Keywords

Introduction

The fungal wall is responsible for the cell shape, provides mechanical protection, and supports the internal osmotic pressure of fungal cells. In addition, it acts as a filter for large molecules, and its rigid structure is useful for penetration into and colonization of insoluble substrates. The cell wall is also the surface of interaction between pathogenic fungi and their host. Indeed the host defense response is usually directed against the cell wall. This structure is not simply a rigid exoskeleton but has the elasticity necessary to permit morphological changes during fungal growth and life cycle.

To build the walls, fungal cells need to synthesize wall components, export them across the plasma membrane, and assemble them outside the cell. The wall is composed basically of polysaccharides (70–90%) and glycoproteins (10–30%). Although composition varies among fungal species, and may even vary within a single fungal isolate, depending upon the growth conditions, most walls have a common structure [1]. When observed by transmission electron microscopy (TEM) the cell walls show a dark external layer formed by glycoproteins and an internal layer more transparent to the electrons, which mainly contains fibrillar polysaccharides (Fig. 12.1). The major fungal wall fibrillar components are: glucose homopolymers, β(1,3)-d-glucan with some β(1,6) branches, that constitutes 48–54% of total cell wall polysaccharides; chitin, a β(1,4)-N-acetylglucosamine polymer; and α(1,3)(1,4)-d-glucan. Chitin accounts for only 1–2% of yeasts wall [2, 3], whereas filamentous fungi, such as Neurospora or Aspergillus, contain 10–20% chitin in their walls [1]. In both yeasts and filamentous fungi, chitin forms microfibrils from interchain hydrogen bonding that have enormous tensile strength and significantly contribute to the overall integrity of the cell wall [4].

Fig. 12.1
figure 00121

Structure and composition of fungal cell wall. Transmission electron micrograph (TEM) of a fission yeast cell. A TEM detail of the cell wall is presented in the lower panel with a scheme of the organization and composition of the two main cell wall layers—electron dense and electron transparent layers—and the inner plasma membrane-bound glycoproteins

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The wall polysaccharides are formed at the plasma membrane by synthase enzymes and extruded into the periplasmic space where they bind to each other [5–7]. The linkages among the different components, which results in a tightly linked network, are generated by transglycosylation [8, 9] and are responsible for the mechanical strength of the cell wall [5–7, 10].

The formation and remodeling of the cell wall involves several biosynthetic pathways and the concerted actions of numerous gene products within the fungal cell. Many of the genes involved in cell wall synthesis or regulation have been cloned by complementation of mutants altered in wall structure or defective in the biosynthesis of cell wall components. Those mutants were isolated in many different ways, reflecting the complexity of functions involved in cell wall integrity and cell viability. Moreover, many of the genes and enzymes critical for assembly and biogenesis of fungal walls remain unidentified or poorly characterized. The main studies on fungal wall composition and biosynthesis have been performed in Saccharomyces cerevisiae [3, 11] but can be extended to other fungi.

Cell Wall Components

Glucan

Glucan is the main structural polysaccharide of the wall, and it represents 50–60% of this structure’s dry weight. The majority of glucan polymers are composed of glucose units with β(1,3) bonds (65–90%), although there are also some β(1,6), β(1,3)(1,4) and β(1,4) glucans. Usually the main backbone is β(1,3)-d-glucan with β(1,6) branches (Fig. 12.2). The β(1,3)-d-glucan is synthesized by a complex of enzymes known as glucan synthases located in the plasma membrane. These enzymes catalyze the formation of linear glucan chains composed of, approximately, 1,500 β(1,3)-bound glucose residues. In these linear chains, new glucose units bind, forming β(1,6) branches in variable proportion depending on the organism—from almost linear to highly branched—which can bind to other glucans, to chitin or to glycoproteins, providing a great mechanical resistance to the wall, which is essential to maintain the fungal cell integrity (see Fig. 12.2).

Fig. 12.2
figure 00122

Schematic representation of the synthesis and organization of the β-glucans forming the central core of the fungal cell wall. Linear β(1,3)-glucan chains are initially synthesized in the plasma membrane by the glucan synthase. Then cell wall transglycosidases form β(1,6)-branched β(1,3)-glucan that is also linked to β(1,6)-glucan through the side chains and to chitin via β(1,4) linkages. Proteins are covalently attached to β(1,6)-glucans (GPI-CWP) through the GPI remnant or to β(1,3)-glucans (PIR-CWP) through a glutamine residue following a transglutaminase reaction

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The genes coding for the putative β(1,3)-d-glucan synthase catalytic subunit were initially identified in S. cerevisiae and named FKS1 and FKS2 [12, 13]. The Fks protein family of β(1, 3)-d-glucan synthase is very well conserved in fungi and plants. Orthologs of these genes have been described in the main fungal genera such as Schizosaccharomyces, Candida, Aspergillus, Cryptococcus or Pneumocystis [1, 3, 14]. Besides the catalytic subunit, fungal glucan synthases (GS) require GTP-bound Rho1 GTPase for their activity [15, 16]. This family of enzymes use uridine-diphospho-glucose (UDP-Glc) as substrate and catalyze the reaction 2 UDP-Glc  →  [Glc-β-1,3-Glc].

A second β-linked glucan contained in most fungal walls is the β(1,6)-glucan. This polymer is shorter than β(1,3)-glucan, it does not form a fibrillar structure, and acts as a flexible glue by forming covalent cross-links to β(1,3)-glucan, chitin, and glycoproteins [6].

Some fungi contain α(1,3)(1,4)-glucan in their cell wall. However, the corresponding in vitro α(1,3)-glucan synthase activity has not been described yet. A putative catalytic subunit was first described in Schizosaccharomyces pombe [17, 18]. Ags1/Mok1 is a multidomain integral membrane protein with a predicted domain highly similar to starch synthase in the inner side and another domain similar to α-amylase and other proteins implicated in glycogen metabolism in the outer side. S. pombe contains five genes coding Ags/Mok proteins, and genomes of other fungi, including several human fungal pathogens in which cell wall α-glucan accounts for around 35% of the total wall polysaccharides, contain sequences of predicted proteins homologous to these genes [19, 20].

Chitin

Chitin is a β(1,4)-linked homopolymer of N-acetylglucosamine present in the cell walls of all fungi studied to date with the exception of S. pombe. Chitin represents 1–2% of the dry weight of the yeast cell wall whereas in the filamentous fungi it can reach up to 10–20% [1]. Chitin is synthesized from N-acetylglucosamine units by the enzyme chitin synthase (CS) that deposits microfibrils of chitin outside of the plasma ­membrane. This family of enzymes use ­uridine-diphosphate-N-acetylglucosamine (UDP-GlcNAc) as substrate and catalyze the reaction 2 UDP-GlcNAc  →  [GlcNAc-β-1,4-GlcNAc]. Chitin biosynthesis has been mainly studied in S. cerevisiae, which has three chitin synthases (CS1–3) responsible for the synthesis of chitin [21] at different times and places during cell growth. The number of chitin synthase genes varies from 1 to 20 according to the fungal species. The large family of chitin synthase (CS) enzymes fall into seven classes according to the evolution of their amino acid sequences [22]. The multiplicity of enzymes suggests that they have redundant roles in chitin synthesis and makes it difficult to find functional significance to the different classes [23].

Glycoproteins

Glycoproteins represent 30–50% of the dry weight of the S. cerevisiae or Candida walls, and around 20% of the dry weight of S. pombe and the filamentous fungi walls. These wall proteins have diverse functions, participating in the maintenance of the cellular form, taking part in adhesion processes, transmitting signals to cytoplasm, and remodeling the components of the wall. The glycoproteins present in the cell wall are extensively modified with both N- and O-linked carbohydrates, predominantly or exclusively formed by mannose residues known as mannan. In some cases, the mannan backbone presents single residues or side chains of different sugars, galactomannan, rhamnomannan, glucogalactomannan, rhamnogalactomannan, etc. [1, 24, 25]. Most cell wall proteins are attached through a glycosylphophatidyl inositol (GPI) remnant to β(1,3)-glucan or chitin, via a branched β(1,6)-glucan linker [11].

Analysis of the Cell Wall Synthases

We will present here the methods described for in vitro measurements of the enzymatic activities responsible for the biosynthesis of the two main structural wall polysaccharides: β(1,3)-glucan synthase (GS) and chitin synthase (CS). Both are integral membrane proteins, localized in the plasma membrane with their catalytic sites facing the inner side of the membrane.

β(1,3)-Glucan Synthase (GS)

The original method to detect the in vitro β(1,3)-glucan synthase activity was described nearly three decades ago [4, 26, 27]. This method has been modified, simplified and improved but essentially the basis of the protocol remains unaltered [28, 29].

Membrane Extracts Preparation

The source of enzyme activity is a crude membrane extract partially purified from the total cell extract.

  1. 1.

    Cell cultures (100 mL) are collected at early log-phase (A600 0.7–1.0) and centrifuged 5 min at 4 °C, 3,000  ×  g (5,000 rpm in a GSA-type rotor).

  2. 2.

    Cells are suspended in 30–40 mL cold buffer A (50 mM Tris–HCl pH 7.5, 1 mM EDTA, and 1 mM β-mercaptoethanol, centrifuged 5 min at 4 °C, 3,000  ×  g (6,000 rpm in a SS34-type rotor), transferred to a 1.5-mL tube (with screw cap), washed with 1 mL cold buffer A (1 min at 16,000  ×  g, 13,200 rpm) and resuspended in 100 μL cold buffer A containing 50 μM GTPγS (GTPγS is more stable than GTP, not hydrolysable and therefore, better GS activator). Glucan synthase is very labile and GTPγS is very useful to preserve the enzyme activity.

  3. 3.

    Cells are broken with glass beads (0.5 mm diameter, filling in all the liquid with glass beads and discarding by gentle drop out the excess of beads not entrapped by liquid capillarity) in a FastPrep apparatus (Q-Biogene, MP Biomedicals, Thermo Scientific) during 15 s at a speed of 6.0 and at 4 °C if possible. Alternatively, the cells can be broken in glass tubes with glass beads by 6 or 7 cycles of vortexing for 30 s and cooling down in ice for another 30 s. Due to enzyme instability, the rest of process must be done at 4 °C and the sample must be kept on ice.

  4. 4.

    Broken material and glass beads are diluted with 30 mL buffer A. Beads and cell debris are removed by low speed centrifugation, 5 min at 4 °C and 3,000  ×  g.

  5. 5.

    The supernatant is then centrifuged at 36,000–38,000  ×  g for 30 min at 4 °C and the membrane pellet is resuspended carefully by using a glass stick and a vortex to extend the membrane material throughout the entire bottom surface of the tube. Then 25 μL of buffer A containing 33% glycerol and 50 μM GTPγS is added and the membranes are homogenized by vortexing with the glass stick throughout the tube surface. The process is repeated with another 25, 50, and 50 μL of the same buffer until the membranes are homogenized in 100–150 μL and stored at −80 °C.

  6. 6.

    A homogeneous emulsion of membrane extract is critical for a reproducible GS assay. The amount of protein is quantified by using the Bradford dye-binding assay (Bio-Rad) with bovine serum albumin as standard. The protein concentration of the enzyme extract is usually kept at 3–5 mg/mL.

GS Assay

The GS mixture contains 5 mM UDP-[14C]-glucose (200 cpm/nmol) (PerkinElmer), 150 μM GTP or GTPγS, 0.75% bovine serum albumin, 2 mM EDTA, 75 mM Tris–HCl pH 8.0, and 5 μL of enzyme extract (15–25 μg of protein) in a total volume of 40 μL. The correct amount of protein in the assay is critical: higher protein concentration does not result in proportional increase of GS activity; therefore, the relative GS activity per milligram of protein decreases.

The reaction mixture is incubated for 30–60 min at 30 °C and stopped by addition of 1 mL of 10% trichloroacetic acid (TCA). The samples are kept at least 30 min at 4 °C, filtered in Whatman GF/C glass fiber filters, and washed three times with 1 mL of 10% TCA and twice with 1 mL of ethanol. The filters are placed into vials, 2 mL of liquid scintillation is added and the radioactivity of the filters is measured in a Beckman scintillation counter. One unit of GS activity is the amount of enzyme that catalyzes the incorporation of 1 μmol of glucose into glucan per min at 30 °C. The specific activity is expressed as milliunits per mg of protein and the reactions are always performed in duplicate. The GS enzyme is very labile and therefore, the data for each GS assay must be reproducible and calculated from at least three to four independent experiments.

When the membrane extract is obtained in the absence of GTPγS, we can measure basal and maximal GS activity by omitting or adding GTP to the reaction mixture. Alternatively, the GS assay may be done at pH 7.0–7.5 and may contain 25–30 mM potassium or sodium fluoride [4, 12, 30–37] and 0.5% Brij-35 [38, 39].

The detergents 2% Tergitol NP-40 (with 2 M NaCl) or 1.0% CHAPS are used for GS fractionation and solubilization of the regulatory subunit [12, 15, 35, 40–43], and 0.5% CHAPS, 0.1% cholesteryl hemisuccinate, or 0.1–0.2% CHAPS, 0.5–1.12% octyl glucoside are used for partial solubilization of GS microsomal fractions [30, 32, 34, 35, 44].

The GS reaction product can be confirmed to be β(1,3)-glucan, not a contaminant product such as glycogen, by degradation with Zymolyase-100T (AMS Biotechnology) or Kitalase (Wako Pure Chemical Industries). Zymolyase-100T is a preparation partially purified by affinity chromatography from Arthrobacter luteus that ­contains β(1,3)-glucanase, protease, and ­mannanase activities, but it does not contain β(1,6)-glucanase and α-glucanase activities. Similarly, Kitalase is a ­preparation from Rhizoctonia solani with β(1,3)-glucanase, ­protease, hemicellulase, pectinase, and amylase activities, but it does not contain α(1,3)-glucanase activity. Kitalase is also named as Lysing enzymes from Rhizoctonia solani (Sigma-Aldrich) or as Yeast Lytic Enzyme from Rhizoctonia solani (MP Biomedicals).

The degradation mixture contains the GS reaction product (40 μL), either 20 μg Zymolyase-100T, 50 mM citrate-phosphate pH 5.6 or 25 μg Kitalase, 50 mM potassium-acetate pH 5.0, and either 0.2% Tween 20 or Triton X-100 (1% detergent produces lower degradation) in a volume of 300 μL. The mixture is incubated 15–24 h at 30 °C with shaking, stopped with 10% TCA and processed as a standard GS assay. Zymolyase or Kitalase degradation in the absence of detergent is not complete, with a 15–20% residual product, likely due to the protection conferred by membrane vesicles. Other enzyme complexes result in only partial degradation of the reaction product.

Microtiter-Based Fluorescence Assay

This method has been described as an alternative to the use of UDP-[14C]-glucose for the GS assay [39]. This method takes advantage of substituting radioactive substrate for the fluorochrome aniline blue that is specific for linear β(1,3)glucan. Aniline binding is proportional to the amount of linear glucan, and it can be measured in a microplate fluorescence reader (excitation at 400 nm, emission at 460 nm). The GS mixture (50 μL total volume, 100 μg of enzyme protein) is similar to that described above except that UDP-[14C]-glucose is omitted and it may contain 0.5% Brij-35. The reactions are performed in microtiter plate wells at 30 °C for 30–60 min and stopped with 10 μL of 6 N NaOH. The glucan product is solubilized by heating at 80 °C for 30 min followed by the addition of 210 μL of aniline blue mix (1 mL contains 400 μL of 0.1% aniline blue, 210 μL of 1 N HCl, and 590 μL of 1 M glycine/NaOH pH 9.5). The plate is incubated at 50 °C for 30 min and at room temperature for another 30 min to allow reaction with the fluorochrome. Then, the fluorescence is quantified in a fluorescence reader. Linear β(1,3)-glucans (such as pachyman, curdlan, or yeast glucan ­dissolved in 1 N NaOH at 80 °C for 30 min) are used as standards, in a reaction mixture containing the same components except the membrane extract.

In Situ GS Assay Using Permeabilized Whole Cells

This method takes advantage of being a more direct enzymatic assay, omitting the steps of membrane extract preparation. In addition, this method can be applied for GS, chitin synthase, or other membrane-bound or cytosolic enzymes. The enzyme activity in permeabilized cells can yield similar or higher activities than those in cell extracts [45, 46]. The procedure is as follows:

  1. 1.

    Early log-phase cells are collected by centrifugation (1,500  ×  g, 5 min) and suspended in 40 mM EDTA, 100 mM β-mercaptoethanol (3.5 mL per g of cells wet weight).

  2. 2.

    The cells are incubated at 30 °C for 30 min with shaking, collected by centrifugation at 3,000  ×  g for 5 min, washed with 5 mL of 1.2 M sorbitol, resuspended (7 mL per g of cells) in 50 mM citrate phosphate pH 6.3, 1 mM EDTA, 1.2 M sorbitol, and incubated at 30 °C for another 30 min with shaking. Similar result can be obtained keeping the cells on ice for 30 min without shaking. Then, the cells are centrifuged at 3,000  ×  g for 5 min, suspended in 30 mL of cold 50 mM Tris–HCl pH 7.5 for osmotic shock, kept on ice for 5 min and centrifuged at 13,000  ×  g for 5 min.

  3. 3.

    The cells are suspended (1–1.5 mL per g of cells) in the buffer of the reaction assay of choice (50 mM Tris–HCl pH 7.5, 33% glycerol for GS and CS assays) and stored at −80 °C for weeks. Under those conditions, more that 90% of the cells are permeabilized (measured by staining with methylene blue).

Alternatively, the cells can be collected, washed with ice-cold water, suspended in cold 50 mM Tris–HCl pH 7.5, 1 mM EGTA, 1 mM β-mercaptoethanol, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 33% glycerol, and kept for 10 min in ice. 3.5 M glycerol can be replaced by 1.2 M glycerol, sorbitol, mannitol, or 1 M KCl. The cells are washed and suspended (1 g of cells wet weight per mL) in cold buffer without glycerol or the corresponding osmolyte [38]. In another ­protocol the cells are collected, washed with 50 mM ­Tris–HCl pH 7.5, 1 mM EDTA, 1 mM DTT, 33% glycerol, resuspended in the same buffer and permeabilized with 2% toluene/methanol (1.1) at 22 °C for 5 min. The cells are washed twice at 4 °C with cold buffer and resuspended in cold buffer (1 g of cells wet weight per mL) [47].

The GS assay contains the same mixture than that of the membrane assay except that the 5 μL of enzyme extract is replaced by 5 μL of permeabilized cells (5 μg of cells wet weight), in a total volume of 40 μL.

Chitin Synthase (CS)

This protocol was developed for S. cerevisiae chitin synthase enzymes CSI, CSII, and CSIII [48]. The in vitro activities of other fungi may be different and the protocols may need to be adapted.

Membrane Extracts Preparation

As for the GS, the source of enzyme activity is a crude membrane extract. The protocol for membrane extract preparation is similar to that described for GS except that the buffer is 50 mM Tris–HCl pH 7.5. Similarly, the membranes are resuspended in the same buffer containing 33% glycerol.

CS Assay

CS is a zymogenic enzyme that must be degraded partially to show its maximal activity. As mentioned previously, three CS activities have been described in S. cerevisiae, corresponding to three different proteins. A method to determine the three activities in the same membrane preparation by the use of several modifications in the reaction conditions has been described [48]. Most of the in vitro CS activity corresponds to CSI. The reaction mixture contains 37 mM Tris–HCl pH 7.5, 4.8 mM magnesium acetate, 5–10 μL of membrane suspension (up to 20 μL) and 2 μL of trypsin at the optimal concentration for activation (0.1–2.0 mg/mL) in a total volume of 40 μL. The mixture is incubated 15 min at 30 °C and the proteolysis is stopped by adding 2 μL of soybean trypsin inhibitor at a concentration 1.5 times that of the used trypsin solution. The tubes are placed on ice and made 1 mM in UDP-[14C]-GlcNAc (400 cpm/nmol) and 32 mM in GlcNAc in a total volume of 46 μL. Samples are incubated for 30–60 min at 30 °C, stopped with 1 mL of 10% TCA, and processed as for the GS assay (see previous). The specific activity is expressed as ηmoles of GlcNAc incorporated per hour and mg of protein.

For CSII and CSIII, the reaction mixture before proteolysis contains 32 mM Tris–HCl pH 8.0, 5 mM cobalt acetate, 20 μL of membrane suspension, 2 μL of trypsin, and 1.1 mM in UDP-[14C]-GlcNAc (400 cpm/nmol) in a total volume of 46 μL. For CSIII, the reaction mixture also contains 5 mM nickel acetate. After proteolysis is stopped, the mixture is made 32 mM in GlcNAc in a volume of 50 μL, incubated for 90 min at 30 °C, and stopped with 1 mL of 10% TCA.

Although the CSI assay detects the three activities, CSII and CSIII are minor contributors and therefore they do not alter significantly the value of CSI activity. However, they can be calculated based in the inhibitor effect of Ni2+ and Co2+. Ni2+ is a powerful inhibitor of CSI and II but has little effect on CSIII and Co2+ stimulates CSII and III but inhibits CSI. In summary:

  • CSI activity (total CS—CSII  +  III): CS assay minus CS assay (+Co2+).

  • CSIII activity: CS assay (+Co2+, +Ni2+).

  • CSII activity (CSII  +  III—CSIII): CS assay (+Co2+) minus CS assay (+Co2+, +Ni2+).

Microtiter-Based Fluorescence Assay

As for GS, an alternative to the use of UDP-[14C]-GlcNAc for the CS assay has been described [49]. The CS reaction is similar but the radioactive UDP-[14C]-GlcNAc is omitted. The wells of a microtiter plate are coated with wheat germ agglutinin (WGA) which binds with high affinity and specificity to chitin. The procedure involves the binding of the synthesized chitin to the WGA-coated surface. Then, horseradish peroxidase–WGA conjugate is added to the mixture. The WGA of the conjugate will bind to the chitin ­previously fixed in the well. The horseradish ­peroxidase activity is measured at 600 nm, and the amount of chitin is calculated using acid-­solubilized chitin as standard. This method is suitable for the three CS activities.

In Situ CS Assay Using Permeabilized Whole Cells

Similar to the procedure described previously for GS [38, 45–47].

Analysis of Cell Wall Polysaccharides

Different methods can be used to analyze the cell wall polysaccharide composition. All the methods, either for a precise analysis or for a rapid estimation of cell wall polymers, require a separation of the wall from the rest of the cell components. In general, the current methods have been adapted for a simple, accurate and rapid analysis of wall polysaccharides and are all based on labeling and fractionation of cell wall polysaccharides using chemical and enzymatic procedures. Basically, the methods available have been established using S. cerevisiae and S. pombe models, although the techniques used can easily be adapted for any organism.

Radioactive Labeling and Fractionation of the Cell Walls

A basic procedure to quantify the cell wall polymers consists in 14C-glucose labeling and fractionation of the cell wall polysaccharides as follows (Fig. 12.3):

Fig. 12.3
figure 00123

Overview of [U-14C]-glucose radioactive labeling and purification of the fungal cell wall

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Cell Wall Labeling

  1. 1.

    Exponentially growing cultures are adjusted to 5  ×  106 cells/mL, 5–7 mL are supplemented with [U-14C]-glucose (3 μCi/mL) (Hartmann Analytic) and incubated for at least one doubling time (3 h) to allow 14C incorporation into the cell. One doubling time means 50% labeling of cell material. If a stronger labeling is required, the cultures can be incubated for longer times, the 14C-glucose can be increased (up to 18 μCi/mL), and the glucose concentration in the culture medium can be reduced to 1 or 0.5%.

  2. 2.

    Cells (1–2  ×  107 cells/mL) are harvested by centrifugation (5,000  ×  g for 5 min).

  3. 3.

    Centrifuged cells from exponentially growing cultures without radioactive glucose (300 μL of a concentrate of 109–1010 cells/mL) are added to minimize lost material.

  4. 4.

    Cells are washed twice with 1 mM EDTA, transferred to 1.5–2.0 mL tubes and resuspended in 1.1 mL of 1 mM EDTA, 1 mM PMSF.

  5. 5.

    Total glucose incorporation is monitored in two 50 μL aliquots added to 1 mL of cold 10% TCA and kept at 4 °C for at least 30 min. Then aliquots are filtered through a fiberglass filter Whatman GF/C, washed three times with 1 mL of 10% TCA, twice with 1 mL of ethanol, and counted in a liquid scintillation counter. Eventually, the samples can be stored in 10% TCA at 4 °C and analyzed with the samples obtained in further steps.

  6. 6.

    The remaining cells (1.0 mL) are centrifuged (5,000  ×  g 5 min), resuspended in 100 μL of 1 mM EDTA, 1 mM PMSF, filled with cold glass beads (0.5 mm diameter) to completely cover the cell suspension and broken in a FastPrep homogenizer (Q-Biogene, MP Biomedicals, Thermo Scientific) during 3× 20 s pulse at a speed of 6.0 and at 4 °C. Complete cell lysis is confirmed by microscopic observation.

  7. 7.

    The broken cells are collected and the glass beads are washed twice with 1 mM EDTA, 1 mM PMSF to collect all residual material.

  8. 8.

    The broken material is centrifuged at 1,500  ×  g for 5 min, washed three times with 5 M NaCl, then again twice with 1 mM EDTA.

  9. 9.

    The cell wall pellet is resuspended in 1.1 mL of 1 mM EDTA, 1 mM PMSF, 0.02% Na azide, and heated at 100 °C for 5 min, to inactivate the intrinsic hydrolytic cell wall enzymes that would interfere the wall analysis.

Total radioactivity incorporated into the cell wall is monitored in two 50-μL aliquots that are added to 1 mL of cold 10% TCA and processed as described previously.

Cell Wall Alkali-Fractionation and Analysis

The most common method for cell wall fractionation is that used for S. cerevisiae [50, 51], which can easily be adapted for other organisms. This method allows the separation of cell wall β-glucan into alkali-soluble and alkali-insoluble fractions. The alkali-soluble fraction contains β(1,3)-glucan, mannan, and some β(1,6)-glucan; and the alkali-insoluble fraction contains chitin and β(1,3)-glucan β(1,6)-glucan linked to the chitin.

  1. 1.

    The cell wall suspension (1 mL) is extracted twice with 6% NaOH for 90 min at 80 °C and centrifuged at 1,500  ×  g for 5 min.

  2. 2.

    The alkali-extracted supernatant is divided into four aliquots of 250 μL.

    1. 2.1.

      Two alkali-extracted aliquots are used to precipitate the mannan with Fehling´s reagent [52] as follows: unlabeled purified mannan from S. cerevisiae (Sigma) is added to the supernatant as carrier (0.1 mL from a stock of 50 mg/mL in water). Fehling´s reagent (2 mL) is then added to the samples, mixed, and left overnight at 4 °C to precipitate the mannan. Fehling’s reagent is freshly prepared for each experiment by adding one volume of reagent B (3.5% CuSO4) to one volume of reagent A (17.3% potassium sodium tartrate dissolved in 12.5% KOH). After centrifugation at 1,500  ×  g for 10 min, the pellet is washed with Fehling´s reagent and solubilized in 20–40 μL of 6 N HCl (drop by drop and mixing until completely solubilized). Then, 100 μL of 50 mM Tris–HCl pH 7.5 is added and the solution is transferred to a vial with 2 mL of liquid scintillation. The tube is washed twice with 100 μL of buffer to collect the residual mannan, which is added to the vial containing liquid scintillation and analyzed (total mannan fraction).

    2. 2.2.

      The other two alkali-extracted aliquots are precipitated with 2 volumes of ethanol, allowed to dry, dissolved in 100 μL of water, collected (washing the tube twice) and analyzed with liquid scintillation as the previous samples (mannan  +  alkali soluble glucan). The difference between both fractions is the alkali-soluble glucan (β1,3  +  β1,6).

  3. 3.

    The alkali-insoluble residue is washed with water several times by centrifugation until it reaches a neutral pH, then suspended in 1.3 mL of water and divided into six aliquots of 200 μL.

    1. 3.1.

      The radioactivity of two aliquots is counted directly (alkali insoluble glucan  +  chitin).

    2. 3.2.

      Two aliquots are incubated for 24–36 h at 30 °C with 25 μg Zymolyase-100T in 50 mM citrate-phosphate pH 5.6, 0.02% Na azide in a volume of 300 μL, and two aliquots are processed similarly but without Zymolyase as a control. After incubation, the four samples are centrifuged and the pellets are washed twice, resuspended in 100 μL of buffer, added to 1 mL of 10% TCA, and processed by filtration in Whatman GF/C glass fiber filters and scintillation counting as described above. The residue remaining after Zymolyase digestion is the chitin fraction. Treatment of the alkali-insoluble fraction with recombinant chitinase from Pyrococcus furiosus (Wako Pure Chemical Industries) is not needed because the remaining residue is the alkali-insoluble glucan (β1,3  +  β1,6), which can be obtained as a difference between fractions.

Alkaline extraction is a widespread procedure for cell wall analysis of many organisms [1, 25, 53–59]. However, the data are only reproducible when maintaining the extraction conditions; the proportions of alkali-soluble and insoluble fractions can change depending on the alkali concentration, temperature, and incubation time. In fact, a process such as storing the cell walls at −20 °C results in total alkali solubility of the cell wall under conditions similar to those described above, showing only a small residue coincident with the chitin fraction.

Chemical fractionation of S. pombe cell wall polysaccharides is similar to that of S. cerevisiae [60], although the cell wall composition is ­different and therefore the results differ. The alkaline extraction procedure is rather harsh in S. pombe, due to the absence of the chitin responsible for the alkali-insoluble maintenance of some β(1,3)-glucan, and to a considerably smaller amount of β(1,6)-glucan than in S. cerevisiae [61, 62]. Therefore, alkaline extraction causes solubilization of nearly all S. pombe cell wall polymers and as a result, more gentle methods involving enzymes capable of specifically digesting one polymer without altering the others, described below, yield more accurate results.

Cell Wall Enzymatic Fractionation and Analysis

This protocol was adapted for S. pombe cell wall. The most commonly used procedure permits quantification of the three major cell wall polymers, β-glucans, α-glucans and galactomannoproteins. Once cell wall labeling and purification has been performed as described above, the procedure is as follows:

  1. 1.

    Half of the cell wall material (500 μL) is divided in:

    1. 1.1.

      Two 100 μL aliquots that are incubated for 24–36 h at 30 °C with shaking with 25 μg Zymolyase-100T in 50 mM citrate-phosphate pH 5.6, 0.02% Na azide in a volume of 300 μL, and two aliquots are processed without enzyme as control. A similar option is the incubation with 100–200 μg of Kitalase in 50 mM potassium-acetate pH 5.0, 0.02% Na azide.

    2. 1.2.

      Two 100 μL aliquots are incubated for 24–36 h at 30 °C with 100 units of Quantazyme (MP Biomedicals) or 100 units of recombinant β-1,3-Glucanase Yeast Lytic Type (Wako Pure Chemical Industries) in 50 mM potassium phosphate monobasic pH 7.5, 60 mM β-mercaptoethanol, 0.02% Na azide in a volume of 300 μL.

    3. 1.3.

      Two 50 μL aliquots are processed without enzyme as control.

  2. 2.

    After incubation, the samples are centrifuged (16,000  ×  g for 3 min). The supernatant is removed and 2  ×  50 μL aliquots are counted directly with 2 mL of liquid scintillation. 1 mL of 10% TCA is added to the pellet and the radioactivity incorporated is determined by filtration and liquid scintillation counting as described previously. As mentioned, Zymolyase-100T and Kitalase contain β(1,3)-glucanase, protease, mannanase and other activities but not α-glucanase activity. Therefore, the residue obtained after Zymolyase-100T or Kitalase digestion is considered α-glucan and the supernatant, β-glucan plus galactomannan. Quantazyme and the β-1,3-Glucanase Yeast Lytic Type are two recombinant endo-β(1,3)-glucanases capable of digesting β(1,3)-glucan without degrading the β(1,6)-glucan or α-glucan [5, 6]. Therefore, the residue obtained after Quantazyme or similar recombinant β(1,3)-glucanase treatment is considered α-glucan plus β(1,6)-glucan and galactomannan, and the supernatant is β(1,3)-glucan.

  3. 3.

    Half of the cell wall material (500 μL) is divided in two aliquots for galactomannan quantification. The wall is solubilized in alkali (6% NaOH) by adding 250 μL of 12% NaOH and heating at 80 °C for 1 h. Then, the galactomannan is precipitated from the alkali-solubilized aliquots with the Fehling’s reagent and quantified as described previously for S. cerevisiae cell walls.

Besides Zimolyase 100T, Kitalase and Quantazyme, there is an ample variety of enzymes and enzyme complexes commercially available that can be used for the cell wall determination of a specific fungal organism. These enzymes can provide information either individually or in combination. In the later case, depending on the cell wall polysaccharides composition and linkages, the order of enzymes can be important for maximal degradation of each enzyme. In addition, the enzymes with exohydrolytic activity present poor or null activity against the cell wall polysaccharides and therefore, all the enzymes to be tested should contain endohydrolytic activity. Some of these enzymes are:

  • β(1-3;1-4)- d -glucan hydrolases such as that from Bacillus subtilis (Biosupplies). This endoglucanase specifically hydrolyzes β-d-glucans containing both β(1,3) and β(1,4)-d-glucosidic linkages in linear sequence. It does not hydrolyze β(1,3)-glucans or β(1,4)-glucans.

  • α(1,4)-glucanases or α-amylases. These enzymes are endoglucanases that hydrolyze α(1,4)-d-glucosidic linkages in polysaccharides containing three or more α(1,4)-linked d-glucose units. Examples of α-amylases that can be used are α-amylase from Bacillus subtilis (heat stable, Sigma), from Bacillus licheniformis (Termamyl-120, heat stable up to 90 °C, Sigma), from Aspergillus oryzae (Taka-Diastase, Taka-Amylase A, Sigma), from Rhizopus sp. (Merck Calbiochem), from porcine pancreas (Sigma), from human pancreas (Merck Calbiochem) or from human saliva (Sigma, Merck Calbiochem). No additional activity for these enzymes has been reported.

  • Chitinases. These enzymes hydrolyze internal linkages in the chitin chain, a linear polymer of β(1,4)-N-Acetyl-d-glucosamine units. A recombinant Chitinase (Wako Pure Chemical Industries) from Pyrococcus furiosus is a thermostable and powerful enzyme. Chitinases from Trichoderma viride (Sigma) and from Streptomyces griseus (Sigma) are combination of exo and endochitinases, but with less efficient hydrolytic activity.

  • Chitosanases. These enzymes catalyze the hydrolysis of β(1,4) linkages between d-glucosamine (GlcN-GlcN) residues in chitosan. Chitosanases from Streptomyces sp. (Sigma; Merck Calbiochem) are available enzymes.

  • Mannanases. These are α-mannosidases that cleave terminal α(1,2), α(1,3) and/or α(1,6)-linked mannose residues in mannan. The supplied enzymes do not contain contaminant protease or glycosidase activities. α(1,2;1,3)-mannosidase, recombinant from Xanthomonas manihotis (Merck Calbiochem) cleaves terminal α(1,2) and α(1,3)-linked mannose residues. Its activity is efficient and reproducible. α(1,6)-mannosidase, recombinant from X. manihotis (ProZyme, AMS Biotechnology), cleaves terminal α(1,6)-linked mannose residues. It is recommended for use after digestion with α(1,2;1,3)-mannosidase for increasing the degradation efficiency. Other available enzymes are α(1,2;1,6)-mannosidase and α(1,2;1.3;1,6)-mannosidase from Canavalia ensiformis, Jack bean (Sigma, ProZyme, AMS Biotechnology), and α(1,2)-mannosidase from Aspergillus saitoi (ProZyme).

  • Proteases such as Proteinase K, recombinant, from Tritirachium album (Roche Applied Science); Protease S, recombinant thermostable from Pyrococcus furiosus (Sigma); Turbo3C protease, recombinant, from human rhinovirus 3C protease; and aminopeptidase T, recombinant, thermostable from Thermus aqualicus (Wako Pure Chemical Industries).

  • Enzymatic complexes. They can be used individually or in combination with other enzymes. The enzymatic composition of some complexes is well characterized although they must be used cautiously because they may contain other not-tested activities. The enzymatic activities of other complexes are not characterized and therefore can only be used to test whether the present and absent activities are helpful for a specific cell wall analysis. We already mentioned Zymolyase-20T and 100T (AMS Biotechnology) and Kitalase (Wako Pure Chemical Industries, City Chemical) also sold as Lysing Enzymes from Rhizoctonia solani (Sigma) or as Yeast Lytic Enzyme from Rhizoctonia solani (MP Biomedicals). Uskizyme (Wako Pure Chemical Industries) is a preparation from Trichoderma sp. with β(1,3)-glucanase, cellulase, protease, and chitinase activities. Westase (Cosmo Bio) is a preparation from the liquid culture supernatant of Streptomyces rochei. This complex contains mainly β(1,3)-glucanase and β(1,6)-glucanase activities according to the specifications sheet of the manufacturer, but minor or absent activities are not reported. Driselase (Sigma, Kyowa Hakko Kogyo) is a crude powder from Basidiomycetes sp. containing laminarinase, xylanase and cellulase activities. Glucanex or lysing enzymes from Trichoderma harzianum (Sigma), previously known as Novozyme-234 (Novozymes Corp., discontinued) contains β-glucanase, cellulase, protease, and chitinase activities. It also contains α(1,3)-glucanase activity, although it is not reported in the specifications sheet. Viscozyme (Sigma, Novozymes Corp.) is a multi-enzyme complex from Aspergillus sp. containing a wide range of carbohydrases, including arabanase, cellulase, β-glucanase, hemicellulase, and xylanase.

Other Cell Wall Chemical Fractionation and Analysis

Other methods that require more time and effort consist of combinations of enzymatic cell wall degradations (and dialysis), chemical degradations, and analytical techniques that permit the determination of the degradation products. These methods usually give more precise information about the type of bonds between the units forming the polymers. Common chemical degradations include alkali solubilization, acid hydrolysis, periodate oxidation, Smith degradation, borohydride reduction, β-elimination, carboxymethylation, and permethylation. Common analytical techniques include determination of reducing sugars, of total sugars, of glucose, of glucosamine, methylation analysis, gas–liquid chromatography, mass spectrometry, paper chromatography, gel filtration (size-exclusion) chromatography, ion-exchange chromatography, thin-layer chromatography (TLC), affinity chromatography, high-performance anionic-exchange chromatography (HPAEC), nuclear magnetic resonance (NMR) spectroscopy, and X-ray diffraction [6, 7, 53, 57, 60–70].

Colorimetric Determination of the Cell Wall Chitin

This is a colorimetric method useful to evaluate the amount of chitin in the cell wall [71]. This method is less precise than [14C] labeling and measurement but it is faster and less toxic.

  1. 1.

    Cell cultures (200 mL) are harvested at early log-phase (A600 of 1.0, aproximately 200 mg wet weight), washed twice with water, and the wet cell pellet is weighed.

  2. 2.

    Cells are resuspended in 1.0 mL water and a volume corresponding to 100 mg of cells is transferred to glass tubes.

  3. 3.

    The walls of 100 mg cells are extracted in 1 mL of 6% KOH at 80 °C for 90 min and the suspension is cooled down, neutralized with 100 μL glacial acetic acid, transferred to a 1.5 mL tube (washing the glass tube with water) and centrifuged 1 min at 16,000  ×  g.

  4. 4.

    The insoluble cell wall material is washed three times with water, resuspended in 600 μL of 50 mM potassium phosphate pH 7.5, and incubated with 1 unit (5 μL) of recombinant chitinase from Pyrococcus furiosus (Wako Pure Chemical Industries) at 85 °C for 2 h. Then, the degraded cell wall material is incubated with 25 μL of Glusulase (PerkinElmer) at 37 °C for 1 h, stopped at 100 °C for 1 min and centrifuged 1 min. Glusulase degrades the chitobiose and small chitin fragments formed by the endo-chitinase treatment into GlcNAc monomers.

  5. 4.

    The amount of GlcNAc is quantified colorimetrically by the Reissig method [72] using different concentrations of GlcNAc (0, 0.02, 0.04, 0.06, 0.08, and 0.1 μmol) as standards. Each reaction contains 250 μL of water, 250 μL of 270 mM potassium tetraborate pH 9.5 and 0, 2, 4, 6, 8 and 10 μL of 10 mM GlcNAc. The samples contain 250 μL of 270 mM potassium tetraborate pH 9.5, 200 or 150 μL of water and 50 or 100 μL respectively of the degraded cell wall material. The samples are boiled (100 °C) during 8 min, cooled down in ice/water and 3 mL of Reissig reagent is added and mixed. The Reissig reagent must be prepared freshly and contains 1 g of 3,5-diamino-benzaldehyde, 1.25 mL of 37% HCl and glacial acetic acid up to 100 mL. The reactions are done in duplicate. The samples are incubated 40 min at 37 °C and the generated color is measured at 585 nm (quartz cuvettes).

Cell Wall β(1,6) Glucan Determination

The most common method for β(1,6)glucan determination consists of alkaline extraction of the cell wall and analysis of the alkali-insoluble β(1,6)-glucan [73, 74], although part of the β(1,6)-glucan is in the alkali-soluble fraction [63, 75].

  1. 1.

    Isolated cell walls from 50 mL cultures are obtained as described in page 9 and Fig 12.3, and extracted three times with 1 mL of 3% NaOH at 75 °C for 1 h (removes mannoproteins and alkali-soluble glucan).

  2. 2.

    The extracted walls are washed once with 1 mL of 100 mM Tris–HCl pH 7.5, once with 1 mL of 10 mM Tris–HCl pH 7.5, resuspended in 1 mL of 10 mM Tris–HCl pH 7.5 and incubated with 1 mg of Zymolyase-100T at 37 °C for 16 h. Approximately 90% of the glucose-containing carbohydrate is released into the supernatant. Zymolyase releases the β(1,6)-glucan to the supernatant.

  3. 3.

    The insoluble material is removed by centrifugation (13,000  ×  g, 15 min) and the supernatant is dialyzed against water (6–8 kDa pore size) for 16 h. The carbohydrate retained after dialysis is the amount of β(1-6)-glucan. Total carbohydrate of each alkali-insoluble fraction (dialysis-retained, Zymolyase-soluble and Zymolyase-insoluble) is measured as hexose by the borosulfuric acid or phenol-sulfuric methods [76, 77]. The alkali-insoluble β(1,6)-glucan is determined as percentage of total carbohydrate (the sum of both the Zymolyase-soluble and insoluble fractions). The alkali-insoluble β(1,3)-glucan is determined as the amount of Zymolyase-soluble material before dialysis subtracted from the amount of alkali-insoluble β(1,6)-glucan.

The alkali-soluble β(1,6)glucan can be detected by immunodetection [75, 78]. The alkali-soluble extract is spotted onto nitrocellulose, dried, blocked with 5% nonfat milk in TBST (10 mM Tris pH 8.0, 150 mM NaCl, 0.05% Tween 20) and probed with anti-β(1,6)-glucan antibody [70]. After antibody binding, the membrane is washed three times with TBST, probed with anti-rabbit IgG horseradish peroxidase secondary antibody, washed again three times with TBST, and visualized with an ECL detection kit (Amersham). The alkali-soluble β(1,3)glucan can also be detected with the same procedure by using commercial monoclonal anti-β(1,3)-­glucan antibodies (Biosupplies) and anti-mouse ­secondary antibody. The amount of β(1,6)glucan or β(1,3)glucan is quantified in dots by using different concentrations of (β(1,6)glucan (pustulan) or β(1,3)-glucan (pachyman, curdlan, or laminarin) as standards, respectively.

β(1,6)-glucan can also be determined by high-performance liquid chromatography (HPLC), gas chromatography, mass spectrometry and NMR analysis of oligosaccharides after alkaline extraction and/or enzymatic degradation of the cell wall polymers.

Determination of Cell Wall Proteins

The cell wall proteins are glycoproteins highly modified with O- and N-linked oligosaccharides, predominantly or exclusively formed by mannose residues known as mannan. In some cases, the mannan backbone presents single residues or side chains of different sugars, galactomannan, rhamnomannan, glucogalactomannan, rhamnogalactomannan, etc. [1, 24, 25].

Most proteins found in the cell wall are water- or detergent-soluble and are usually secreted to the medium. Some few cell wall proteins are covalently linked to the polysaccharides and can be divided into two groups, proteins covalently attached to β(1,3)glucan (Pir proteins) through a glutamine residue, an alkali-labile linkage that can be extracted by a mild alkali treatment (30 mM NaOH at 4 °C for 16 h); and proteins covalently attached by a GPI anchor to the β(1,6)glucan of a β(1,6)/β(1,3)glucan core that can be removed by β(1,3)glucanases or β(1,6)glucanases [1, 59, 79–82].

The SDS-soluble proteins are extracted by hot SDS-mercaptoethanol treatments, twice with 50 mM Tris–HCl pH 7.8–8.0, 2% SDS, 100 mM EDTA, and 40 mM β-mercaptoethanol for 5–15 min at 100 °C [70, 83, 84]. The proteins are concentrated and the SDS removed by precipitation with 9 volumes of cold acetone at −20 °C for 2 h. The proteins are dried, resuspended and analyzed by SDS-PAGE or Western blot.

The covalently attached cell wall proteins can be purified by three methods [82, 84]:

  1. 1.

    The proteins covalently attached to β(1,3)glucan are released by treatment with 30 mM NaOH at 4 °C for 16 h. The reaction is stopped by adding acetic acid to neutrality followed by dialysis.

  2. 2.

    The GPI-attached proteins are released by degradation of the GPI anchor by treatment with undiluted pyridine hydrofluoride at 24 °C for 16 h. HF-pyridine is removed by dialysis. Treatment with recombinant endo-β(1,6)-glucanase (noncommercial) also releases the GPI proteins.

  3. 3.

    Both groups of covalently attached proteins can be released by treatment with recombinant endo-β(1,3)-glucanase (MP Biomedicals; Wako Pure Chemical Industries), followed by dialysis.

The mannan oligosaccharides can be analyzed directly or after protease treatment by enzymatic treatment with different mannosidases. As the glycoproteins are highly glycosylated and this may interfere with the electrophoresis protein analysis, the N-linked oligosaccharides can be removed by treatment with endoglycosidase H (Boehringer, Roche, New England Biolabs) [70, 85]. The proteins can be analyzed by SDS-PAGE or Western blot with specific antibodies or with the lectin concanavalin A (Sigma) that specifically binds to the mannan region [81, 86]. Additionally, the cell wall proteins can be biotin-labeled directly in the cell prior to cell wall purification, with the biotinylation reagent Sulfo-NHS-LC-Biotin (Pierce) in 50 mM potassium phosphate pH 8.0 by incubation for 90 min on ice. The proteins are analyzed by Western blot and visualized with streptavidin-horseradish peroxidase conjugate (Pierce) [87, 88].

The mannan can be analyzed by incorpo‑ration of radioactive orthophosphate into N-mannosylated glycoproteins. The cell wall is purified and extracted with SDS to obtain part of the mannan. Then the insoluble material is treated with β(1,3)glucanases to release the rest of the mannan. The phosphate is bound as mannose-6-phosphate to both soluble and covalently linked cell wall mannoproteins [83, 89]. The phosphorylated cell wall oligosaccharides can be characterized after hydrolysis in trifluoracetic acid (TFA) by Quaternary aminoethyl (QAE)-Sephadex A50 chromatography, Bio-Gel P2 chromatography, HPAEC, electrospray ionization tandem mass spectrometry (ESI-MS-MS), and methylation analysis with gas chromatography–mass spectrometry [83].

Nonquantitative Methods for Analysis of Cell Wall Polysaccharides

Other methods can also be used in order to detect cell wall differences in structure or composition without the need of precise polymer quantification. Among them, those more used are briefly described as follows.

Sensitivity of Cells to Enzymatic Degradation

This method is used for a rough analysis of the cell wall state and is used to corroborate other results that suggest an altered cell wall [29, 90]. It is also used as a screening for mutations affecting the cell wall. Common enzymes used in this procedure are Zymolyase-100T in 50 mM citrate/phosphate buffer pH 5.6, Kitalase in 50 mM potassium-acetate pH 5.0, or Glucanex (Sigma) in any of these buffers. Zymolyase-100T and Kitalase mainly degrade β(1,3)-glucan and mannoproteins, whereas Glucanex degrades the entire cell wall. Increased sensitivity of the cells to these enzymes can be due to different causes such as a decrease in the amount of cell wall β-glucan or an increase in the permeability of the cell wall to the enzymes, which can be caused by a decrease in the cell wall surface glycoproteins or an altered cell wall structure. A specific cell wall-related mutant strain may show different sensitivities to Zymolyase, Kitalase and Glucanex, depending on the importance that the α-glucan may have in maintaining cell integrity in this mutant strain. The sensitivity to degradation using α(1,3)-glucanase has never been assayed due to the lack of a commercially available enzyme, although the purification of recombinant α(1,3)-glucanases (mutanases) from Penicillium purpurogenum and Trichoderma harzianum has been described [91].

Hypersensitivity or Resistance to Cell Wall Biosynthesis Inhibitors

This method can be used to detect changes in the cell wall composition or structure, and to detect mutations in the enzymes involved in cell wall synthesis; it is also used in the analysis of genetic interactions between genes that might be specifically related to cell wall biosynthesis and cell integrity. The main inhibitors are:

  • Calcofluor white used to stain chitin. Calcofluor also binds to linear β(1,3)glucan with high affinity when chitin is not present, as in the case of S. pombe [92]. The binding of this dye perturbs the wall structure at low concentration and halts cell growth at high concentration.

  • Echinocandins (caspofungin, micafungin and anidulafungin, available for clinical use) are a family of antifungal drugs that specifically inhibit both, the in vitro β(1,3)-glucan synthase activity and the in vivo β(1,3)-glucan synthesis [28, 93–97]. Other families of β(1,3)-glucan synthase inhibitors like papulacandins and the acidic terpenoid enfumafungin are also used [29].

  • Polyoxins and Nikkomycins are chitin synthase inhibitors [23, 94, 96] or recently discovered β(1,6)glucan synthase inhibitors can also be used for specific assays [98, 99].

  • 2-deoxi-d-glucose can be used as competitor of glucose for assays of cell wall biosynthesis defects.

Fluorescence Microscopy

Direct observation of the cell wall using microscopy techniques is an important method for cell wall studies. Fluorochromes, lectins, and antibodies that stain a specific cell wall polysaccharide can be used for cell wall fluorescence microscopy analysis. The most commonly used are:

  • Calcofluor white (Sigma) (25 μg/mL final concentration) stains the cell wall chitin and in its absence specifically stains linear β(1,3)glucan. Similarly, aniline blue (0.5 mg/mL final concentration) (Biosupplies), specifically stains linear β(1,3)glucan, although its affinity for the cell wall β(1,3)glucan of growing poles is lower than that of Calcofluor.

  • Lectins against the mannan of cell wall glycoproteins such as Concanavalin A, which binds mannose residues, the lectin from Bandeiraea simplicifolia that recognizes specifically terminal galactose residues or wheat germ agglutinin (WGA) that recognize specifically chitin. They can be used for immunofluorescence analysis bound to fluorescein isothiocyanate (FITC) (Sigma). Lectins are added (200 μg/mL) to the cells, in culture medium or in phosphate buffer solution (PBS) if the cells are fixed. After 15 min in dark, cells are washed and resuspended for microscopy observation. For the observation of chitin, since it is internal to the cell wall, WGA-FITC binding and visualization requires a previous mild alkali extraction of the cell wall. The same lectins, labeled with colloidal gold, can be used for immunoelectron microscopy.

  • Antibodies raised against specific proteins or polymers can also be used (see below). These antibodies are combined with secondary fluorochrome-labeled antibodies for immunofluorescence studies.

Electron Microscopy (EM)

The techniques for EM are numerous and have advanced greatly during the last years:

Transmission Electron Microscopy

Cells fixed with glutaraldehyde have been used to study the cell wall and septum structures of fungal cells. This technique permits observation of the cell wall as a three-layered structure of polysaccharides with different electron densities (see Fig. 12.1) [92, 100–109]. Negative staining for TEM with uranyl acetate is an alternative method for visualization of fibrils network and other cell wall structures [110–114].

Scanning Electron Microscopic (SEM)

Cells fixed with glutaraldehyde are used to observe the cell surface clearly and with high fidelity. An improved technique is ultra-high-resolution low-voltage SEM (UHR-LVSEM) [103, 110–113, 115–118]. SEM microscopy can be coupled with lectins or antibodies labeled with colloidal gold particles. This technique served to detect a cell wall surface completely filled with particles specific for the mannan carbohydrate of glycoproteins [116], and to detect the β(1,3)-glucan in the bud scars of S. Cerevisiae [1].

Atomic Force Microscopy (AMF)

This technique is used to measure the mechanical properties of the fungal cell wall macromolecules [1, 113, 119, 120].

Cryoscanning and Cryosectioning Electron Microscopy

This technique uses cryofixation to physically immobilize the specimen [113]. An improved method combines high-pressure freezing with ultra-low temperature and low-voltage SEM (ULT-LVSEM). It is useful to analyze fractured and coated cell samples, allowing the observation of a fine plane and the ultrastructure of both external and internal cell components [121].

Transmission Immunoelectron Microscopy (IEM)

Cells are fixed with paraformaldehyde plus glutaraldehyde. This technique has improved with the method of cryofixation by high-pressure freezing followed by freeze-substitution to retain the antigenicity [122]. Specific mono- and polyclonal antibodies in combination with secondary antibodies conjugated with 10-nm gold particles have been used to locate the different types of β-glucans [122]. The currently described antibodies against different wall polysaccharides are:

  • Anti-β(1,3)-glucan [123] (Biosupplies), a murine monoclonal antibody without cross-reactivity with β(1,4)-glucans or β(1,3;1,4)-glucans.

  • Anti-β(1,3;1,4)-glucan (Biosupplies). It is a murine monoclonal antibody without cross-reactivity with β(1,3)-glucans [124].

  • Anti-β(1,6)-glucan. This rabbit antiserum specifically recognizes this polymer in S. cerevisiae [70, 125] and S. pombe [122] but is not commercially available.

  • Anti-β(1,6)-branched-β(1,3)-glucan [122, 126] is a rabbit antiserum obtained against grifolan (a type of β(1,6)-branched-β(1,3)-glucan) and does not reacts with linear β(1,3)-glucan. It is suggested that the hapten site of the antibody is the monoglucosyl β(1,6)-glucan-branched moiety of β(1,3)-glucan. It is not commercially available.

  • Anti-α(1,3)-glucan. A polyclonal anti-α(1,3)-glucan has been used to analyze the cell wall α(1,3)-glucan but is not commercially available [64, 127]. Additionally, the monoclonal IgM antibody MOPC-104E (Sigma, Abcam) has been used to detect the α(1,3)-glucan in Histoplasma capsulatum yeast cell walls. This antibody specifically recognizes α(1,3)-glucan because only α(1,3)-linked and not β-linked glycosyl polysaccharides can block the antibody and because the cell walls lacking α(1,3)-glucan of an avirulent strain of Histoplasma capsulatum are not recognized by the antibody [128–130].

  • Anti-GFP murine monoclonal antibody (JL-8 anti-GFP; BD Biosciences, Sigma) has been used to detect GFP-fused cell wall or plasma membrane proteins with secondary gold-labeled anti-mouse secondary antibodies [131].

The lectins mentioned previously, Conca‑navalin A, which binds mannose residues, and wheat germ agglutinin (WGA), which binds GlcNAc residues, are also used for IEM studies when labeled with colloidal gold particles [109, 132–134].