Mucin-Bacterial Binding Assays 429
429
35
Mucin-Bacterial Binding Assays
Nancy A. McNamara, Robert A. Sack, and Suzanne M. J. Fleiszig
1. Introduction
Surface epithelia throughout the body are covered by mucus, a protective secretion
that serves as a selective physical barrier between the epithelial cell plasma membrane
and the extracellular environment. Mucin, the glycoprotein constituent of mucus, has
been shown to bind bacteria at mucosal surfaces that line the lung, gut, bladder, oral
cavity, and eye (1–7). Since bacterial binding to an epithelial cell surface is generally
thought to be an important prerequisite for infection (8), the interaction between bac-
teria and mucin, together with normal mucosal clearance mechanisms, is believed to
act as a defense against infection by inhibiting bacterial adherence to the underlying
epithelial cell surface.
In support of mucin’s role as a nonspecific defense mechanism, malfunctions in the
production and/or clearance of mucin have been implicated in the etiology of many
diseases. This has led to the development of methods that can be used to study the effects
of disease and various interventions (e.g., drugs and medical devices) on the ability of
mucin to protect the underlying tissue. In this chapter, we present methods that can be
used to examine the interaction between bacteria and mucin, as well as the extent to
which this interaction serves to protect the epithelial cell surface from bacterial invasion.
We have used these methods to study the interactions of Pseudomonas aeruginosa with
the ocular surface in both human and animal models; however, they can also be used to
test bacterial/mucin interactions in other tissues with other organisms.
2. Materials
2.1. Preparation and Analysis
of Human Precorneal Tear Film Components
1. Noninvasive corneal irrigation chamber (9,10).
2. 0.9% NaCl (autoclaved).
3. Spectra/Por

®
cellulose ester membranes, 500 Da molecular weight (MW) cut-off (Fisher
Scientific, Fair Lawn, NJ).
4. BCA Protein assay kit (Pierce, Rockford, IL).
From:
Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The Mucins
Edited by: A. Corfield © Humana Press Inc., Totowa, NJ
430 McNamara et al.
5. Precast, 4–20% Tris-HCl Ready Gel (Bio-Rad, Hercules, CA) for sodium dodecyl sul-
fate-polyacrylamide gel electrophoresis (SDS-PAGE).
6. Silver stain kit (Bio-Rad).
7. Coomasie blue R-250 (Sigma, St. Louis, MO).
2.2. Isolation and Analysis of Human Ocular Sialoglycoprotein
1. Glass microcapillary tubes (calibrated, fire-polished, disposable, 10 µL capacity).
2. SW 4000 size-exclusion column.
3. 0.5 M NaCl, 0.1 M phosphate buffer, pH 5.0.
4. 20% Methanol (v/v).
5. 3-kDa Cutoff centrifugal ultrafilters (Filtron, Northborough, MA).
6. Precast, 4% and 4-20% Tris-glycine gels for SDS-PAGE (Novex, San Diego, CA).
7. Kaleidoscope™ (Bio-Rad) and See-Blue ™ (Novex) molecular weight ladders, 6-250 kDa.
8. Human lysozyme, IgG, lactoferrin, albumin and sIgA (Sigma).
9. Pre-stained molecular weight markers IgM (990 kDa) and thyroglobulin (669 kDa)
(Calbiochem
®
, La Jolla, CA).
10. Coomasie brilliant blue R-250 (Sigma).
11. Periodate silver and Alcian blue (AB) stains (Sigma).
12. Immobilon P membranes (Millipore, Bedford, MA).
13. Sialyl-Lewis epitope (clone 258-11413, O.E.M. Concepts, Toms River, NJ).
2.3. Preparation of Bacteria

1. P. aeruginosa (strain 6294, serogroup O6).
2. Trypticase soy agar (TSA) plates (PML Microbiologicals, Wilsonville, OR).
3. Phosphate-buffered saline (PBS), pH 7.4 (Sigma).
4. MacConkey agar plates (PML Microbiologicals).
5. Spectrophotometer.
2.4. Microtiter Plate Assay of Bacterial Adherence
1. Linbro/Titertek 96-well microtiter plate (ICN Biomedicals, Aurora, OH).
2. PBS, pH 7.4 (Sigma).
3. 0.50% Triton X-100 (LabChem, Pittsburgh, PA).
4. MacConkey agar plates (PML Microbiologicals).
5. Bovine submaxillary gland (BSG) mucin (Sigma).
2.5. Assay to Confirm Adherence of Mucin to Microtiter Wells
1. Same as Subheading 2.4., step 1 (Linbro/Titertek 96-well microtiter plate [ICN
Biomedicals]).
2. PBS, pH 7.4 (Sigma).
3. 1% Bovine serum albumin (BSA) in PBS (w/v).
4. Biotinylated wheat germ agglutinin (WGA) (Vector, Burlington, CA).
5. PBS/Tween: 0.25 mL Tween-20 (Sigma) in 500 mL PBS.
6. PBS/Tween:Streptavidin conjugated alkaline phosphatase (Jackson Immuno Research,
West Grove, PN), 500:1 (v/v).
7. Development solution: 5 mMp-nitrophenyl phosphate (NPP) in 0.1 M alkaline buffer
solution (Sigma).
8. Stop solution: 2 M Na
2
CO
3
(2.12 g Na
2
CO
3

/10 mL distilled water).
Mucin-Bacterial Binding Assays 431
2.6. Assay to Measure Bacterial Invasion of Corneal Epithelial Cells
2.6.1. Preparation of Rabbit Corneal Epithelial Cell Cultures
1. Cell culture-treated 96-well plates (Fischer).
2. Modified supplemental hormone essential medium (SHEM) containing 10 µg/mL of
bovine pituitary extract (11).
2.6.2. Gentamicin Survival Assay
1. Buffered minimal essential medium (BMEM): 9.53 g of MEM (Cellgro™) plus 2.2 g of
sodium bicarbonate per liter of distilled water (pH to 7.4).
2. Gentamicin sulfate (BioWhittaker, Walkersville, MD).
3. 0.25% Triton X-100 (LabChem).
4. BSG mucin (Sigma).
3. Methods
3.1. Preparation and Analysis
of Human Precorneal Tear Film Components
1. Collect precorneal tear film components (TFCs) from the ocular surface of human eyes
using a noninvasive corneal irrigation chamber by irrigating each cornea for 30 s with 10 mL of
sterile saline using a metered pump as previously described (9,10).
2. Remove cells and debris by centrifuging the eyewash samples three times at 6000 rpm for
15 min.
3. Dialyze the final supernatant against several changes of distilled water at 4°C and con-
centrate to 200 µL using vacuum centrifugation.
4. Determine the protein content of each 200 µL eyewash sample using the BCA protein
assay kit (12).
5. Separate tear film proteins using a precast 4–20% Tris-HCl gel, and visualize using a
silver staining procedure that is ideal for staining polysaccharides and highly glycosylated
proteins as recommended by the manufacturer (Bio-Rad) (13).
6. Counter-stain the silver-stained gels with 200 mL of 0.1% Coomassie brilliant blue
R-250 in 25% methanol/7.5% acetic acid for 1 h, destain overnight, and then photograph

in color as previously described (14) (see Note 1).
3.2. Isolation and Analysis of Human Ocular Sialoglycoprotein
1. Collect closed-eye tear samples (which are rich in high molecular weight sialoglycoprotein)
(15) from four human subjects over a period of several weeks as previously described (16).
2. Pool samples and centrifuge at 11,000 rpm in a refrigerated Eppendorf microfuge for
30 min, then repeat. Store the resultant supernatants at –70°C until needed.
3. Separate the supernatant isocratically in 15-µL aliquots on a SW 4000 size exclusion
column in 0.5 M NaCl, 0.1 M phosphate buffer (pH 5.0), at a flow rate of 0.25 mL/min,
while monitoring the eluent at 254 nm.
4. Concentrate each fraction using a 3-kDa centrifugal ultrafilter. To establish the elution
profile for all the major tear proteins, run fractions under both reducing and non-reducing
conditions at 125 V for one hour on a precast, 4–20% Tris-glycine gel for SDS-PAGE.
Use Kaleidoscope™ and See-Blue™ MW ladders, as well as human lysozyme, IgG,
lactoferrin, albumin, and sIgA as standards.
5. Using this method, the high molecular weight glycoprotein fractions are recovered slightly
after the void volume in the first peak, which elutes off the HPLC column at approxi-
432 McNamara et al.
mately 24 min. To prepare the high molecular weight glycoprotein, collect the initial
HPLC fraction, concentrate to 100 µL by centrifugal ultrafiltration, dilute 1:1 with HPLC
solvent, and separate into 15-µL aliquots.
6. Run high molecular weight glycoprotein fraction under both reducing and nonreducing
conditions on a precast 4% Tris-glycine gel with pre-stained molecular weight markers
IgM (990 kDa) and thyroglobulin (669 kDa).
7. To detect sialoglycoprotein (SG), periodate treat gel and stain with alcian blue in 3%
acetic acid as previously described (17).
8. For further characterization, transfer glycoprotein overnight onto Immobilon P in 20%
methanol (v/v) at 30 V, followed by 80 V for 1 h.
9. Probe with a panel of antibodies to specific known mucins, their core proteins, and com-
mon sugar epitopes (see Note 2).
3.3. Preparation of Bacteria for Binding Assay

1. Grow bacteria overnight at 37
°
C on TSA plates.
2. Wash bacteria three times in PBS by centrifugation at 7000 rpm for 5 min (9).
3. Prepare the inoculum by resuspending the washed bacteria into PBS until the optical
density at 650 nm reaches 0.1 (equivalent to 1 × 10
8
cfu/mL).
4. Quantify the starting inoculum used in each experiment (typically 1 × 10
6
cfu/mL) by
serially diluting the sample and plating 10 µL (in duplicate) on MacConkey agar.
3.4. Microtiter Plate Assay of Bacterial Adherence
3.4.1. To Determine Whether or Not Bacteria Bind
to Human Tear Film or Mucin (
see
Note 3)
1. Coat microtiter wells overnight at 37°C with 100 µL of tear film or mucin sample. Pretreat
control wells with PBS which does not promote bacterial adherence to these wells (5,7).
2. Wash wells four times with PBS to remove non-adherent material.
3. Prepare bacterial inoculum containing 1 × 10
6
cfu/mL in PBS (10 µL of 1 × 10
8
cfu/mL +
990 µL PBS).
4. Add inoculum containing 30 µL of 1 × 10
6
cfu/ml P. aeruginosa 6294 to all wells.
5. Incubate plate at 37°C for 30 min.

6. Aspirate bacteria with a sterile pipette and wash wells 20 times with PBS to remove
nonadherent bacteria.
7. Dislodge adherent bacteria from the well surface by adding 300 µL of 0.5% Triton X.
8. Incubate plate at 37°C for 30 min.
9. Vigorously stir each well with a sterile pipet and perform a viable count by plating 10 µL
(in duplicate) on MacConkey agar.
3.4.2. To Determine Whether or Not Mucin Blocks Bacterial Adherence
to Known Bacterial Binding Factors (
see
Note 4)
1. Coat microtiter wells overnight at 37°C with 100 µL of known bacterial binding factor
(e.g., TFCs or BSG mucin).
2. Wash wells four times with PBS to remove nonadherent material.
3. Coat microtiter wells with 100 µL of SG for 18 h at 37°C. Treat control wells with 100 µL of
PBS instead of SG since PBS does not affect bacterial binding to either TFCs or BSG mucin.
4. Prepare bacterial inoculum containing 1 × 10
5
cfu/mL in PBS (1 µL of 1 × 10
8
cfu/mL +
999 µL PBS).
5. Add inoculum containing 30 µL of 1 × 10
5
cfu/mL P. aeruginosa 6294 to all wells.
Mucin-Bacterial Binding Assays 433
6. Incubate plate at 37°C for 30 min.
7. Wash wells 20 times with PBS to remove nonadherent bacteria.
8. Dislodge adherent bacteria from the well surface by adding 300 µL of 0.5% Triton X.
9. Incubate plate at 37°C for 30 min.
10. Vigorously stir each well with a sterile pipet and perform a viable count by plating 10 µL

(in duplicate) on MacConkey agar.
3.4.3. To Determine Whether or Not Treating Bacteria with Mucin Blocks
Their Ability to Adhere to Known Bacterial Binding Factors (
see
Note 4)
1. Coat microtiter wells overnight at 37°C with 100 µL of known bacterial binding factor
(e.g., TFCs or BSG mucin).
2. Wash wells four times with PBS to remove nonadherent material.
3. Prepare bacterial inoculum containing 2 × 10
5
cfu/mL in PBS (2 µL of 1 × 10
8
cfu/mL +
998 µLl PBS).
4. Prepare starting inoculum by mixing 100 µL of 2 × 10
5
cfu/mLP. aeruginosa (prepared
above) with 100 µL of SG and incubate at 37°C for 1 h. Prepare inoculum for controls by
mixing 100 µL of 2 × 10
5
cfu/mL bacteria with 100 µL of PBS (see Note 5).
5. Add 30 µL of the starting inoculum containing P. aeruginosa 6294 (1 × 10
5
cfu/mL) and
either SG or PBS (control) to wells.
6. Incubate plate at 37°C for 30 min.
7. Wash wells 20 times with PBS to remove nonadherent bacteria.
8. Dislodge adherent bacteria from the well surface by adding 300 µL of 0.5% Triton X.
9. Incubate plate at 37°C for 30 minu.
10. Vigorously stir each well with a sterile pipet and perform a viable count by plating 10 µL

(in duplicate) on MacConkey agar.
3.5. Assay to Confirm Adherence of Mucin to Microtiter Wells
1. Coat 96-well microtiter plate overnight at 37°C with several dilutions of SG, TFC, and
BSG mucin.
2. Wash wells 24 times with PBS.
3. Block for 2 h at room temperature (or overnight at 4°C) by adding 200 µL of 1% BSA/
PBS to each well.
4. After blocking, wash wells twice with PBS/Tween and incubate with 100 µL of 5 µg/mL
biotinylated wheat germ agglutinin (WGA) for 45 min at room temperature (wrap plate in plas-
tic). WGA is a plant lectin that binds specifically to sialic acid residues, and thus, serves as a
probe for quantifying the amount of sialylated glycoprotein that is bound to the microtiter well.
5. Wash wells six times with PBS/Tween.
6. Detect WGA-bound biotin by adding 100 µL of PBS/Tween:streptavidin-peroxidase
solution to each well and incubating the wrapped plate at 37°C for 45 min.
7. Wash wells six times with PBS/Tween.
8. Add 100 µL of development solution (NPP in alkaline buffer) and incubate at room tem-
perature. Detect the sialoglycoprotein-lectin-enzyme complexes by adding the enzyme sub-
strate (NPP) which is converted to a colored product in the presence of enzyme. Immediately
begin monitoring the intensity at 405 nm using a standard ELISA reader. Readings should
be taken every 2 min since development occurs quickly. Controls consist of ovalbumin
treated wells incubated with the WGA and/or streptavidin system alone (18,19).
9. Stop the reaction by adding 10 µL of stop solution. This step can be omitted if the absorbance
is monitored continuously following the addition of development solution (see Note 6).