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. 2011 Aug;17(4):137-41.
doi: 10.1016/j.anaerobe.2011.05.017. Epub 2011 Jun 2.

The human commensal Bacteroides fragilis binds intestinal mucin

Julie Y Huang  1 , S Melanie Lee, Sarkis K Mazmanian
Affiliations

The human commensal Bacteroides fragilis binds intestinal mucin

Julie Y Huang et al. Anaerobe. 2011 Aug.

Abstract

The mammalian gastrointestinal tract harbors a vast microbial ecosystem, known as the microbiota, which benefits host biology. Bacteroides fragilis is an important anaerobic gut commensal of humans that prevents and cures intestinal inflammation. We wished to elucidate aspects of gut colonization employed by B. fragilis. Fluorescence in situ hybridization was performed on colonic tissue sections from B. fragilis and Escherichia coli dual-colonized gnotobiotic mice. Epifluorescence imaging reveals that both E. coli and B. fragilis are found in the lumen of the colon, but only B. fragilis is found in the mucosal layer. This observation suggests that physical association with intestinal mucus could be a possible mechanism of gut colonization by B. fragilis. We investigated this potential interaction using an in vitro mucus binding assay and show here that B. fragilis binds to murine colonic mucus. We further demonstrate that B. fragilis specifically and quantitatively binds to highly purified mucins (the major constituent in intestinal mucus) using flow cytometry analysis of fluorescently labeled purified murine and porcine mucins. These results suggest that interactions between B. fragilis and intestinal mucin may play a critical role during host-bacterial symbiosis.

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Figures

Fig. 1
Fig. 1
Colon tissue section from B. fragilis and E. coli dual-colonized Swiss Webster mouse. Epifluorescence image of bacteria visualized by FISH, and the epithelial cells counterstained with DAPI (blue) to visualize DNA. Both E. coli (green) and B. fragilis (red) are found in the lumen but only B. fragilis is found in the mucus layer.
Fig. 2
Fig. 2
B. fragilis binds intestinal mucus. (A) Number of B. fragilis (in CFUs) recovered after one hour incubation in wells with an immobilized mucus layer, an immobilized BSA layer, or buffer only. Of the 1×108 CFUs incubated, 1.6×106 (1.6%) bound to immobilized mucus. The CPM1 mutant binds mucus similarly to wild-type bacteria. These data are representative of four independent trials. (B) Number of bacteria recovered from mucus binding assay after a 2 hour pre-incubation with different concentrations of excess mucus. These data are representative of three independent trials.
Fig. 3
Fig. 3
Schematic of the mucin purification protocol. Briefly, crude mucus was scraped from the colon and cecum of conventionally colonized 8-week-old male Swiss Webster mice into 0.1 M NH4HCO3, 0.5 M NaCl, and a cocktail of protease inhibitors on ice. The sample was then homogenized and centrifuged at 45,000 × g for 45 minutes at 4°C. Non-soluble material was removed before centrifuging again at 45,000 × g for another 45 minutes at 4°C. The supernatant was taken and dialyzed against 10 mM Tris pH 8.0 + 150 mM NaCl for about 24 hours. Next, the sample was sonicated at eight 15-second pulses with intermediate 1 minute cooling on ice on a Brason Sonicator at speed 3 to break up large aggregates and then centrifuged once more at 45,000 × g for 45 minute. Next HPLC was employed whereby the supernatant was size fractionated on a XK 26/70 column containing Sephacryl S-400 resin (equilibrated in 10 mM Tris, pH 8.0). The void volume (which contained the large mucin glycoproteins) was collected and dialyzed against water for about 36 hours and then lyophilized. The lyophilized glycoproteins were resuspended in a solution containing RNase A and DNase I and digested for 2 hours at room temperature. After the digestion, the sample was centrifuged at 27,000 × g for 30 minutes at 4 °C and the supernatant was dialyzed against phosphate buffered saline (PBS) for 36 hours. Cesium chloride was added to the dialyzed supernatant to a final concentration of 0.54 g/ml, and then centrifuged at 160,000 × g for 72 hours. One milliliter fractions were collected and analyzed with the Pro Q Emerald Glycoprotein Staining Kit to determine which fractions contained the purified mucins. The mucin-containing fractions were pooled, dialyzed against water for 24 hours, lyophilized, and then stored at -20°C. Positive fractions from gel filtration chromatography were identified by absorbance readings at 280 nm. CsCl fractions and final product were assayed to contain mucin by glycoprotein staining (data not shown).
Fig. 4
Fig. 4
B. fragilis binds soluble murine and porcine mucin. (A) Flow cytometry plots indicating percentage of B. fragilis bound to fluorescently labeled murine colonic mucin. Cells were either pre-incubated with BSA or not (1°), and secondary incubations were with fluorescently labeled BSA or mucin (denoted by asterisk). Percentages represent bacteria bound to fluorescently-labeled mucin relative to total number of bacteria analyzed per sample. These data are representative of two independent trials. (B) Percentage of B. fragilis bound to fluorescently labeled porcine mucin with no pre-incubation. These data are representative of two independent trials. (C) Percentage of B. fragilis bound to fluorescently labeled porcine gastric mucin following pre-incubation with unlabeled mucin (left) or unlabeled BSA (right). Porcine mucin was purchased from a commercial source and purified as described in Figure 3 from the RNase/DNase digestion step. These data are representative of two independent trials.
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