Many bacteria including pathogens, commensals, and mutualisms utilize glycosphingolipids (GSLs) at the cell surface as receptors for initial infection or colonization in host animals (Karlsson KA. 1989; Svensson M et al. 2003). The host specificity of bacteria depends on, at least in part, the structure of the carbohydrate and ceramide moieties of GSLs. Determination of the specificity of bacteria adhering to GSLs could improve understanding of bacterial infections and diseases. This report describes a TLC-based method for identifying a GSLs as bacterial receptors (Chisada S et al. 2005; Matsunaga N et al. 2011). |
| Category | Roles of glycans during microbial infection |
| Protocol Name | Assay of the binding of bacteria to glycosphingolipids |
Authors
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Shin-ichi Chisada
*
Aquatic Animal Health Division, National Research Institute of Aquaculture, Fisheries Research Agency
Naoyuki Matsunaga
Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University
Makoto Ito
*
Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University
*To whom correspondence should be addressed.
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Reagents
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GSLs: purified from appropriate animal tissues or purchased from commercial sources |
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Thin-layer chromatography (TLC) plate: plastic TLC plate pre-coated with Silica Gel 60 |
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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. |
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Chromogenic reagents: orcinol-H2SO4 reagent for acidic/neutral GSLs, resorcinol-HCL reagent for acidic GSLs. |
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Phosphate buffer: 1.5mM KH2PO4, 14 mM Na2HPO4, 2.7mM 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) |
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Liquid medium: Suitable medium for bacterial growth, e.g. 1% polypeptone, 0.2% yeast extract, 0.05% MgSO47H2O, 75% filtrated seawater, 25% distilled water, pH7.2, for vibrios |
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Instruments
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TLC Developing chamber lined with a filter paper |
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Imaging plate (Fuji Film Co., Kanagawa, Japan) |
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Imaging analyzer FLA-5000 (Fuji Film Co., Kanagawa, Japan) |
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| Methods |
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1. |
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Apply GSLs*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. * is explained in Note. |
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Develop the plates using an appropriate solvent*2 to near their top. |
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Dry the plates completely with warm air using a hair dryer. |
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Visualize the GSLs on one plate using the chromogenic procedure*3. |
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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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Dry the plate completely at room temperature. |
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Metabolic labeling of bacteria with 35S-methionine |
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Culture the bacteria in 1 ml of liquid medium overnight with appropriate conditions*4 for the bacteria used. |
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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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Incubate the bacteria at optimum temperature*5 for 24 h. |
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Co-incubation of a GSL-loading TLC plate with 35S-labeled bacteria |
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Collect 35S-labeled bacterial cells in a centrifugation tube as a pellet by centrifugation (6,000 g for 5 min). |
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Add phosphate buffer*6 to the pellet and prepare bacterial suspension (2.0-3.0 x 1011 CFU/ml, 2.0-5.0 x 105 cpm/ml). |
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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*5 with gentle shaking. |
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4. |
Detection of GSLs adhering to bacteria |
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Wash the plate three times (each for 30 sec) with phosphate buffer*6. |
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Cover the TLC plate with plastic wrap. |
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Expose the wrapped plate to an imaging plate for 10 h. |
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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.
*1Each 30 nmol
*2Chloroform/methanol/water (60:35:8, v/v/v)
*3Orcinol-H2SO4 reagent
*4At 25oC with liquid medium (1% polypeptone, 0.2 % yeast extract, 0.05 % MgSO47H2O, 75 % filtrated seawater, 25 % distilled water, pH7.2)
*5At 25oC
*6Containing 3% NaCl
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| Figure & Legends |
Figure & Legends
Figure 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. |
| Copyrights |
 Attribution-Non-Commercial Share Alike
This work is released underCreative Commons licenses
|
| Date of registration：2012-11-09 15:38:47 |
Karlsson KA (1989) Animal GSLs as membrane attachment sites for bacteria. Annu Rev Biochem. 58: 309-350. [PMID : 2673013]
Svensson M, Frendeus B, Butters T, Platt F, Dwek R, Svanborg C. (2003) Glycolipid depletion in antimicrobial therapy. Mol Microbiol. 47: 453-461. [PMID : 12519195]
Chisada S, Horibata Y, Hama Y, Inagaki M, Furuya N, Okino N, Ito M. (2005) The glycosphingolipid receptor for Vibrio trachuri in the red sea bream intestine is a GM4 ganglioside which contains 2-hydroxy fatty acids. Biochem Biophys Res Commun. 333: 367-373. [PMID : 15979459]
Matsunaga, N., Chisada, S., et al. (2011) Glycosphingolipid receptors for pathogenic vibrios in intestines of mariculture fish. Fisheries Science. 77: 583-590. |
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