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Assay of the binding of bacteria to glycosphingolipids
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Assay of the binding of bacteria to glycosphingolipids

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Introduction Protocol References Credit lines
Category
Roles of glycans during microbial infection
Protocol Name

Assay of the binding of bacteria to glycosphingolipids

Authors
Chisada, Shin-ichi *
Laboratory of Bioresources, National Institute for Basic Biology, National Institutes of Natural Sciences

Matsunaga, Naoyuki
Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University

Ito, Makoto *
Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University
*To whom correspondence should be addressed.
KeyWords
Reagents

GSLs: purified from appropriate animal tissues or purchased from commercial sources

Thin-layer chromatography (TLC) plate: plastic TLC plate pre-coated with Silica Gel 60

Developing solvent system: chloroform/methanol/0.02% CaCl2 (5:4:1, v/v/v) for acidic/neutral GSLs, chloroform/methanol/water (60:35:8, v/v/v) for neutral GSLs.

Chromogenic reagents: orcinol-H2SO4 reagent for acidic/neutral GSLs, resorcinol-HCL reagent for acidic GSLs.

Diethyl ether

n-Hexane

Polyisobutylmethacrylate

PBS

Phosphate buffer: 1.5 mM KH2PO4, 14 mM Na2HPO4, 2.7 mM KCl, pH 7.2, containing NaCl (the concentration of NaCl depends on bacteria used, i.e., 0.85% for terrestrial bacteria and 2–3% for marine bacteria)

Bacteria

Liquid medium: Suitable medium for bacterial growth, e.g. 1% polypeptone, 0.2% yeast extract, 0.05% MgSO47H2O, 75% filtrated seawater, 25% distilled water, pH 7.2, for vibrios

[35S]methionine

Instruments

5 or 10 μL microsyringe

TLC Developing chamber lined with a filter paper

Hybridization bag

Shaking incubator

Imaging plate (FujiFilm, Tokyo, Japan)

Imaging analyzer FLA-5000 (FujiFilm)

Methods
1.

TLC of GSLs

1) 

 Apply GSLs (see Note 1) (10–60 nmol) onto two TLC plates, one for the visualization of GSLs by chemical reagents and the other for co-incubation with bacteria.

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2) 

 Develop the plates using an appropriate solvent (see Note 2) to near their top.

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3) 

 Dry the plates completely with warm air using a hair dryer.

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4) 

 Visualize the GSLs on one plate using the chromogenic procedure (see Note 3).

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5) 

 Dip the other plate in diethyl ether/n-hexane (1/5, v/v) containing 0.5% (w/v) polyisobutylmethacrylate for 30 sec.

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6) 

 Dry the plate completely at room temperature.

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2.

Metabolic labeling of bacteria with 35S-methionine

1) 

 Culture the bacteria in 1 mL of liquid medium overnight with appropriate conditions (see Note 4) for the bacteria used.

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2) 

 Add 50 μL of the bacterial culture to 5 mL of a sterile fresh medium containing [35S]methionine (2.0 mCi/mL).

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3) 

 Incubate the bacteria at optimum temperature (see Note 5) for 24 h.

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3.

Co-incubation of a GSL-loading TLC plate with 35S-labeled bacteria

1) 

 Collect 35S-labeled bacterial cells in a centrifugation tube as a pellet by centrifugation (6,000 × g for 5 min).

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2) 

 Add phosphate buffer (see Note 6) to the pellet and prepare bacterial suspension (2.0–3.0 × 1011 CFU/mL, 2.0–5.0 × 105 cpm/mL).

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3) 

 Co-incubate a GSL-loading TLC plate and 0.3 mL/TLC plate cm2 of the bacterial suspension in a plastic hybridization bag for 24 h at optimum temperature (see Note 5) with gentle shaking.

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4.

Detection of GSLs adhering to bacteria

1) 

 Wash the plate three times (each for 30 sec) with phosphate buffer (see Note 6).

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2) 

 Cover the TLC plate with plastic wrap.

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3) 

 Expose the wrapped plate to an imaging plate for 10 h.

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4) 

 Determine the activity of bacteria to bind each GSL by scanning the plate with an imaging analyzer.

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Notes

Conditions for the assay shown in Fig. 1 are described below.

  1. Each 30 nmol
  2. Chloroform/methanol/water (60:35:8, v/v/v)
  3. Orcinol-H2SO4 reagent
  4. At 25°C with liquid medium (1% polypeptone, 0.2% yeast extract, 0.05 % MgSO47H2O, 75% filtrated seawater, 25% distilled water, pH 7.2)
  5. At 25°C
  6. Containing 3% NaCl


Figure & Legends

Figure & Legends

 

Fig. 1 V. anguillarum NBRC12710 adhered to GM4 and GM3 on the TLC plate.

 

(a) TLC showing GSLs (GSLs) visualized with orcinol-H2SO4 reagent. Various GSLs were developed on a TLC plate with chloroform/methanol/water (60/35/8, v/v/v). The plate was visualized with orcinol-H2SO4 reagent. (b) TLC autoradiogram showing GSLs adhering to 35S-labeled V. anguillarum NBRC12710. GSLs were developed on a TLC plate under the same conditions as in panel (a). The plate was incubated with 35S-labeled V. anguillarum NBRC12710, and visualized by autoradiography as described in the Methods. In both panels, the left lane contained authentic LacCer, GM3 and GM1a from the top and the right lane contained authentic GalCer, sulfatide and GM4 from the top. The amount of each GSL was 30 nmol.

This figure was obtained and modified from Fisheries Science. 77, 2011, 583–590, "Glycosphingolipid receptors for pathogenic vibrios in intestines of mariculture fish", Matsunaga N., Chisada S., Fujioka H., Takashima K., & Okino N. Springer Japan.

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