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

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 10 6 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.Pseudomonas aeruginosa is a gram-negative opportunistic bacterium capable of causing severe corneal infection. There are at least two types of P. aeruginosa isolated from infected corneas; those that invade corneal epithelial cells (5) and those that cause ExoU-dependent cytotoxicity from an extracellular location (3,4,8,9). Both invasive and cytotoxic strains can target surface cells on intact corneas in vitro (7, 10), yet neither can infect healthy corneas in vivo, suggesting that protective factors are absent in in vitro assays. The most obvious of these are tear fluid and the mechanical effects of blinking. Shear stresses generated by blinking motions of the eyelids reduce, but do not eliminate, P. aeruginosa cytotoxicity (C. Lakkis Defensins have bactericidal activity against a wide variety of organisms, including gram-negative bacteria, and have been found in small but detectable quantities in tears (13). Other tear components can alter behavior of P. aeruginosa; e.g....

Extensive characterization of human tear fluid collected using different techniques unravels the presence of novel lipid amphiphiles

“…In line with this, lyso-PC has been reported to increase the bactericidal activity of neutrophils ( 36 ); while lyso-PEs were shown to possess antifungal and antibacterial activity in the housefl y ( 37 ). Therefore, the physiological signifi cance of the tear lysophospholipidome seems to coincide with that of the tear proteome, which comprises a predominance of metabolites with microbicidal properties ( 38 ).…”

Lipidomic analysis of human tear fluid reveals structure-specific lipid alterations in dry eye syndrome

“…1B ). Also, the propensity of GluCers to aggregate forming highly ordered gel domains has been shown to increase the order of fl uid membranes ( 21 ). In addition, the trapping Fig.…”

Pattern recognition receptors in microbial keratitis

“…Although many P. aeruginosa strains grow readily in undiluted tear fluid despite its antimicrobial components such as lactoferrin and lysozyme, 75,76 tear fluid can still protect corneal epithelial cells against them. 77,78 This is thought to be due to the tear fluid acting directly upon corneal epithelial cells to make them more resistant to P. aeruginosa invasion and cytotoxicity. 74 Should conditions enable P. aeruginosa to breach the corneal epithelium, its lipopolysaccharide (LPS) and Figure 1 Assembly and activation of the inflammasome.…”