involved in human tear fluid protection against P. aeruginosa invasion. Whether SP-D plays other roles in the regulation of other innate or adaptive immune responses at the ocular surface, as it does in the airways, remains to be explored.

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Modification of Pseudomonas aeruginosa Interactions with Corneal Epithelial Cells by Human Tear Fluid

Infection and Immunity ◽

10.1128/iai.71.7.3866-3874.2003 ◽

2003 ◽

Vol 71 (7) ◽

pp. 3866-3874 ◽

Cited By ~ 45

Author(s):

Suzanne M. J. Fleiszig ◽

Mary S. F. Kwong ◽

David J. Evans

Keyword(s):

Pseudomonas Aeruginosa ◽

Epithelial Cells ◽

Bacterial Motility ◽

Tear Fluid ◽

Blue Staining ◽

Bacteriostatic Activity ◽

Corneal Epithelial Cells ◽

Corneal Epithelial ◽

Swimming Motility

ABSTRACT Both cytotoxic and invasive strains of Pseudomonas aeruginosa can damage corneal epithelial cells in vitro, but neither can infect healthy corneas in vivo. We tested the hypothesis that whole human tear fluid can protect corneal epithelia against P. aeruginosa virulence mechanisms. Cultured corneal epithelial cells were inoculated with 106 CFU of one of 10 strains of P. aeruginosa (five cytotoxic, five invasive)/ml with or without reflex tear fluid collected from the conjunctival sacs of human volunteers. Cytotoxicity was assessed by observation of trypan blue staining and measurement of lactate dehydrogenase release; invasion was quantified by using gentamicin survival assays. Tear fluid retarded growth of only 50% of the P. aeruginosa strains (three of five invasive strains, two of five cytotoxic strains) yet protected corneal cells against invasion by or cytotoxicity of 9 of 10 strains. The only strain resistant to the tear cytoprotective effects was susceptible to tear bacteriostatic activity. Dilution of tear fluid threefold significantly reduced cytoprotection, while bacteriostatic activity prevailed with dilutions beyond 100-fold. Sulfacetamide (1 mg/ml) with bacteriostatic activity matching that of tear fluid was less cytoprotective than tear fluid (80% protection with tear fluid, 48% with sulfacetamide). Video microscopy revealed bacterial chain formation in both tear fluid and sulfacetamide, but tear fluid also blocked bacterial swimming motility. After prolonged tear contact, bacteria regained normal growth rates, swimming motility, and cytotoxic activity, suggesting a breakdown of protective tear factors. Boiled tear fluid lost bacteriostatic activity and effects on bacterial motility but retained cytoprotective function. These results suggest that human tear fluid can protect corneal epithelial cells against P. aeruginosa virulence mechanisms in a manner not dependent upon bacteriostatic activity or effects on bacterial motility. Whether overlapping tear film components are involved in these defense functions is to be determined.

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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

Infection and Immunity ◽

10.1128/iai.00173-09 ◽

2009 ◽

Vol 77 (6) ◽

pp. 2392-2398 ◽

Cited By ~ 38

Author(s):

James J. Mun ◽

Connie Tam ◽

David Kowbel ◽

Samuel Hawgood ◽

Mitchell J. Barnett ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Ocular Surface ◽

Surfactant Protein ◽

Tear Fluid ◽

Bacterial Invasion ◽

Surfactant Protein D ◽

Infection Model ◽

Strain Pao1 ◽

Corneal Epithelial

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 107 or 109 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 109 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.

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Expression of surfactant protein D in human corneal epithelial cells is upregulated byPseudomonas aeruginosa

FEMS Immunology & Medical Microbiology ◽

10.1111/j.1574-695x.2008.00461.x ◽

2008 ◽

Vol 54 (2) ◽

pp. 177-184 ◽

Cited By ~ 13

Author(s):

Minjian Ni ◽

Connie Tam ◽

Amrisha Verma ◽

Reuben Ramphal ◽

Samuel Hawgood ◽

...

Keyword(s):

Epithelial Cells ◽

Surfactant Protein ◽

Surfactant Protein D ◽

Corneal Epithelial Cells ◽

Corneal Epithelial ◽

Human Corneal Epithelial Cells

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Exposure of human corneal epithelial cells to contact lenses in vitro suppresses the upregulation of human β-defensin-2 in response to antigens of Pseudomonas aeruginosa

Experimental Eye Research ◽

10.1016/j.exer.2007.04.001 ◽

2007 ◽

Vol 85 (1) ◽

pp. 142-153 ◽

Cited By ~ 24

Author(s):

I.A. Maltseva ◽

S.M.J. Fleiszig ◽

D.J. Evans ◽

S. Kerr ◽

S.S. Sidhu ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Epithelial Cells ◽

Contact Lenses ◽

Corneal Epithelial Cells ◽

Corneal Epithelial ◽

Human Corneal Epithelial Cells

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Pseudomonas aeruginosa invasion of and multiplication within corneal epithelial cells in vitro.

Infection and Immunity ◽

10.1128/iai.63.10.4072-4077.1995 ◽

1995 ◽

Vol 63 (10) ◽

pp. 4072-4077 ◽

Cited By ~ 111

Author(s):

S M Fleiszig ◽

T S Zaidi ◽

G B Pier

Keyword(s):

Pseudomonas Aeruginosa ◽

Epithelial Cells ◽

Corneal Epithelial Cells ◽

Corneal Epithelial

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FlhA, a Component of the Flagellum Assembly Apparatus of Pseudomonas aeruginosa, Plays a Role in Internalization by Corneal Epithelial Cells

Infection and Immunity ◽

10.1128/iai.69.8.4931-4937.2001 ◽

2001 ◽

Vol 69 (8) ◽

pp. 4931-4937 ◽

Cited By ~ 55

Author(s):

Suzanne M. J. Fleiszig ◽

Shiwani K. Arora ◽

Rajana Van ◽

Reuben Ramphal

Keyword(s):

Pseudomonas Aeruginosa ◽

Epithelial Cell ◽

Epithelial Cells ◽

Vector Control ◽

Plasmid Vector ◽

Wild Type ◽

Corneal Epithelial Cells ◽

Corneal Epithelial

ABSTRACT Pseudomonas aeruginosa invades various epithelial cell types in vitro and in vivo. The P. aeruginosa genome possesses a gene (flhA) which encodes a protein that is believed to be part of the export apparatus for flagellum assembly and which is homologous to invA of Salmonella spp. Because invA is required for invasion ofSalmonella spp., a role for flhA in P. aeruginosa invasion was explored using cultured rabbit corneal epithelial cells. An flhA mutant of P. aeruginosa strain PAO1 was constructed and was shown to be nonmotile. Complementation with flhA in transrestored motility. Corneal cells were infected for 3 h with the wild type (PAO1), the flhA mutant, the flhA mutant complemented with flhA in trans, anflhA mutant containing the plasmid vector control, or anfliC mutant (nonmotile mutant control). Invasion was quantified by amikacin exclusion assays. Both the flhA and the fliC mutants invaded at a lower level than the wild-type strain did, suggesting that both fliC andflhA played roles in invasion. However, loss of motility was not sufficient to explain the reduced invasion by flhAmutants, since centrifugation of bacteria onto cells did not restore invasion to wild-type levels. Unexpectedly, the flhA mutant adhered significantly better to corneal epithelial cells than wild-type bacteria or the fliC mutant did. The percentage of adherent bacteria that invaded was reduced by ∼80% for the flhAmutant and ∼50% for the fliC mutant, showing that only part of the role of flhA in invasion involvesfliC. Invasion was restored by complementing theflhA mutant with flhA in trans but not by the plasmid vector control. Intracellular survival assays, in which intracellular bacteria were enumerated after continued incubation in the presence of antibiotics, showed that although flhAand fliC mutants had a reduced capacity for epithelial cell entry, they were not defective in their ability to survive within those cells after entry. These results suggest that the flagellum assembly type III secretion system plays a role in P. aeruginosainvasion of epithelial cells. Since the flhA mutants were not defective in their ability to adhere to corneal epithelial cells, to retain viability at the cell surface, or to survive inside epithelial cells after entry, the role of flhA in invasion of epithelial cells is likely to occur during the process of bacterial internalization.

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