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Novel entries in a fungal biofilm matrix encyclopedia.

Zarnowski R, Westler WM, Lacmbouh GA, Marita JM, Bothe JR, Bernhardt J, Lounes-Hadj Sahraoui A, Fontaine J, Sanchez H, Hatfield RD, Ntambi JM, Nett JE, Mitchell AP, Andes DR - MBio (2014)

Bottom Line: Newly described, more abundant polysaccharides included α-1,2 branched α-1,6-mannans (87%) associated with unbranched β-1,6-glucans (13%) in an apparent mannan-glucan complex (MGCx).Importance: This report is the first to decipher the complex and unique macromolecular composition of the Candida biofilm matrix, demonstrate the clinical relevance of matrix components, and show that multiple matrix components are needed for protection from antifungal drugs.The availability of these biochemical analyses provides a unique resource for further functional investigation of the biofilm matrix, a defining trait of this lifestyle.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Infectious Diseases, University of Wisconsin, Madison, Wisconsin, USA.

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Related in: MedlinePlus

Chromatographic fractionation and NMR analysis of carbohydrates from the C. albicans biofilm extracellular matrix. (A) Initial fractionation employing flash size exclusion chromatography on the HiPrep 26/10 Desalting Sephadex G-25 Fine column. The matrix sample was separated into the Man/Glc-rich HMW fraction (38.3%) and the Ara-rich LMW fraction (61.7%). Proteins were detected at 280 nm. (B) Fractionation of the HMW fraction on the weak anion exchanger HiPrep 16/10 DEAE FF column resulted in separation of uncharged protein-free polysaccharides from charged proteins and glycoproteins. Polysaccharides were eluted in the flowthrough fraction (shown as FT), whereas multiple (glyco) protein peaks, D1 to D8, were separated in the applied salt gradient (dashed line with larger spaces) as reflected by increasing conductivity (dashed line with smaller spaces). (C) Purification of neutral polysaccharides in gel filtration chromatography on the HighPrep 16/60 Sephacryl S-300 HR column. This procedure yielded a total of 22 individual polysaccharide-positive peaks, F1 to F22. (D) Size exclusion column calibration with a set of Leuconostoc species dextran standards at the following molecular weights: 100, 70, 40, 25, and 6. Positions of the select purified polysaccharides, F2, F5, F10, F14, F16, and F17, are indicated by reverse triangles. (E) Comparison of the 500-MHz 1H NMR spectra of representative purified matrix neutral polysaccharides, F2, F5, F10, F14, F16, and F17, from the C. albicans biofilm matrix.
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fig2: Chromatographic fractionation and NMR analysis of carbohydrates from the C. albicans biofilm extracellular matrix. (A) Initial fractionation employing flash size exclusion chromatography on the HiPrep 26/10 Desalting Sephadex G-25 Fine column. The matrix sample was separated into the Man/Glc-rich HMW fraction (38.3%) and the Ara-rich LMW fraction (61.7%). Proteins were detected at 280 nm. (B) Fractionation of the HMW fraction on the weak anion exchanger HiPrep 16/10 DEAE FF column resulted in separation of uncharged protein-free polysaccharides from charged proteins and glycoproteins. Polysaccharides were eluted in the flowthrough fraction (shown as FT), whereas multiple (glyco) protein peaks, D1 to D8, were separated in the applied salt gradient (dashed line with larger spaces) as reflected by increasing conductivity (dashed line with smaller spaces). (C) Purification of neutral polysaccharides in gel filtration chromatography on the HighPrep 16/60 Sephacryl S-300 HR column. This procedure yielded a total of 22 individual polysaccharide-positive peaks, F1 to F22. (D) Size exclusion column calibration with a set of Leuconostoc species dextran standards at the following molecular weights: 100, 70, 40, 25, and 6. Positions of the select purified polysaccharides, F2, F5, F10, F14, F16, and F17, are indicated by reverse triangles. (E) Comparison of the 500-MHz 1H NMR spectra of representative purified matrix neutral polysaccharides, F2, F5, F10, F14, F16, and F17, from the C. albicans biofilm matrix.

Mentions: Monosugar analysis of the total biofilm matrix revealed four relatively abundant monosaccharides, arabinose (Ara), mannose (Man), glucose (Glc), and xylose (Xyl), which accounted for 47.9%, 20%, 12.5%, and 12.6% of the total carbohydrate pool. Initial size exclusion chromatography of the crude matrix yielded two major pools: a high-molecular-weight (HMW) fraction and low-molecular-weight (LMW) fraction that contained 38.3% and 61.7% of the matrix material, respectively (Fig. 2A). Of the total matrix carbohydrates, 27.1% separated in the LMW pool, which contained mostly Ara (52.2%) with smaller amounts of Man, Xyl, and Glc (13.8%, 13.8%, and 12.9%, respectively). The HMW pool contained 21.6% of the total carbohydrate and consisted primarily of Man (89.2%) and Glc (8.6%).


Novel entries in a fungal biofilm matrix encyclopedia.

Zarnowski R, Westler WM, Lacmbouh GA, Marita JM, Bothe JR, Bernhardt J, Lounes-Hadj Sahraoui A, Fontaine J, Sanchez H, Hatfield RD, Ntambi JM, Nett JE, Mitchell AP, Andes DR - MBio (2014)

Chromatographic fractionation and NMR analysis of carbohydrates from the C. albicans biofilm extracellular matrix. (A) Initial fractionation employing flash size exclusion chromatography on the HiPrep 26/10 Desalting Sephadex G-25 Fine column. The matrix sample was separated into the Man/Glc-rich HMW fraction (38.3%) and the Ara-rich LMW fraction (61.7%). Proteins were detected at 280 nm. (B) Fractionation of the HMW fraction on the weak anion exchanger HiPrep 16/10 DEAE FF column resulted in separation of uncharged protein-free polysaccharides from charged proteins and glycoproteins. Polysaccharides were eluted in the flowthrough fraction (shown as FT), whereas multiple (glyco) protein peaks, D1 to D8, were separated in the applied salt gradient (dashed line with larger spaces) as reflected by increasing conductivity (dashed line with smaller spaces). (C) Purification of neutral polysaccharides in gel filtration chromatography on the HighPrep 16/60 Sephacryl S-300 HR column. This procedure yielded a total of 22 individual polysaccharide-positive peaks, F1 to F22. (D) Size exclusion column calibration with a set of Leuconostoc species dextran standards at the following molecular weights: 100, 70, 40, 25, and 6. Positions of the select purified polysaccharides, F2, F5, F10, F14, F16, and F17, are indicated by reverse triangles. (E) Comparison of the 500-MHz 1H NMR spectra of representative purified matrix neutral polysaccharides, F2, F5, F10, F14, F16, and F17, from the C. albicans biofilm matrix.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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fig2: Chromatographic fractionation and NMR analysis of carbohydrates from the C. albicans biofilm extracellular matrix. (A) Initial fractionation employing flash size exclusion chromatography on the HiPrep 26/10 Desalting Sephadex G-25 Fine column. The matrix sample was separated into the Man/Glc-rich HMW fraction (38.3%) and the Ara-rich LMW fraction (61.7%). Proteins were detected at 280 nm. (B) Fractionation of the HMW fraction on the weak anion exchanger HiPrep 16/10 DEAE FF column resulted in separation of uncharged protein-free polysaccharides from charged proteins and glycoproteins. Polysaccharides were eluted in the flowthrough fraction (shown as FT), whereas multiple (glyco) protein peaks, D1 to D8, were separated in the applied salt gradient (dashed line with larger spaces) as reflected by increasing conductivity (dashed line with smaller spaces). (C) Purification of neutral polysaccharides in gel filtration chromatography on the HighPrep 16/60 Sephacryl S-300 HR column. This procedure yielded a total of 22 individual polysaccharide-positive peaks, F1 to F22. (D) Size exclusion column calibration with a set of Leuconostoc species dextran standards at the following molecular weights: 100, 70, 40, 25, and 6. Positions of the select purified polysaccharides, F2, F5, F10, F14, F16, and F17, are indicated by reverse triangles. (E) Comparison of the 500-MHz 1H NMR spectra of representative purified matrix neutral polysaccharides, F2, F5, F10, F14, F16, and F17, from the C. albicans biofilm matrix.
Mentions: Monosugar analysis of the total biofilm matrix revealed four relatively abundant monosaccharides, arabinose (Ara), mannose (Man), glucose (Glc), and xylose (Xyl), which accounted for 47.9%, 20%, 12.5%, and 12.6% of the total carbohydrate pool. Initial size exclusion chromatography of the crude matrix yielded two major pools: a high-molecular-weight (HMW) fraction and low-molecular-weight (LMW) fraction that contained 38.3% and 61.7% of the matrix material, respectively (Fig. 2A). Of the total matrix carbohydrates, 27.1% separated in the LMW pool, which contained mostly Ara (52.2%) with smaller amounts of Man, Xyl, and Glc (13.8%, 13.8%, and 12.9%, respectively). The HMW pool contained 21.6% of the total carbohydrate and consisted primarily of Man (89.2%) and Glc (8.6%).

Bottom Line: Newly described, more abundant polysaccharides included α-1,2 branched α-1,6-mannans (87%) associated with unbranched β-1,6-glucans (13%) in an apparent mannan-glucan complex (MGCx).Importance: This report is the first to decipher the complex and unique macromolecular composition of the Candida biofilm matrix, demonstrate the clinical relevance of matrix components, and show that multiple matrix components are needed for protection from antifungal drugs.The availability of these biochemical analyses provides a unique resource for further functional investigation of the biofilm matrix, a defining trait of this lifestyle.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Infectious Diseases, University of Wisconsin, Madison, Wisconsin, USA.

Show MeSH
Related in: MedlinePlus