Isolation of conjunctival mucin and differential interaction with Pseudomonas aeruginosa strains of varied pathogenic potential

Experimental Eye Research 77 (2003) 699–710 www.elsevier.com/locate/yexer

Isolation of conjunctival mucin and differential interaction with Pseudomonas aeruginosa strains of varied pathogenic potential Lina Panayiota Aristoteli*, Barbara Bojarski, Mark D.P. Willcox Cooperative Research Centre for Eye Research and Technology, University of New South Wales, Sydney, NSW 2052, Australia Received 28 February 2003; accepted in revised form 14 August 2003

Abstract The purpose of the study was to investigate the adhesion of Pseudomonas aeruginosa strains with varying pathogenic potential to purified ocular mucin. Bovine conjunctival mucin was purified by three sequential density gradient centrifugation steps. Immobilised mucin was probed with biotin-labelled bacteria isolated from different contact lens events and quantified by densitometry. Bacterial pili were identified by electron microscopy. The results indicate that purified ocular mucin consisted of a polydisperse high molecular weight population containing at least one species of goblet cell origin and was associated with a 97 kDa mucin-associated protein. Three pathogenic P. aeruginosa strains, Paer1 (57·5 ^ 10·8 £ 10 6 CFU ml21; contact lens induced acute red eye (CLARE)), 6294 (127·0 ^ 4·7 £ 106 CFU ml21; microbial keratitis) and Paer25 (60·5 ^ 11·3 £ 106 CFU ml21; CLARE) exhibited a significantly higher level of adhesion to mucin than the negative control, E. coli (14·3 ^ 9·6 £ 106 CFU ml21) ðp , 0·005Þ: The remaining P. aeruginosa isolates, Paer3 (asymptomatic patient), Paer12 (microbial keratitis) and ATCC15442 (standard environmental strain) did not significantly differ in their mucin adhesion from the negative control. The majority of bacterial strains tested contained pili; thus differences in mucin adhesion observed could not be solely explained by pili status. In conclusion, P. aeruginosa isolates exhibit differential adhesion patterns to purified ocular mucin. It is proposed that more avid mucin-adhering strains are given the opportunity to adhere and subsequently penetrate the mucous layer of the tear film to initiate pathogenesis. q 2003 Elsevier Ltd. All rights reserved. Keywords: Pseudomonas aeruginosa; mucin; glycoprotein; conjunctiva; contact lenses

1. Introduction Microbial keratitis is an infection of the cornea induced by bacteria, fungi or parasites and produces excavation of the corneal epithelium, Bowman’s layer and stroma, with infiltration of inflammatory cells and tissue necrosis (Holden et al., 2000). Clinical symptoms of microbial keratitis include pain, severe redness of the eye, discharge and excess tear secretions, photophobia, and swollen lids (Holden et al., 2000). Since the 1980s, the predominant risk factor for bacterial keratitis has been contact lens wear (Liesegang, 1997; Wong et al., 1997; Schaefer et al., 2001), where up to 36% of bacterial keratitis patients are contact * Corresponding author. Address: Dr Lina Panayiota Aristoteli, HRI, 145-147 Missenden Road, Camperdown, Sydney, NSW 2050, Australia. E-mail address: [email protected] (L.P. Aristoteli). 0014-4835/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. DOI:10.1016/j.exer.2003.08.007

lens wearers (Schaefer et al., 2001). Pseudomonas aeruginosa has been isolated from most clinical studies investigating the aetiology of microbial keratitis, in some cases as the predominant bacterium isolated (Chatterjee et al., 1995; Wong et al., 1997) and in others as the main gram-negative bacterium (Kunimoto et al., 1998, 2000; Schaefer et al., 2001). P. aeruginosa may also be responsible for contact lens induced acute red eye (CLARE), a non-destructive inflammatory response to overnight contact lens wear (Holden et al., 1996). A CLARE patient experiences irritation or pain to the eye, excessive tear secretion, photophobia, and the eye appears red; symptoms that are soon alleviated after removal of the contact lens from the eye (Holden et al., 2000). The mucous layer is the major component of the precorneal tear film (Nichols et al., 1985; Prydal et al., 1992; Chen et al., 1997), and consists largely of O-linked

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mucin glycoproteins that have been hypothesised to bind antibacterial constituents such as sIgA, lactoferrin and lysozyme for their anchorage in the tear film (Corfield et al., 1997; Argu¨eso and Gipson, 2001). Mucin thus forms the scaffold of an ocular protection network in the tear film, facilitating the adhesion and removal of bacteria from the eye during blinking. The mucin layer also protects the underlying epithelium where the removal of mucous from the ocular surface by N-acetylcysteine resulted in an increase in the adherence of P. aeruginosa to the cornea (Fleiszig et al., 1994). The eye characteristically contains both secretory and membrane-bound mucins that are believed to interact for optimal tear film function. Secretory-type MUC5AC mucin primarily originates from goblet cells in the conjunctiva and is secreted in the tear film, constituting the major gelforming mucin of the tear film (Jumblatt et al., 1999). Low quantities of MUC2 (McKenzie et al., 2000) and a soluble form of MUC4 (Pfugfelder et al., 2000) have also been identified in tear secretions. Such conjunctival-derived secretory mucins are characteristically oligomeric containing a subunit structure, and consist of a number of distinct mucin species differing in molecular weight and charge (Berry et al., 1996; Hicks et al., 1997; Ellingham et al., 1999). Ocular membrane-bound mucins, MUC1 (Inatomi et al., 1995) and MUC4 (Pfugfelder et al., 2000), are anchored to the conjunctival and corneal epithelium, forming the glycocalyx. However, the presence of MUC4 specifically in the corneal epithelium is debatable (Inatomi et al., 1996), and only low amounts of MUC1 have been detected on the superficial corneal epithelium using antibody probes (Inatomi et al., 1995; Pfugfelder et al., 2000). Other mucins such as MUC3, MUC5B or MUC6 do not seem to be major mucins of the ocular epithelium (Inatomi et al., 1996; Gipson and Inatomi, 1998). Perturbations to the precorneal tear film, including the mucin layer, may disrupt the functional defence mechanism of the eye and allow microbial infection. Thus, the aim of the present work was to purify and characterise conjunctival mucin, compare the adhesion of P. aeruginosa contact lens isolates from various ocular responses to this purified mucin source and examine the role of bacterial pili in this process.

2. Materials and methods 2.1. Mucin extraction Bovine conjunctival mucin (BCM) was purified by three sequential caesium chloride density gradients on the basis of a previous method (Berry et al., 1996). Briefly, bovine eyes were delivered promptly on ice from the local abattoir and the conjunctiva was dissected and flooded with cold extraction buffer. The extraction buffer consisted of 4 M guanidine hydrochloride (Ultrapure; ICN Biomedicals,

Aurora, OH, USA) in PBS with 1 mM phenylmethylsulphonylfluoride (PMSF; ICN Biomedicals), 5 mM ethylenediaminetetraacetic acid (EDTA, ChemSource, Taren Point, NSW), 0·1 mg ml21 soybean trypsin inhibitor (ICN), 5 mM N-ethylmaleimide (Sigma Chemical Company, St Louis, MO, USA), 10 mM benzamidine – HCl monohydrate (ICN) and 0·02% (w/v) sodium azide (Fluka Chemie AG, Buchs, Switzerland). The tissue was gently scraped with a microscope slide or metal scraper and the ocular extract was stirred overnight at 48C. Following clarification of the extract by centrifugation at 12 000g for 1 hr at 48C, the protein concentration of the supernatant was adjusted to 1– 5 mg ml21. The protein concentration was determined with a bicinchoninic protein assay (Pierce, Rockford, IL, USA). 2.2. Density gradient ultracentrifugation Solid caesium chloride (Ultrapure, 99·999%; ICN) was added to the extract to a density of approximately 1·4 g ml21. Extracts were centrifuged in a Vti 50 rotor (Beckman, Palo Alto, CA, USA) at 150 000g for 18 –24 hr at 108C (Sorvall Discovery 100, Newtown, CT, USA). After centrifugation, 1 ml fractions were aspirated from the top of the tube. After pooling mucin-containing fractions, the density was readjusted to 1·4 g ml21 if necessary, and the sample was rerun under identical conditions. Mucin-rich samples were again pooled, and dialysed against filtered 0·5 M guanidine hydrochloride in PBS. For the final (third) caesium chloride run, the density was adjusted to approximately 1·5 g ml21 and the samples centrifuged as stated above. After analysis, mucin fractions were pooled and dialysed exhaustively against Milli-Q water. Purified mucin was freeze-dried and stored at 2 208C. 2.3. Analysis of fractions During mucin purification, the density of each fraction was established by weighing and the presence of mucin was detected in each fraction by slot blot analysis using a nitrocellulose membrane (0·2 mm pure nitrocellulose, BioRad, Hercules, CA, USA) immobilised in a slot blot apparatus (Hoefer Pharmacia Biotech, San Francisco, CA, USA). Periodic acid-Schiff (PAS) staining was performed (Thornton et al., 1989) and membranes were also stained with wheat germ agglutin (WGA) lectin by a modification of a prior procedure (Gravel and Golaz, 1996). In this case, the membrane was blocked with PBS þ 0·5% Tween-20 (v/v) (Sigma) (PBST) for 1 hr and incubated with 1 mg ml21 biotinylated Triticum vulgaris lectin (WGA; Sigma) in PBST for 2 hr. The membrane was washed 6 £ 10 min in PBST and incubated for 1 hr in ExtrAvidin-peroxidase (Sigma) at a dilution of 1:2000 in PBST. Further 3 £ 10 min washes in PBST, and then 3 £ 10 min washes in PBS alone were conducted. Peroxidase was developed by a solution consisting of 80 ml PBS, two tablets of 3,30 diaminobenzidine

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tetrahydrochloride (DAB; 10 mg tablet21, Sigma) dissolved in 10 ml methanol, two tablets of 4-chloro-1-naphthol (4CN; 30 mg tablet21, Sigma) dissolved in 10 ml methanol and 40 ml hydrogen peroxide solution (30% solution BDH Laboratory Supplies, Poole, UK). The peak density of membranes was calculated using the GelDoc imager (BioRad) and the Quantity One software (Bio-Rad). Fraction samples from the third caesium chloride run only were additionally stained for nucleic acid (Tseng et al., 1987), using 1% Gills Haematoxylin, to identify fractions containing nucleic acid contamination, and also probed with mucin antibodies for a more precise determination of mucin-containing fractions. For mucin antibody staining, the membrane was dried for half an hour and subsequently blocked with 0·5% (w/v) casein/10% foetal calf serum (v/v) in Tris –buffered saline (TBS; 10 mM Tris, 0·15 M NaCl, pH 7·4) for 2 hr. After washing three times for 10 min each in TBS þ 0·05% (v/v) Tween-20, the membrane was incubated in a 1/100 dilution of 19M1 (f-binding epitope of AM1 anti-mucin antibody, Bara et al., 1991; donated by Professor Jacques Bara, U-482 INSERM, Hoˆpital St Antoine, Paris, France) in blocking buffer for 2 hr. The blot was washed three times for 10 min in TBS þ 0·05% (v/ v) Tween-20 and incubated in a 1/1000 dilution of peroxidase conjugated goat F(AB0 )2 fragment to mouse IgG (whole molecule) (ICN) in blocking buffer for 1 h. Further three 10 min washing steps in TBS were conducted and the membrane was finally developed with DAB and 4CN as described above. 2.4. Agarose electrophoresis and blotting A 1% (w/v) agarose (Agarose MP, Roche Diagnostics GmbH, Mannheim Germany) gel was prepared in electrophoresis running buffer (40 mM Tris – acetate, 1 mM EDTA, pH 8·0, 0·1% (w/v) SDS) (Thornton et al., 1995). Purified BCM, PGM (Type III; Sigma) and open eye tears (Sack et al., 1992) was applied to the gel under reducing and nonreducing conditions, where reduced samples contained 4 mM dithiothreitol (DTT) and were heated at 1008C for 5 min. Electrophoresis was performed at 30 V constant voltage at room temperature until the dye front was near the edge of the gel (approximately 24 hr). Samples were transferred to a nitrocellulose membrane by capillary blotting using 4 £ standard sodium citrate buffer as the transfer buffer (4 £ SSC; 0·6 M NaCl, 0·06 M sodium citrate, pH 7·0) overnight (Thornton et al., 1989). The membrane was allowed to dry for 30 min at room temperature after which the membrane was briefly washed three times in PBS. For the detection of immobilised mucin, the blot was probed with biotin-labelled WGA lectin as previously described. 2.5. SDS – PAGE electrophoresis Mucin solutions in PBS were diluted with 4 £ SDS – PAGE sample buffer (0·2 g SDS (Sigma), 4 ml glycerol

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(Ajax, NSW, Australia), 4 ml 4% (w/v) bromophenol blue, 2·5 ml 1 M Tris – HCl pH 6·8, Milli-Q water to 10 ml) under reducing and non-reducing conditions. Samples were loaded onto a 4 – 15% ready-made gel (Bio-Rad) with tank running buffer (0·025 M Tris, 0·192 M glycine and 1% (w/v) SDS) and electrophoresis commenced at 100 V constant voltage at room temperature. Gels were subsequently silver stained (Morrissey, 1981). 2.6. Immunohistochemistry of bovine conjunctival tissue Fresh bovine conjunctival tissue was immediately fixed in 10% neutral buffered formalin for 2 hr at room temperature and placed in PBS overnight at 48C. It was necessary to control the time of fixation, as this is a factor affecting the intensity and pattern of staining, leading to inconsistencies (Walsh and Jass, 2000). As the fornix region contained a relatively high density of goblet cells in the canine conjunctiva (Moore et al., 1987), the fornix of the bovine conjunctiva was used for immunohistochemistry and staining procedures. The tissue was subsequently embedded in paraffin by routine procedures and stained with PAS reagent to verify the presence of goblet cells in each set of sections. For immunohistochemistry, 3 mm sections were cut and transferred to silane-coated slides (Sigma). Slides were immersed in two changes of xylene for 3 min each, two 100% ethanol changes for 1 min each, and in 70% ethanol for 1 min. Slides were immersed in Milli-Q for 5 – 10 min and incubated in antigen retrieval solution (0·01 M citric acid, pH 6·0) for 20– 30 min in a 1008C water bath. After cooling, slides were washed with Milli-Q water and flooded with TBS þ 0·05 % (v/v) Tween-20, pH 7·6 (TBST) for 5 min to equilibrate the tissue. The LSABw2 kit, HRP (DAKO Corporation, Carpinteria, CA, USA) was used with modifications to the recommended procedure to cater for the particular application. After quenching endogenous peroxidase and washing with TBST, the primary antibody and negative control were incubated with the sections for 30 min at room temperature. The primary antibody was ascites fluid of 21M1 (f-binding epitope of AM1 anti-mucin antibody) (Bara et al., 1991), like 19M1, a monoclonal antibody against in part, the cysteine-rich poorly glycosylated Cterminal domain of the MUC5AC gene (Bara et al., 1998), a kind donation by Professor Jacques Bara. The antibody was used at a working concentration of 10 –100 mg ml21 IgG in a protein-based antibody diluent (DAKO). The negative control was mouse non-immune serum diluted in the same antibody diluent (DAKO) to achieve the equivalent concentration of the working primary antibody concentration. For a more conservative approach, the lower dilution of mouse serum was assumed. The remainder of the procedure was carried out according to the kit instructions. Sections were immediately lightly counterstained with haematoxylin and sections were taken through

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graded ethanol steps and xylene before mounting the slides with DPX (BDH Laboratory Supplies). 2.7. Adhesion of P. aeruginosa strains to BCM 2.7.1. Bacterial strains and culture conditions P. aeruginosa strains used for this study included Paer1 and Paer25, originally isolated from CLARE events, Paer3 from an asymptomatic contact lens wearer, and Paer12 and 6294 from contact lens associated microbial keratitis. Clinical P. aeruginosa strains tested were non-mucoid with two of serogroup O1 (Paer1 and Paer3); one was serogroup O4 (Paer25); and two strains were of serogroup O6 (Paer12 and 6294) (Thuruthyil et al., 2001). ATCC 15442 is a standard environmental strain. To account for possible non-specific adhesion, E. coli K12 was used as the negative control. This strain was obtained from the School of Microbiology and Immunology, University of New South Wales culture collection and documented not to bind to porcine gastric mucin (PGM) (Nelson et al., 1990). The positive control was Paer1 as it was able to adhere to fibronectin, a sialylated glycoprotein (Baleriola-Lucas and Willcox, 1998), and bovine conjunctival epithelial cells (Willcox et al., 1998). P. aeruginosa strains were obtained from the culture collection at the Cooperative Research Centre for Eye Research and Technology (CRCERT) and were stored in tryptone soya broth (TSB; Oxoid, Basingstoke, Hampshire, UK) supplemented with 30% glycerol (Ajax, NSW, Australia) at – 868C. As strains may lose pathogenicity over extended periods of culture, isolates were routinely sub-cultured only once on chocolate blood agar plates (Oxoid, Australia, West Heidelberg, Victoria) at 358C for 18 hr, after initial removal from frozen storage. Bacteria were harvested from the plates with PBS, washed three times in PBS by centrifugation at 2000g for 10 min. To ensure biotin-labelling of a viable bacterial population in the same stage of growth, the OD660 nm of each suspension was adjusted to 0·1with TSB and the strains were brought to the mid-log-phase of growth, then washed three times in PBS for labelling with biotin. 2.8. Slot blot adhesion assay Bacterial strains were labelled with biotin at a concentration of 0·5 mg biotin (EZ-Linke Sulfo-NHS-LC-Biotin; Pierce, Rockford, IL, USA) per ml of bacterial suspension and incubated with immobilised mucin. For this, highly purified BCM (15 mg) was immobilised onto nitrocellulose using a slot blot apparatus and the membrane dried between two filter papers for 30 min at room temperature. The blot was blocked for 2 hr with 0·5% (w/v) casein (technical grade from bovine milk, Sigma) in PBS, washed three times briefly in PBS and incubated with biotin labelled bacterial isolates for 2 hr. Unbound bacteria were removed from the membrane by washing three times for 10 min each in PBS

and the membranes were incubated for one hour in ExtrAvidin-peroxidase (Sigma) at a dilution of 1:2000 in PBS þ 0·1% (v/v) Tween-20. The membrane was washed three times and developed for biotin. The development solution consisted of 80 ml PBS, two tablets of tetrahydrochloride (DAB; 3,30 diaminobenzidine 10 mg tablet21, Sigma) dissolved in 10 ml methanol, two tablets of 4-chloro-1-naphthol (4CN; 30 mg tablet21, Sigma) dissolved in 10 ml methanol and 40 ml hydrogen peroxide solution. Densitometry was performed using a calibrated GS-710 scanning densitometer (Bio-Rad) and the volume analysis tools of the Quantity One software (Bio-Rad). A standard curve was also constructed for each P. aeruginosa strain comparing the density value of biotinylated bacteria at serial dilutions with the bacterial concentration (CFU ml21) of that particular dilution. 2.9. Statistics Statistics were performed using the statistical package SPSS 10·0 for Windows. The mean adhesion counts (CFU ml21) from three independent experiments ðn ¼ 3Þ were compared for differences in adhesion between the seven bacterial strains using One-Way ANOVA. The Levene test was also conducted to confirm equal variance between groups, as this was an assumption required for ANOVA analyses. Post-hoc comparisons using Bonferroni allowed the determination of the level of significance between groups. Significance was taken at a p-value of less than 0·05. 2.10. Electron microscopy evaluation of bacterial ultrastructure P. aeruginosa may bind to glycoconjugates by pili (Rudner et al., 1992; Comolli et al., 1999), bacterial surface appendages involved in adhesion. To examine the presence or absence of pili in the tested bacterial strains, P. aeruginosa strains were grown to mid-log phase and labelled with biotin under the same conditions as in the mucin adhesion assay. A 6 ml drop of the bacterial suspension that had been pre-mixed with 2% phosphotungstic acid was placed on a carbon-coated grid, for 5 min. The solution was removed with filter paper, the grid allowed to dry, and examined under a Philips CM100 transmission electron microscope.

3. Results 3.1. Bovine conjunctival mucin purification Approximately 400 bovine eyes were dissected for the purification of conjunctival mucin used in the present study. The staining of fractions by PAS and WGA from the first

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gradient step in 4 M guanidine hydrochloride revealed the presence of at least three distinct species in the BCM preparation (results not shown). Fractions were pooled from the start of the first low-density peak onwards, that included all three mucin populations. PAS staining for the second round of caesium chloride ultracentrifugation displayed two predominant subpopulations of mucin species with additional staining at the highdensity edge of the gradient (Fig. 1(A)). The first ‘low’ density population comprised a small peak, or shoulder in one case, at a peak density of approximately 1·35 g ml21 and a major ‘high density’ peak was located at a density of

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approximately 1·40 g ml21. Interestingly, WGA staining also showed evidence of two major mucin populations (Fig. 1(A)). However, PAS exhibited low intensity staining for the low-density peaks but relatively pronounced staining at the higher density peaks and vice versa with WGA. The ‘high density’ mucin peaks, corresponding to a density range of 1·35– 1·5 g ml21, were pooled. The final density gradient in 0·5 M guanidine hydrochloride was used to remove nucleic acid contamination. PAS and WGA staining of fractions exhibited a small peripheral peak at 1·42 g ml21 (1·4 –1·45 g ml21) and a broad peak at a density of 1·52 g ml21 (1·46 –1·56 g ml21; Fig. 1(B)). WGA

Fig. 1. Density gradient profiles of bovine conjunctival mucin. (A) Staining and density of fractions from the second run in 4 M GuHCl. Fractions having a density of 1·35–1·5 g ml21 were pooled as indicated. (B) Pooled fractions from the second run in 4 M GuHCl were dialysed in 0·5 M GuHCl and recentrifuged at a starting density of 1·5 g ml21. Region 2 was pooled to constitute the purified BCM preparation. V, PAS; S, WGA; X, density. Arrows indicate distinct mucin populations.

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Table 1 Staining profile of mucin fractions Stain

Epitope

Dilution

Region 1

Region 2

Region 3

Periodic acid-Schiff (PAS) Wheat germ agglutin (WGA) Gills haematoxylin AM1 antibody

Vicinal glycols GlcNAc, sialic acid Nucleic acid C-terminal MUC5AC

– 1 mg ml21 1% (w/v) 1/100

þþ þ þþ þ – –

þ þ þþ þ þ þþ þ þ þ þþ

þþ þ þþ þþ þþ þþ

After the third run in 0·5 M GuHCl, individual fractions were probed with the stains indicated using a slot blot assay. Overall staining trends for individual fractions within the three regions indicated in Fig. 1B are summarised as –, no staining; þ, weak staining; þ þþ þ, maximal staining.

additionally had a peak at 1·4 g ml21, and both WGA and PAS contained two peaks greater than 1·55 g ml21, probably representing nucleic acid and/or proteoglycan material. Further, WGA, unlike PAS, stained low density fractions of the gradient. Mucin antibody staining with 19M1 detected the presence of mucin from a density of 1·4– 1·56 g ml21 (Table 1). The presence of nucleic acid was detected from fraction 29 onwards (Table 1). Thus, fractions 10 – 28 (region 2) were pooled, which stained with mucin antibody, PAS and WGA, but hardly stained for nucleic acid. 3.2. Agarose electrophoresis Agarose electrophoresis was conducted with lectin blotting for further analysis of mucin products. Specifically, to compare migration of purified BCM under reducing and non-reducing conditions, and to compare the mucin profile of purified conjunctival mucin with that of commercial PGM and open eye tears. Although mucins migrate on an agarose gel according to both their size and intrinsic charge, a molecular weight marker was included on the gel as a point of reference (Tygat et al., 1995). BCM exhibited a migration profile typical of mucins as a high molecular weight, slow migrating polydisperse smear (Fig. 2). There was a slight change in migration of BCM upon reduction, indicating the presence of disulphide bonds, characteristic of secretory type mucins. A distinct small molecular weight band was present in both reduced and non-reduced BCM. In comparison with the other mucins analysed, BCM had a faster migration than commercial PGM, possibly due to the aggregating nature of the latter that may impede migration through the gel. Tears

showed mucin species of greater migration than those found in PGM and BCM, indicating smaller mucins or more charged mucin species (Fig. 2, lanes 2 and 5). 3.3. SDS – PAGE electrophoresis To assess the purity of the conjunctival mucin preparation, SDS – PAGE electrophoresis of purified BCM was conducted. When comparing the protein profile of the conjunctival extract (Fig. 3(A)) and after one caesium chloride density gradient (Fig. 3(B)), with the end product (Fig. 3(C)), extensive purification of mucin was observed. SDS – PAGE analysis of purified BCM demonstrated a high molecular weight component at the top of the gel greater than 200 kDa (Fig. 3(C)). In addition, there was a prominent band between 78 and 125 kDa that was present in both reduced and non-reduced samples, that in all likelihood corresponds to a mucinassociated protein. Interestingly, a similar 97 kDa protein was also documented in purified human conjunctival mucin preparations (Ellingham et al., 1999). Several additional light staining bands were also observed (Fig. 3(C)), indicating the slight presence of nonmucin components. Thus the results suggest that the sample was extensively purified mucin with no appreciable low molecular weight contaminants present. 3.4. Immunohistochemistry of BCM tissue Immunohistochemistry using the same mucin antibody that reacted with the purified mucin fractions pooled (Table 1) was used to determine the cellular origin of the end product. PAS staining of the fornical region of

Fig. 2. WGA staining of 1% (w/v) agarose blot comparing PGM (Lanes 1 and 4), open eye tears (Lanes 2 and 5) and BCM (Lanes 3 and 6) under reducing (Lanes 1–3) and non-reducing conditions (Lanes 4–6). Arrowheads indicate the position of the small molecular weight band present in BCM.

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the conjunctiva indicated the presence of goblet cells (Fig. 4(A) and (B)), consisting of multiple mucin-containing globules upon higher magnification (Fig. 4(B)). Immunohistochemistry using the mucin antibody to MUC5AC revealed specific staining of most, but not all, of goblet cells (Fig. 4(C)) with no staining of the negative control (Fig. 4(D)). In some cases, the apical surface of the conjunctiva was additionally stained by PAS and MUC5AC antibody. Thus, the presence of MUC5AC in bovine conjunctival goblet cells relates to its relevance as a model for the study of human ocular mucin and also suggests that the BCM purified with this antibody has a predominantly goblet cell origin. 3.5. Adhesion of P. aeruginosa strains to mucin

Fig. 3. Protein profile of reduced BCM at various stages of purification. The protein profiles under non-reducing conditions were essentially the same. (A) Crude extract in 4 M GuHCl; (B) pooled samples after the first CsCl run in 4 M GuHCl; (C) BCM preparation after three sequential CsCl runs. Samples were applied to 4–15% SDS–PAGE and stained with silver. Arrow indicates the position of putative 97 kDa mucin associated protein.

The slot blot adhesion assay for the adhesion of P. aeruginosa strains to purified BCM was independently repeated three times, and the results obtained were consistent and repeatable (Fig. 5). Assessment for nonspecific interactions also included the adhesion of strains to bovine serum albumin (Sajjan et al., 1992) and binding of free biotin label to mucin, both of which were negligible in the current assay (results not shown). An equality of variance between the seven bacterial groups could be

Fig. 4. (A) and (B) PAS staining of sections from the lower temporal fornix of the bovine conjunctiva. Magnification (A) 250 £ ; (B) 1000 £ . Arrows indicate goblet cells. (C) and (D) Localisation of mucin in goblet cells of the lower temporal fornix in the bovine conjunctiva. (C) AM1/MUC5AC mucin antibody. Magnification 500 £ (D) Non-immune mouse serum. Magnification 250 £ . Arrows indicate presence of goblet cells.

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Fig. 5. Graphical comparison of the adhesion of P. aeruginosa strains to purified BCM using the biotinylation slot blot assay. Data presented are the mean and standard deviation of three independent experiments (except Paer25, Section 3) *Significant difference in adhesion to mucin compared with the negative control, E. coli ðp , 0·005Þ: BCM; CFU ml21, colony forming units ml21.

Table 2 The relation between the bacterial source of isolation and presence of pili on the adhesion of P. aeruginosa strains to ocular mucin P. aeruginosa strain

Source of isolation

Adhesion to BCMa

Pili

Flagella

Paer1 Paer3 Paer12 ATCC 15442 6294 Paer25

CLARE Asymptomatic Microbial keratitis Environmental Microbial keratitis CLARE

Medium Low Low Low High Medium

Yes Yes Yes Yes No Yes

Yes Yes Yes Yes Yes Yes

CLARE, contact lens induced acute red eye; BCM, bovine conjunctival mucin. a Determined from quantitative values in slot blot adhesion assay (Section 3).

assumed as tested by the Levene test ðp ¼ 0·5Þ after removing one outlier from the series of experiments with Paer25. One-way ANOVA (without the outlier) showed significant differences in the adhesion of P. aeruginosa strains to ocular mucin ðF ¼ 57·289; p , 0·001Þ: To identify which P. aeruginosa isolates differed significantly from the negative control E. coli K12, post-hoc comparisons using the conservative Bonferroni analysis were used. Three P. aeruginosa strains, Paer1 (57·5 ^ 10·8 £ 106 CFU ml 21), 6294 (127·0 ^ 4·7 £ 10 6 CFU ml 21) and Paer25 (60·5 ^ 11·3 £ 106 CFU ml21) had a significantly higher level of adhesion to mucin than the negative control, E. coli (14·3 ^ 9·6 £ 106 CFU ml21) (p , 0·005; Fig. 5). Furthermore, 6294 had a significantly higher adhesion than all other bacterial strains tested (p , 0·001; Fig. 5), thus its designation as a strain with ‘high’ adhesion, as summarised in Table 2. Paer1 and Paer25 were designated as strains

Fig. 6. Electron micrographs of representative P. aeruginosa strains (A) Paer3 and (B) Paer12 under conditions used for the adhesion assay.

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having ‘medium’ adhesion to mucin while the remaining P. aeruginosa isolates were assigned as bacteria exhibiting ‘low’ adhesion to mucin as they did not significantly differ in their mucin adhesion from the negative control (Table 2). In comparing the extent of mucin adhesion to the clinical origin of the particular P. aeruginosa strain (Table 2), there may be a correlation between the adhesion of a strain to mucin and its pathogenic potential. The adhesion of pathogenic strains (Paer1, 6294 and Paer25) to mucin was of a high or medium level. In contrast, non-pathogenic P. aeruginosa strains isolated from non-disease outcomes, Paer3 and the environmental strain (ATCC 15442), did not have an appreciable ability to adhere to mucin. However, Paer12, also a pathogenic isolate, had a low adhesion to mucin. 3.6. Electron microscopy evaluation of bacterial ultrastructure As indicated in Fig. 6 and Table 2, all of the bacterial isolates tested were piliated under the current experimental conditions except for 6294. It therefore seems that the presence of bacterial pili alone was not a determinative factor for the differential levels of mucin adhesion observed. All P. aeruginosa strains tested possessed flagella.

4. Discussion The present study aimed to purify and characterise bovine conjunctival mucin, a secretory ocular mucin source, and to compare the adhesion of P. aeruginosa isolates to this purified mucin. The results indicate that contact lens isolates of P. aeruginosa significantly vary in their ability to adhere to ocular mucin ðp , 0·001Þ and bacterial surface appendages, pili, were not solely determinative for levels of mucin adhesion. The BCM purified in the current study was polydisperse and contained a subunit structure, consistent with secretorytype mucins. The mucin was purified by three sequential caesium chloride gradients, the gold standard for mucin purification, and mucin was probed using PAS, WGA and a MUC5AC antibody. Mucin banded at densities typical to that of conjunctival preparations at 1·35– 1·5 g ml21, was devoid of nucleic acids and was pure from most non-mucin proteins by SDS – PAGE electrophoresis. Immunohistochemistry was used to observe the cellular origin of purified BCM. Specificity of the MUC5AC antibody to mucin was confirmed by its reactivity with purified mucin fractions from the gradient (Table 1). Probing the bovine conjunctival epithelium with the same antibody revealed a predominantly goblet cell staining profile, in line with the nature of secretory MUC5AC mucin. There was a notable non-reducible band at about 97 kDa that was also observed in SDS –PAGE analysis of purified human conjunctival mucin (Ellingham et al., 1999). Its

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repeated presence in purified conjunctival preparations suggests it may be a mucin-associated protein. Indeed, a 97 kDa glycosylated mucin binding protein (MBP) was recently characterised in the rat gastric mucosa, and postulated to be a membrane-bound protein linked to mucin that is responsible for the tight adhesion of the mucous gel onto epithelial cells (Slomiany et al., 2001). It is also possible that the 97 kDa glycoprotein is a fragment from the N-terminal or C-terminal cysteine-rich region of the mucin peptide (Khatri et al., 1998). N-terminal sequence data of the 97 kDa band may identify a specific region of the mucin polypeptide corresponding to this protein. The results of bacterial adhesion to purified BCM presented in the current study emphasise two major points concerning the adhesion of bacteria to mucin at the molecular level. The first was that certain P. aeruginosa strains, such as 6294, do have the capacity to adhere specifically to ocular mucin. It seems likely that mucin carbohydrates were involved in the adhesion process, as P. aeruginosa strains bind to Lea, Ley, Lex, 30 -sulpho-Lex and Gal(a1-2)Galb determinants, with the highest affinity for sialyl-Lewisx epitopes (Scharfman et al., 1999). However, the results are in contrast with studies documenting low adhesion of P. aeruginosa strain 6294 to purified human tear sialoglycoprotein (McNamara et al., 2000) and rat corneal mucin (Fleiszig et al., 1994), but supports studies showing P. aeruginosa adhesion to respiratory mucins (Vishwanath and Ramphal, 1984, 1985; Ramphal et al., 1991). It is likely that the discrepancy in adhesion of 6294 to ocular mucin is related to differences in techniques. The purified mucin in the Fleiszig studies was pooled at a density of 1·40– 1·42 g ml21 (Fleiszig et al., 1994); thus, possibly not all mucin species were present in this preparation, including mucins that may be able to bind to bacteria. Moreover, their corneal mucin adhesion data do not reconcile with their mucin inhibition experiments, as 6294 adhered greater to the non-mucin fraction of the corneal preparation while only the mucin fraction was able to inhibit 6294 adhesion to the cornea (Fleiszig et al., 1994). The present adhesion data of various P. aeruginosa strains are in good agreement with our previous work using PGM (Aristoteli and Willcox, 2001). Commercial PGM, like BCM, contains MUC5AC secretory type mucins (Jumblatt et al., 1999), while rabbit ocular mucin and PGM have similar monosaccharide compositions (Tseng et al., 1987), likely to be involved in bacterial binding. Common antigenic epitopes are also evident by the crossreaction of antibodies against the non-glycosylated regions of the peptide core of MUC5AC between PGM and ocular mucin (Huang and Tseng, 1987; Jumblatt et al., 1999). An exception is Paer3 that consistently bound at a medium level to PGM but a low level to BCM. Perhaps Paer3 may have a distinct mucin receptor compared with the other P. aeruginosa isolates that may be sensitive to slight

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differences in carbohydrate conformation between the two mucins. The second point for consideration is that the repertoire of P. aeruginosa strains tested exhibited differential adhesion to mucin. This highlights the possibility that because the pathogenic outcome is dependent on the interaction between host and microbe, the inherent mucin adhesion capabilities of the P. aeruginosa strain infecting the eye may be a contributing factor. Evidence suggests that more virulent microbial strains exhibit a higher level of mucin adhesion than relatively avirulent strains (Mantle and Husar, 1993; Vimal et al., 2000). In the present study, it was interesting to note that both non-pathogenic isolates (Paer3 and ATCC 15442) did not adhere to mucin while three out of four pathogenic strains (Paer1, 6294 and Paer25) did adhere. Increased adhesion of bacteria to ocular mucin would readily eliminate the bacterium from the eye as the tear film is replenished and tears exit the eye. However, the application of a contact lens may cause delayed tear clearance (Paugh et al., 2001) and tear film instability (Creech et al., 1998). It is possible that in this alteration of tear defence mechanisms, the effect of bacterial adhesion to mucin takes on a more important role, much like the colonisation of P. aeruginosa in the relatively stagnant mucous of cystic fibrosis patients. The bacterium would need to obtain a foothold in the mucin layer for subsequent degradation of the tear film and glycocalyx by the bacterium’s protease machinery, and translocation to the underlying epithelial cells. The role of pili in the adhesion of P. aeruginosa isolates to purified ocular mucin was investigated. Adhesive pili promote the adhesion of bacteria to host cells (Farinha et al., 1994). P. aeruginosa pili are of Type IV, forming tangled rope-like bundles with each bundle abundantly comprised of smaller filaments (Finlay and Caparon, 2000). Strain 6294 was found to be non-piliated, consistent with a previous study (Fleiszig et al., 1992) while the other strains tested were piliated. The results suggest that the presence of pili alone does not dictate levels of adhesion to mucin. Even though pili may mediate the adherence of some P. aeruginosa isolates to mucin, its presence in other strains does not infer mucin-binding abilities, as ATCC 15442 and Paer12 expressed pili under the current experimental conditions, but both revealed significantly low adhesion to mucin. Recent studies suggest a role for the flagella, in particular the flagellar cap protein (FliD) in the adhesion of P. aeruginosa to mucin (Arora et al., 1998; Arora et al., 2000), where two types of FliD proteins in P. aeruginosa recognise different mucin carbohydrate epitopes (Scharfman et al., 2001). The P. aeruginosa strains tested in the current study contained flagella, however, future work examining the FliD protein from P. aeruginosa isolates may indicate potential correlations with the different levels of adhesion to mucin observed between isolates.

In conclusion, the present study aimed to purify secretory mucin and investigate microbial factors that may contribute to ocular pathogenesis by defeat of the ocular mucin barrier. This work has shown that certain contact lens isolates of P. aeruginosa have the capability to adhere to highly purified mucin and strains significantly differ in their intrinsic ability to adhere to mucin. In addition, the presence of pili on P. aeruginosa isolates was not determinative for mucin adhesion. It is proposed that under normal circumstances, ocular mucin inhibits P. aeruginosa from adhering to the underlying corneal epithelial cells; however, with contact lens wear, as the tear exchange is greatly reduced under the lens, the more avid mucin-adhering strains are given the opportunity to adhere and subsequently penetrate the mucous layer to initiate pathogenesis.

Acknowledgements This study was supported in part by grants from the Australian Federal Government under the Cooperative Research Centres Programme, and the American Optometric Foundation. The authors would like to thank Dr Anthony Corfield for advice concerning mucin purification procedures and Dr Jacques Bara for his kind donation of mucin antibodies.

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