December 2002
Volume 43, Issue 12
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Cornea  |   December 2002
Pseudomonas aeruginosa Binds to Extracellular Matrix Deposited by Human Corneal Epithelial Cells
Author Affiliations
  • Miechia A. Esco
    From the Department of Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, Michigan.
  • Linda D. Hazlett
    From the Department of Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, Michigan.
  • Michelle Kurpakus-Wheater
    From the Department of Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, Michigan.
Investigative Ophthalmology & Visual Science December 2002, Vol.43, 3654-3659. doi:
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      Miechia A. Esco, Linda D. Hazlett, Michelle Kurpakus-Wheater; Pseudomonas aeruginosa Binds to Extracellular Matrix Deposited by Human Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2002;43(12):3654-3659.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To measure the effect of extracellular matrix substrate, pH, and O2 on Pseudomonas aeruginosa binding.

methods. Extracellular matrix substrates were prepared from human corneal epithelial cells cultured in 2% or 20% O2. P. aeruginosa strains ATCC 19660 or PAO1 (suspended in pH 7.0 or 7.5 buffer) were cultured on extracellular matrix substrates in 2% or 20% O2. The mean number of adherent bacteria per counted per field ± SEM (n = 15) was determined for combinations of bacteria, extracellular matrix substrate, pH, and O2. Binding in the presence of antibodies directed against laminin-5 was also measured.

results. Extracellular matrix substrates produced by cells cultured in 20% O2, combined with an environment of pH 7.0, provided the least favorable conditions for binding of strain 19660. In contrast, extracellular matrix substrates produced by cells cultured in 2% O2, combined with an environment of pH 7.0, provided the most favorable conditions for binding of strain 19660. Binding of PAO1, however, as a function of extracellular matrix substrate and pH, did not similarly compare with binding of strain 19660. Antibodies against laminin-5 chains served to increase the number of strain 19660 bacteria bound to extracellular matrix substrates compared with the control.

conclusions. The extracellular matrix secreted by hypoxic corneal epithelial cells is a substrate for binding of P. aeruginosa. Results in previous studies have shown that hypoxic extracellular matrix contains less laminin-5 protein than normoxic matrix. The antibody studies in this report suggest that the decrease in laminin-5 content in hypoxic matrix, relative to matrix secreted by normoxic corneal epithelium, may be responsible for increased bacterial adhesion.

An intact layer of epithelium firmly attached to the underlying basement membrane creates a barrier that is essential for the cornea to maintain proper vision. The epithelial barrier, along with elements provided by the tear film, plays a role in preventing bacterial binding, colonization, and infectivity of the corneal surface and stromal spread of the bacteria. Microbial infections that disrupt the integrity of corneal epithelial cell–extracellular matrix, if left untreated, will result in cellular loss, inflammatory reactions, and subsequent decreased visual acuity. 
Pseudomonas aeruginosa is a virulent opportunistic pathogen responsible for a particularly destructive corneal disease. 1 2 3 4 5 It is opportunistic and does not infect tissues unless normal host–defense mechanisms are impaired. The bacteria does not readily adhere to or infect the intact corneal epithelium. 6 7 Experimental evidence confirms that P. aeruginosa does not bind to intact adult mouse cornea. Adhesion is initiated only in experimentally injured tissue. 6 8 9 In clinical settings P. aeruginosa keratitis may be preceded by corneal injury 1 or perturbations due to contact lens usage. Injury or experimental scarification may alter the physiochemical state of the epithelial cell membrane or expose cell-surface binding sites, to more readily allow bacterial ligands to interact with ocular binding sites. Scanning electron microscopy of wounded corneas has demonstrated that P. aeruginosa adheres to injured epithelium and bare stroma along the edges of a linear corneal abrasion, but not to the normal-appearing regions of the cornea. 6 11  
Depending on severity, wounding of tissue may expose the basement membrane, and indeed in studies using a debridement model of epithelial cells that leaves the basement membrane intact, more bacteria adhered to surfaces of wounded areas than to unwounded corneas. 7 However, any combination of environmental factors that disrupt normal cell structure or function may also result in denudation of epithelial cells and subsequent exposure of the basement membrane, potentially providing a substrate for bacterial adherence. 12 13  
One such environmental factor that may alter the integrity of the epithelial cell barrier is hypoxia, or reduced oxygen levels. Acute hypoxia has been shown to alter significantly the levels of laminin-5, a major extracellular matrix (basement membrane) adhesion protein secreted by human keratinocytes in culture. 14 Our previous study has shown that hypoxia also decreases the level of laminin-5 secreted into the extracellular matrix deposited by human corneal epithelial cell (HCECs) in culture. 15 Because laminin-5 is essential to epithelial cell–extracellular matrix integrity, we have suggested based on previous studies that reduced laminin-5 levels in corneal extracellular matrix in culture and in basement membrane in the intact eye may serve to disrupt homeostatic cell–matrix adhesion and thus upregulate apoptotic pathways. 15 Hypoxia therefore, may provide a catalyst for basement membrane exposure in intact cornea through apoptotic loss of overlying epithelium. 
The detrimental effects of hypoxia on the cornea are not limited to disruption of cell–matrix interactions. Biochemical changes within and outside the cell have also been observed. For example, the in vivo exposure of human eyes to 95% nitrogen-5% CO2 for 1 hour not only creates an obvious hypoxic environment, but causes the pH of the corneal environment to decline to approximately the same level (pH 7.16 ± 0.05) that occurs in the closed eye of the contact lens wearer. 16 A relationship between pH and bacterial binding has been demonstrated by Chen et al. 17 who showed that alterations in environmental pH can significantly affect adherence of P. aeruginosa to cultured human corneal epithelium. 
We hypothesize that the protein composition of the extracellular matrix secreted by hypoxic corneal epithelial cells, coupled with the slight acidosis characteristic of hypoxia, combine to create an environment particularly conducive to adhesion of P. aeruginosa. The present study was designed to test this hypothesis by examining the synergistic effects of the extracellular matrix produced by HCECs, levels of environmental oxygen, and pH levels on P. aeruginosa binding. 
Methods
Antibodies
Monoclonal antibody (mAb) 17 specific for the 145-kDa β3 chain of laminin-5 was purchased from Transduction Laboratories (Lexington, KY). mAb D4B5 was purchased from Chemicon International, Inc. (Temecula, CA). This antibody recognizes both the unprocessed (155-kDa) and processed (105-kDa) forms of the γ2 chain of laminin-5. mAb P3H92, a function-inhibiting antibody to laminin-5, was also purchased from Chemicon. 
Preparation of Extracellular Matrix Substrates
HCECs were purchased from Cascade Biologics (Portland, OR) as cryopreserved secondary cultures obtained from a 17-year-old male donor. HCECs are not a transformed cell line. The transfected HCEC line 10.014 pRSV-T (HCET) was a generous gift of Sherry Ward (The Gillette Company, Gillette Medical Evaluation Laboratories, Boston, MA). In separate experiments, HCECs (at passage 3) or HCETs (at passage 16) were plated into wells of a six-well plate at an initial density of 96,000 cells/well. HCECs were cultured in serum-free culture medium (EpiLife; Cascade Biologics) supplemented with 0.06 M calcium chloride and human corneal growth supplement, as recommended by the manufacturer. HCETs were cultured in serum-free keratinocyte growth medium (KGM; BioWhittaker, Walkersville, MD). One group of plates was maintained in normoxic conditions by culture in a conventional humidified tissue culture incubator at 37°C, 20% O2, 5% CO2, and 75% N2. A second group of plates was maintained at 37°C in hypoxic conditions by culture in an atmosphere of 2% O2, 4.9% CO2, and 93.1% N2 using a humidified environmental chamber (Coy Laboratory Products, Ann Arbor, MI). An oxygen analyzer was used to maintain the O2 level at 2% by regulating the flow of a calibrated mixture of 95% N2 and 5% CO2 into the chamber. 
After the HCETs or HCECs reached confluence, the epithelial cell layer was lysed by a 10-minute treatment with sterile 0.1 N ammonium hydroxide. 18 The extracellular matrix substrate (ECMS) remaining on the culture dish was washed extensively with sterile phosphate-buffered saline to remove all cell debris. The ECMS prepared from HCECs or HCETs cultured in 20% O2 is termed normoxic matrix, and represents the control condition. The ECMS prepared from HCECs or HCETs cultured in 2% O2 is termed hypoxic matrix, and represents the experimental condition. Before application of the bacteria, disks of approximately 1 cm in diameter were prepared from six-well culture plates as a substrate for the subsequent bacterial adhesion assay and quantitative measurements by scanning electron microscopy. 
Bacterial Strains and Preparation of Inoculum
Both P. aeruginosa cytotoxic strain 19660 from the American Type Culture Collection (ATCC, Rockville, MD) and invasive strain PAO1 (Pseudomonas Genetic Stock Center, East Carolina University School of Medicine, Greenville, NC) were used for adhesion assays. Each strain was used in separate experiments. A loopful of culture was retrieved from a tryptic soy broth (TSB)-peptone slant, TSB-peptone medium was inoculated, and the cultures were incubated at 37°C for 18 hours to an optic density at 540 nm (OD540) of approximately 1.6. The cultures were pelleted by centrifugation at 6000g for 10 minutes at 15°C, washed with sterile saline (0.85% NaCl, pH 7.2) and resuspended in D-PBS+ (phosphate-buffered saline containing 0.9 mM Ca2+ and 0.5 mM Mg2+) adjusted to pH 7.0 or 7.5. 17 When bacteria suspended in pH 7.5 are used, it is referred to as binding assay pH 7.5. pH 7.5 represents the control pH condition. Similarly, when bacteria suspended in pH 7.0 are used, it is referred to as binding assay pH 7.0. pH 7.0 represents the experimental pH condition and is a model of the acidosis that often accompanies hypoxia. 
Bacterial Adhesion Assay
A 10-μL aliquot of strain 19660 containing 1.0 × 107 colony-forming units (CFU) suspended in D-PBS+ at pH 7.0 or 7.5 was placed on the plastic disks of hypoxic or normoxic matrix. Similarly, a 10-μL aliquot of PAO1 (1.0 × 107 CFU) suspended in D-PBS+ at pH 7.0 or 7.5 was placed on hypoxic or normoxic matrix. Bacteria were placed on matrix substrates and incubated for 1 hour at 37°C in the hypoxic chamber (2% O2 assay conditions). In separate but concurrently run experiments, bacteria placed on matrix substrates were also incubated for 1 hour at 37°C in the conventional tissue culture incubator (20% O2 assay conditions). One hour was chosen as the assay time point, because it allows sufficient time for bacteria in the inoculum to bind to the matrix. Between 2 and 6 hours, binding does not increase enough to alter experimental results significantly. After 6 to 8 hours, however, it becomes difficult to control for the increased number of bacteria due to replication of the original inoculum or to sources of contamination. 19  
Scanning Electron Microscopy
After the 1-hour incubation, matrix substrates were washed in 0.1 M phosphate buffer (pH 7.4) to remove any nonadherent bacteria. Matrix substrates were processed for scanning electron microscopy by fixation for 1.5 hours in a 1:1:1 solution of 2% aqueous osmium tetroxide (OsO4), 2.5% glutaraldehyde, and 0.1 M phosphate buffer (pH 7.4). After fixation, the substrates were washed in phosphate buffer and dehydrated in a graded series of ethanols. 
The specimens were critical point dried with CO2 used as a transition agent, mounted on specimen stubs with conductive silver paint, and gold coated to approximately a 30-nm thickness. Specimens were examined by scanning electron microscope (840A; JEOL, Peabody, MA). For each experimental condition, five randomly selected fields per disc were photographed at a magnification of ×6000. Adherent bacteria were quantitated by counting three fields of 80 mm2 on each of the five micrographs generated for each experimental condition (n = 15 for calculation of the mean ± SEM and statistical analysis). 
Antibody Treatment
mAbs 17, D4B5, and P3H92 were separately diluted in PBS to a concentration of 50 or 100 μg/mL. In one experiment, each mAb was incubated with normoxic matrix for 1 hour before application of 1.0 × 107 CFU strain 19660 (suspended in D-PBS+ at pH 7.5). In a second experiment, each mAb was mixed with 1.0 × 107 CFU strain 19660 (suspended in D-PBS+ at pH 7.5) for 1 hour before application on normoxic matrix. In both experiments, the bacteria were incubated in an atmosphere of 20% O2 during the assay. As a control, experiments were run similarly using an irrelevant isotype-matched mAb at a concentration of 100 μg/mL. 
The results of the bacterial adhesion assay with or without the addition of mAb are expressed as the mean number of bacteria per counted field ± SEM as a function of the type of bacteria, ECMS, pH of the bacterial suspension, and % O2 available to the bacteria during the assay. The data were analyzed with unpaired Student’s t-test with a two-tailed hypothesis and P ≤ 0.05. 
Results
Binding of P. aeruginosa Strains ATCC 19660 and PAO1 to Extracellular Matrix Substrates
To determine potential synergistic effects of the ECMSs deposited by hypoxic corneal epithelium, the acidosis characteristic of hypoxia, and altered levels of oxygen on bacterial adhesion, an assay was conducted with a scanning electron microscopy protocol. The results of the adhesion assay were analyzed as a function of the ECMS and binding assay pH condition. The cellular source of the matrix and the amount of oxygen provided to the bacteria during the adhesion assay were constants in the analyses and are presented as such in Figures 1 and 2
The binding characteristics of P. aeruginosa strain 19660 were consistent under the experimental conditions chosen for this study, regardless of the cellular source of the ECMS or the amount of oxygen provided to the bacteria during the adhesion assay (Figs. 1 and 2) . Based on the experimental outcome, we arbitrarily defined bacterial adhesion to normoxic matrix and binding assay pH 7.0 as “low” and adhesion to hypoxic matrix and binding assay pH 7.0 as “high.” By our arbitrary classifications, adhesion to either matrix in binding assay pH 7.5 could in general be considered “moderate.” Statistical comparison of strain 19660 binding was performed only between the defined “low” and “high” conditions of the assay. This type of analysis revealed that the conditions of hypoxic matrix and binding assay pH 7.0 resulted in a significantly higher adhesion of strain 19660, compared with normoxic matrix and binding assay pH 7.0 (P < 0.05 for all comparisons shown in Figs. 1 and 2 , probabilities are shown in the figure legends). 
We did not observe adhesion characteristics of strain 19660 with PAO1, as a direct function of ECMS and binding assay pH. When PAO1 was exposed to 20% O2 during the adhesion assay the highest level of binding was achieved on hypoxic matrix and binding assay pH 7.5 (Fig. 1) . This was not different from binding to normoxic matrix and binding assay pH 7.0, but was significantly higher than adhesion to normoxic matrix and binding assay pH 7.5 (P ≤ 0.02 for comparisons in Fig. 1 ; exact values are provided in the figure legend). 
When PAO1 was exposed to 2% O2 during the incubation period, binding was generally higher on hypoxic matrix compared with normoxic matrix (Fig. 2) . However, no pattern with respect to binding assay pH on the effect of PAO1 adhesion to ECMSs could be discerned. For example, when analyzing hypoxic matrix derived from HCECs, adhesion of PAO1 was significantly higher in binding assay pH 7.5 (P = 0.0009). However, for hypoxic matrix derived from HCETs, binding of PAO1 was significantly higher at pH 7.0 (P = 0.0330). 
In general, the total number of bacteria of both strain 19660 and PAO1 adherent to ECMSs was greater in 20% environmental O2 than in 2% environmental O2. We do not believe, however, that this difference in binding was due to the hypoxia-mediated death of the bacteria during the time of the adhesion assay. P. aeruginosa can generate adenosine triphosphate (ATP) and thrive in an anaerobic environment, if arginine is available. 20 Arginine in sufficient concentration could be derived from proteolytic degradation of the proteins found in the ECMSs to allow the bacteria to survive the 1-hour incubation required to complete the adhesion assay. 
Effect of Antibodies on the Binding of P. aeruginosa ATCC 19660 to Normoxic Matrix
We have demonstrated that levels of laminin-5 in the ECMS deposited by HCECs in culture are altered by hypoxia. 15 Because bacterial binding to hypoxic matrix was increased compared with binding to normoxic matrix, we hypothesize that laminin-5 may play a role in adhesion by actually inhibiting such binding. Therefore, to gain more information regarding the potential role of laminin-5 in mediating binding of P. aeruginosa to ECMSs, we blocked or inhibited the interactions of laminin-5-bacteria by using mAb to the β3 and γ2 chains of laminin-5 or a function-inhibiting antibody to laminin-5 (Fig. 3) . For mAb 19562 to laminin-5 γ2 chain and function-blocking mAb P3H92, bacterial adhesion was enhanced in an antibody-concentration dependent manner compared with binding in the presence of an irrelevant mAb control (exact probabilities are given in the legend to Fig. 3 ). Incubation of the normoxic matrix with mAb enhanced bacterial adhesion to a higher degree than did mixing the mAb with the bacteria. Significantly more bacterial binding was similarly noted in the presence of mAb 17 to the β3 chain of laminin-5, regardless of whether the antibodies were incubated on normoxic matrix before the assay or incubated with the bacteria. However, in the case of mAb to the β3 chain, more binding was observed when the antibodies and bacteria were mixed. 
Discussion
Opportunistic pathogens such as P. aeruginosa can readily adhere to basement membrane components, including fibronectin, collagen type IV, and laminin. 21 22 23 24 P. aeruginosa binds readily to extracellular matrix exposed as a result of tissue damage in patients with cystic fibrosis 25 26 27 or after bronchial infection. 13 In vitro studies using respiratory epithelium also demonstrate the ability of P. aeruginosa to bind to ECMSs. 21 28 The studies in this report extend previous observations by examining binding of P. aeruginosa to corneal epithelial cell extracellular matrix. The present study is novel, in that this is the first report of the effects of altered extracellular matrix composition, resulting from hypoxia, on bacterial binding. 
For P. aeruginosa strains 19660 and PAO1, we observed enhanced binding on ECMSs produced by hypoxic HCECs. In general, strain 19660 demonstrated more significant differences in binding characteristics when the adhesion assay was performed at pH 7.0. In contrast, PAO1 showed significant differences in binding characteristics when the adhesion assay was performed at pH 7.5. A study by Chen et al. 17 showed that there were no differences in the binding of either strain 19660 or PAO1 to the enucleated mouse eye when bacteria were suspended in buffer at pH 7.0 or 7.5. Their study, however, was performed on freshly enucleated and intact eyes, and thus examined “normal” epithelium and basement membrane. The present study attempted to determine the synergistic effects of ECMSs, pH, and concentration of O2—best performed in an in vitro model. From these in vitro studies we conclude that when the binding substrate (i.e., extracellular matrix in cultured cells or basement membrane in intact cornea) and O2 levels are altered from “normal” conditions, pH can differentially affect the ability of strain 19660 and PAO1 to bind to the cornea. Our analyses of bacterial adhesion were conducted after a 1-hour incubation period, because longer incubation times can be complicated by bacterial proliferation or cross-contamination. We cannot speculate on whether increased adhesion times would have affected the outcome of our study. 
The extracellular matrix deposited by corneal epithelial cells is a complex of collagens, laminins, and other glycoproteins. Although we have not completed an exhaustive analysis of the protein composition of corneal epithelial cell matrix, we have demonstrated that less laminin-5 is incorporated in hypoxic matrix than in normoxic matrix. 15 This relative decrease in laminin-5 content in hypoxic matrix, as observed in Western blot analysis, occurs after at least 3 days, usually between 5 and 7 days, of exposure to hypoxia. The dual observations of less laminin-5 protein and increased bacterial binding to hypoxic matrix lead to the conclusion that the presence of laminin-5 in extracellular matrix may actually be inhibitory to adhesion of P. aeruginosa. This notion is supported by the antibody data, which demonstrate that inhibiting bacteria–laminin-5 interactions serves to enhance binding to normoxic matrix. 
Our hypothesis regarding the relationship between hypoxia, apoptosis, laminin-5 content, and bacterial adhesion is summarized in the schematic diagram shown in Figure 4 . As a consequence of hypoxia (3 to 7 days’ exposure), less laminin-5 is produced by corneal epithelial cells, resulting in reduced laminin content in the basement membrane. Within that same 3- to 7-day time frame, epithelial cells respond to the absence of oxygen and the loss of cell–basement membrane adhesion by undergoing apoptosis. Localized cell death now exposes the basement membrane hat has previously been altered by hypoxia to contain subnormal levels of laminin-5. Opportunistic bacteria such as P. aeruginosa may subsequently bind to the exposed matrix. 
Plotkowski et al. 21 have speculated that affinity for laminin may be of biological significance in the pathogenesis of P. aeruginosa infection of injured tissues. However, they analyzed laminin-1, not laminin-5. Similarly pathogens other than Pseudomonas can bind to laminin-1. 29 30 31 For example, a surface adhesin on Mycobacterium leprae binds to laminin-2 and facilitates entry into Schwann cells. 32 These studies certainly suggest that laminins function to promote bacterial adhesion to extracellular matrix, not to inhibit it, as evidence from our studies suggests. 
Laminin-5 is biochemically different from the other known laminin isoforms. For example the α3, β3, and γ2 chains that form laminin-5 are significantly smaller than the corresponding laminin-1 or -2 chains. 33 It is covalently associated with laminin-6 and -7 in extracellular matrix, and these three laminins are the only isoforms that contain the truncated 160-kDa α3 chain. 33 It is reasonable to hypothesize that the unique molecular characteristics of laminin-5 may carry over to a unique role in regulating bacterial adhesion to exposed extracellular matrix. However, we cannot exclude the possibility that the loss of laminin-5 from the matrix alters the supramolecular structure of the substrate, because the disassembly of the laminin-5/-6/-7 complex may expose binding sites for bacteria that would be masked in a “normal” basement membrane. 
Laminin-5 is also unique because it is the only human laminin for which all three chains have a demonstrated role in a genetic disorder. 34 Mutations in the α3, β3, or γ2 chains of laminin-5 underlie the junctional forms of epidermolysis bullosa, an inherited skin disorder characterized by the splitting of the epidermis from the dermis at the level of the basement membrane. The basement membrane that is exposed by denudation of the epidermis has no functional laminin-5. Most significant is that cutaneous infections with P. aeruginosa are a major complication of this disease. 35  
Invasin is a surface protein associated with Yersinia pseudotuberculosis. Invasin can bind to the integrin α3β1. In doing so, it displaces laminin-5 from the integrin. The attachment of cell surface integrins to Yersinia through invasin leads to internalization of the bacterium by the host cell. These host–bacteria interactions do not need laminin-5. In essence, the bacteria removes laminin-5 from the adhesion site by competitive binding to host cell surface receptors. It remains to be determined whether Pseudomonas possesses similar surface proteins or similar mechanisms to exclude or remove functional laminin-5 adhesion sites. 
 
Figure 1.
 
Adhesion of P. aeruginosa to ECMS 20% O2. Bacterial strains ATTC 19660 and PAO1 were allowed to adhere to normoxic matrix and hypoxic matrix in the presence of 20% O2. Top: extracellular matrix produced by HCECs (non-transformed). Strain 19660 showed a significant increase in binding on the hypoxic matrix produced by HCECs in binding assay pH 7.0, compared with normoxic matrix in binding assay pH 7.0 (P < 0.0001). PAO1 showed a significant increase in binding on hypoxic matrix produced by HCECs at pH 7.5 compared with normoxic matrix at pH 7.5 (P = 0.0050). Bottom: extracellular matrix produced by HCETs (a virally transformed HCEC line). Strain 19660 showed a significant increase in binding on hypoxic matrix produced by HCETs in binding assay pH 7.0 compared with normoxic matrix in binding assay pH 7.0 (P = 0.0003). PAO1 showed a significant increase in binding on hypoxic matrix produced by HCECs at pH 7.5 compared with normoxic matrix at pH 7.5 (P = 0.0200).
Figure 1.
 
Adhesion of P. aeruginosa to ECMS 20% O2. Bacterial strains ATTC 19660 and PAO1 were allowed to adhere to normoxic matrix and hypoxic matrix in the presence of 20% O2. Top: extracellular matrix produced by HCECs (non-transformed). Strain 19660 showed a significant increase in binding on the hypoxic matrix produced by HCECs in binding assay pH 7.0, compared with normoxic matrix in binding assay pH 7.0 (P < 0.0001). PAO1 showed a significant increase in binding on hypoxic matrix produced by HCECs at pH 7.5 compared with normoxic matrix at pH 7.5 (P = 0.0050). Bottom: extracellular matrix produced by HCETs (a virally transformed HCEC line). Strain 19660 showed a significant increase in binding on hypoxic matrix produced by HCETs in binding assay pH 7.0 compared with normoxic matrix in binding assay pH 7.0 (P = 0.0003). PAO1 showed a significant increase in binding on hypoxic matrix produced by HCECs at pH 7.5 compared with normoxic matrix at pH 7.5 (P = 0.0200).
Figure 2.
 
Adhesion of P. aeruginosa to ECMS in 2% O2. Strains 19660 or PAO1 were allowed to adhere to hypoxic or normoxic matrix in the presence of 2% O2. Top: extracellular matrix produced by HCECs. Strain 19660 showed a significant increase in binding on hypoxic matrix produced by HCECs in binding assay pH 7.0, compared with normoxic matrix in binding assay pH 7.0 (P < 0.0001). PAO1 showed a significant increase in binding on hypoxic matrix produced by HCECs at pH 7.5 compared with normoxic matrix at pH 7.0 (P = 0.0009). Bottom: extracellular matrix produced by HCETs. Strain 19660 showed a significant increase in binding on hypoxic matrix produced by HCETs in binding assay pH 7.0 compared with normoxic matrix in binding assay pH 7.0 (P = 0.0160). PAO1 showed a significant increase in binding on hypoxic matrix produced by HCECs at pH 7.0, compared with normoxic matrix at pH 7.0 (P = 0.0330).
Figure 2.
 
Adhesion of P. aeruginosa to ECMS in 2% O2. Strains 19660 or PAO1 were allowed to adhere to hypoxic or normoxic matrix in the presence of 2% O2. Top: extracellular matrix produced by HCECs. Strain 19660 showed a significant increase in binding on hypoxic matrix produced by HCECs in binding assay pH 7.0, compared with normoxic matrix in binding assay pH 7.0 (P < 0.0001). PAO1 showed a significant increase in binding on hypoxic matrix produced by HCECs at pH 7.5 compared with normoxic matrix at pH 7.0 (P = 0.0009). Bottom: extracellular matrix produced by HCETs. Strain 19660 showed a significant increase in binding on hypoxic matrix produced by HCETs in binding assay pH 7.0 compared with normoxic matrix in binding assay pH 7.0 (P = 0.0160). PAO1 showed a significant increase in binding on hypoxic matrix produced by HCECs at pH 7.0, compared with normoxic matrix at pH 7.0 (P = 0.0330).
Figure 3.
 
Effect of anti-laminin-5 mAb on adhesion of strain 19660 to normoxic matrix produced by HCECs. For this assay, bacteria interacted with normoxic matrix in an environment of 20% O2. mAbs were presented to the bacteria in two ways: They were mixed with the bacteria before application on normoxic matrix (M) or they were incubated with normoxic matrix before addition of bacteria (O). mAb concentrations were 50 μg/mL (50) or 100 μg/mL (100). Top: mAb 19562 against laminin-5 γ2 chain significantly enhanced bacterial binding to normoxic matrix, regardless of whether mAb was mixed with bacteria (P = 0.0008 for 50:M and P < 0.0001 for 100:M, compared with the irrelevant mAb control) or incubated on the normoxic matrix (P < 0.0001 for 50:O and P < 0.0001 for 100:O compared with the control). Middle: the function-inhibiting mAb P3H92 significantly enhanced bacterial binding to normoxic matrix, regardless of whether mAb was mixed with bacteria (P = 0.0500 for 50:M and P = 0.0132 for 100:M, compared with irrelevant mAb control) or incubated on the matrix (P = 0.0003 for 50:O and P < 0.0001 for 100:O compared with control). Bottom: mAb 17 to laminin-5 β3 chain significantly enhanced bacterial binding normoxic matrix, regardless of whether mAb was mixed with bacteria (P < 0.0001 for 100:M compared with irrelevant mAb control) or incubated on the matrix (P = 0.0020 for 100:O compared with control).
Figure 3.
 
Effect of anti-laminin-5 mAb on adhesion of strain 19660 to normoxic matrix produced by HCECs. For this assay, bacteria interacted with normoxic matrix in an environment of 20% O2. mAbs were presented to the bacteria in two ways: They were mixed with the bacteria before application on normoxic matrix (M) or they were incubated with normoxic matrix before addition of bacteria (O). mAb concentrations were 50 μg/mL (50) or 100 μg/mL (100). Top: mAb 19562 against laminin-5 γ2 chain significantly enhanced bacterial binding to normoxic matrix, regardless of whether mAb was mixed with bacteria (P = 0.0008 for 50:M and P < 0.0001 for 100:M, compared with the irrelevant mAb control) or incubated on the normoxic matrix (P < 0.0001 for 50:O and P < 0.0001 for 100:O compared with the control). Middle: the function-inhibiting mAb P3H92 significantly enhanced bacterial binding to normoxic matrix, regardless of whether mAb was mixed with bacteria (P = 0.0500 for 50:M and P = 0.0132 for 100:M, compared with irrelevant mAb control) or incubated on the matrix (P = 0.0003 for 50:O and P < 0.0001 for 100:O compared with control). Bottom: mAb 17 to laminin-5 β3 chain significantly enhanced bacterial binding normoxic matrix, regardless of whether mAb was mixed with bacteria (P < 0.0001 for 100:M compared with irrelevant mAb control) or incubated on the matrix (P = 0.0020 for 100:O compared with control).
Figure 4.
 
The hypothesized relationship between laminin-5, hypoxia, apoptosis, and bacterial adhesion in the cornea.
Figure 4.
 
The hypothesized relationship between laminin-5, hypoxia, apoptosis, and bacterial adhesion in the cornea.
The authors thank Sharon McClellan, Beth Szliter, and Ron Barrett for expert technical assistance in the completion of this project. 
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Figure 1.
 
Adhesion of P. aeruginosa to ECMS 20% O2. Bacterial strains ATTC 19660 and PAO1 were allowed to adhere to normoxic matrix and hypoxic matrix in the presence of 20% O2. Top: extracellular matrix produced by HCECs (non-transformed). Strain 19660 showed a significant increase in binding on the hypoxic matrix produced by HCECs in binding assay pH 7.0, compared with normoxic matrix in binding assay pH 7.0 (P < 0.0001). PAO1 showed a significant increase in binding on hypoxic matrix produced by HCECs at pH 7.5 compared with normoxic matrix at pH 7.5 (P = 0.0050). Bottom: extracellular matrix produced by HCETs (a virally transformed HCEC line). Strain 19660 showed a significant increase in binding on hypoxic matrix produced by HCETs in binding assay pH 7.0 compared with normoxic matrix in binding assay pH 7.0 (P = 0.0003). PAO1 showed a significant increase in binding on hypoxic matrix produced by HCECs at pH 7.5 compared with normoxic matrix at pH 7.5 (P = 0.0200).
Figure 1.
 
Adhesion of P. aeruginosa to ECMS 20% O2. Bacterial strains ATTC 19660 and PAO1 were allowed to adhere to normoxic matrix and hypoxic matrix in the presence of 20% O2. Top: extracellular matrix produced by HCECs (non-transformed). Strain 19660 showed a significant increase in binding on the hypoxic matrix produced by HCECs in binding assay pH 7.0, compared with normoxic matrix in binding assay pH 7.0 (P < 0.0001). PAO1 showed a significant increase in binding on hypoxic matrix produced by HCECs at pH 7.5 compared with normoxic matrix at pH 7.5 (P = 0.0050). Bottom: extracellular matrix produced by HCETs (a virally transformed HCEC line). Strain 19660 showed a significant increase in binding on hypoxic matrix produced by HCETs in binding assay pH 7.0 compared with normoxic matrix in binding assay pH 7.0 (P = 0.0003). PAO1 showed a significant increase in binding on hypoxic matrix produced by HCECs at pH 7.5 compared with normoxic matrix at pH 7.5 (P = 0.0200).
Figure 2.
 
Adhesion of P. aeruginosa to ECMS in 2% O2. Strains 19660 or PAO1 were allowed to adhere to hypoxic or normoxic matrix in the presence of 2% O2. Top: extracellular matrix produced by HCECs. Strain 19660 showed a significant increase in binding on hypoxic matrix produced by HCECs in binding assay pH 7.0, compared with normoxic matrix in binding assay pH 7.0 (P < 0.0001). PAO1 showed a significant increase in binding on hypoxic matrix produced by HCECs at pH 7.5 compared with normoxic matrix at pH 7.0 (P = 0.0009). Bottom: extracellular matrix produced by HCETs. Strain 19660 showed a significant increase in binding on hypoxic matrix produced by HCETs in binding assay pH 7.0 compared with normoxic matrix in binding assay pH 7.0 (P = 0.0160). PAO1 showed a significant increase in binding on hypoxic matrix produced by HCECs at pH 7.0, compared with normoxic matrix at pH 7.0 (P = 0.0330).
Figure 2.
 
Adhesion of P. aeruginosa to ECMS in 2% O2. Strains 19660 or PAO1 were allowed to adhere to hypoxic or normoxic matrix in the presence of 2% O2. Top: extracellular matrix produced by HCECs. Strain 19660 showed a significant increase in binding on hypoxic matrix produced by HCECs in binding assay pH 7.0, compared with normoxic matrix in binding assay pH 7.0 (P < 0.0001). PAO1 showed a significant increase in binding on hypoxic matrix produced by HCECs at pH 7.5 compared with normoxic matrix at pH 7.0 (P = 0.0009). Bottom: extracellular matrix produced by HCETs. Strain 19660 showed a significant increase in binding on hypoxic matrix produced by HCETs in binding assay pH 7.0 compared with normoxic matrix in binding assay pH 7.0 (P = 0.0160). PAO1 showed a significant increase in binding on hypoxic matrix produced by HCECs at pH 7.0, compared with normoxic matrix at pH 7.0 (P = 0.0330).
Figure 3.
 
Effect of anti-laminin-5 mAb on adhesion of strain 19660 to normoxic matrix produced by HCECs. For this assay, bacteria interacted with normoxic matrix in an environment of 20% O2. mAbs were presented to the bacteria in two ways: They were mixed with the bacteria before application on normoxic matrix (M) or they were incubated with normoxic matrix before addition of bacteria (O). mAb concentrations were 50 μg/mL (50) or 100 μg/mL (100). Top: mAb 19562 against laminin-5 γ2 chain significantly enhanced bacterial binding to normoxic matrix, regardless of whether mAb was mixed with bacteria (P = 0.0008 for 50:M and P < 0.0001 for 100:M, compared with the irrelevant mAb control) or incubated on the normoxic matrix (P < 0.0001 for 50:O and P < 0.0001 for 100:O compared with the control). Middle: the function-inhibiting mAb P3H92 significantly enhanced bacterial binding to normoxic matrix, regardless of whether mAb was mixed with bacteria (P = 0.0500 for 50:M and P = 0.0132 for 100:M, compared with irrelevant mAb control) or incubated on the matrix (P = 0.0003 for 50:O and P < 0.0001 for 100:O compared with control). Bottom: mAb 17 to laminin-5 β3 chain significantly enhanced bacterial binding normoxic matrix, regardless of whether mAb was mixed with bacteria (P < 0.0001 for 100:M compared with irrelevant mAb control) or incubated on the matrix (P = 0.0020 for 100:O compared with control).
Figure 3.
 
Effect of anti-laminin-5 mAb on adhesion of strain 19660 to normoxic matrix produced by HCECs. For this assay, bacteria interacted with normoxic matrix in an environment of 20% O2. mAbs were presented to the bacteria in two ways: They were mixed with the bacteria before application on normoxic matrix (M) or they were incubated with normoxic matrix before addition of bacteria (O). mAb concentrations were 50 μg/mL (50) or 100 μg/mL (100). Top: mAb 19562 against laminin-5 γ2 chain significantly enhanced bacterial binding to normoxic matrix, regardless of whether mAb was mixed with bacteria (P = 0.0008 for 50:M and P < 0.0001 for 100:M, compared with the irrelevant mAb control) or incubated on the normoxic matrix (P < 0.0001 for 50:O and P < 0.0001 for 100:O compared with the control). Middle: the function-inhibiting mAb P3H92 significantly enhanced bacterial binding to normoxic matrix, regardless of whether mAb was mixed with bacteria (P = 0.0500 for 50:M and P = 0.0132 for 100:M, compared with irrelevant mAb control) or incubated on the matrix (P = 0.0003 for 50:O and P < 0.0001 for 100:O compared with control). Bottom: mAb 17 to laminin-5 β3 chain significantly enhanced bacterial binding normoxic matrix, regardless of whether mAb was mixed with bacteria (P < 0.0001 for 100:M compared with irrelevant mAb control) or incubated on the matrix (P = 0.0020 for 100:O compared with control).
Figure 4.
 
The hypothesized relationship between laminin-5, hypoxia, apoptosis, and bacterial adhesion in the cornea.
Figure 4.
 
The hypothesized relationship between laminin-5, hypoxia, apoptosis, and bacterial adhesion in the cornea.
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