Pseudomonas aeruginosa is an opportunistic bacterial pathogen implicated in a variety of devastating conditions. Its flexibility as a pathogen is attributed to a myriad of virulence factors and regulatory elements that respond to prevailing environmental conditions. ExoS and ExoT are type III secreted effector proteins, regulated by the transcriptional activator ExsA, that can inhibit invasion of epithelial cells by cytotoxic strains of P. aeruginosa. This study sought to understand why invasive strains, which can secrete both ExoS and ExoT, still invade epithelial cells. The results showed that LasA and elastase (LasB), which are regulated by the Las and Rhl quorum-sensing systems, modulated P. aeruginosa invasion. Mutation of lasA and/or lasB reduced P. aeruginosa invasion, which was not fully restored by extracellularly added LasB, P. aeruginosa conditioned medium containing LasA and LasB, or EGTA pretreatment of cells. This indicated that protease effects on invasion involved factors additional to tight junction disruption and subsequent alterations to cell polarity. Upon mutation of lasA and/or lasB, steady-state levels of ExoS and ExoT were increased in culture medium of P. aeruginosa grown under conditions stimulatory for these toxins. The increase in ExoS was significantly correlated with reduced invasion. In vitro experiments showed that purified LasB degraded recombinant ExoS. Taken together, these studies suggest a mechanism by which invasive strains can synthesize inhibitors of invasion, ExoS and ExoT, yet still invade epithelial cells. By this mechanism, LasA and LasB decrease the levels of the toxins directly or indirectly, and thus reduce inhibition of invasion.

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....

Pseudomonas aeruginosa keratitis is an acute sight-threatening infection. We previously reported that human tear fluid could protect individual human corneal epithelial cells in vitro against invasion by and cytotoxicity due to clinical and laboratory isolates of P. aeruginosa and that the protective mechanism was independent of bacteriostatic activity. In the present study, we examined the effects of human tear fluid in vivo. Tears were collected from healthy human volunteers and were studied in vivo in mice. The effects on the virulence of both invasive and cytotoxic clinical isolates of P. aeruginosa were examined. Tear fluid was found to reduce the severity of disease when corneas were challenged with cytotoxic bacteria immediately after scratch injury, and it completely protected against susceptibility to infection by a cytotoxic strain in a model in which corneas were infected during the healing process 6 h after scratching. Visible protection correlated with the inhibition of bacterial colonization 1, 4, and 48 h postinoculation. Tear fluid also significantly reduced the severity of infections caused by invasive P. aeruginosa in the 6-h-healing model. This result also coincided with significantly reduced bacterial colonization at 48 h. In vitro, human tear fluid significantly reduced the ability of invasive and cytotoxic bacteria to translocate across corneal epithelia and increased transepithelial resistance with or without bacterial inoculation. These data show that human tear fluid can protect against P. aeruginosa corneal infection in vivo and that the mechanism likely involves enhanced epithelial barrier function in addition to protection of individual epithelial cells against bacterial internalization and cytotoxicity.Pseudomonas aeruginosa can cause microbial keratitis associated with soft contact lens wear or corneal injury (4,14). Clinical and laboratory isolates of this opportunistic bacterial pathogen are capable of using multiple virulence strategies to cause disease, including adherence to and invasion of epithelial cells, intoxication of epithelial cells and other cell types using type III secretion mechanisms, and extracellular secretion of potentially damaging toxins, enzymes, and proteases (1,8,9,12,15,23,24). Despite the presence of these traits and a plethora of genes devoted to virulence regulation, resistance to antimicrobial agents, and environmental adaptation, P. aeruginosa cannot infect the healthy cornea in vivo (22). Our longterm research goals are to understand the mechanisms involved in corneal resistance to infection and the circumstances that compromise resistance, with a view toward developing preventive and therapeutic measures for infections of the cornea and other sites.We have shown previously that corneal epithelial cells in vitro are vulnerable to invasion by P. aeruginosa and to the type III secretion-dependent cytotoxicity of P. aeruginosa, irrespective of whether they are in cell culture or on the surface of whole eyeballs (5, 7, 10). Thus, we have been working tow...

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P. aeruginosa bacteria bind more efficiently to closed-eye tears than to open-eye tears. The mechanism by which tears bind bacteria and protect against invasion does not require SG/mucin, as this fraction of closed-eye tears does not contain either activity.