The Cell Wall of Fusarium oxysporum

Sugar analysis of isolated cell walls from three formae speciales of Fusarium oxysporum showed that they contained not only glucose and (N-acetyl)-glucosamine, but also mannose, galactose, and uronic acids, presumably originating from cell wall glycoproteins. Cell wall glycoproteins accounted for 50–60% of the total mass of the wall. X-ray diffraction studies showed the presence of α-1,3-glucan in the alkali-soluble cell wall fraction and of β-1,3-glucan and chitin in the alkali-insoluble fraction. Electron microscopy and lectin binding studies indicated that glycoproteins form an external layer covering an inner layer composed of chitin and glucan.     

Molecular organization of the alkali-insoluble fraction of Aspergillus fumigatus cell wall

Physical and biological properties of the fungal cell wall are determined by the composition and arrangement of the structural polysaccharides. Cell wall polymers of fungi are classically divided into two groups depending on their solubility in hot alkali. We have analyzed the alkali-insoluble fraction of the Aspergillus fumigatus cell wall, which is the fraction believed to be responsible for fungal cell wall rigidity. Using enzymatic digestions with recombinant endo-beta-1,3-glucanase and chitinase, fractionation by gel filtration, affinity chromatography with immobilized lectins, and high performance liquid chromatography, several fractions that contained specific interpolysaccharide covalent linkages were isolated. Unique features of the A. fumigatus cell wall are (i) the absence of beta-1,6-glucan and (ii) the presence of a linear beta-1, 3/1,4-glucan, never previously described in fungi. Galactomannan, chitin, and beta-1,3-glucan were also found in the alkali-insoluble fraction. The beta-1,3-glucan is a branched polymer with 4% of beta-1,6 branch points. Chitin, galactomannan, and the linear beta-1, 3/1,4-glucan were covalently linked to the nonreducing end of beta-1, 3-glucan side chains. As in Saccharomyces cerevisiae, chitin was linked via a beta-1,4 linkage to beta-1,3-glucan. The data obtained suggested that the branching of beta-1,3-glucan is an early event in the construction of the cell wall, resulting in an increase of potential acceptor sites for chitin, galactomannan, and the linear beta-1,3/1,4-glucan.     

The cell wall architecture of Candida albicans wild-type cells and cell wall-defective mutants

In Candida albicans wild-type cells, the beta1, 6-glucanase-extractable glycosylphosphatidylinositol (GPI)-dependent cell wall proteins (CWPs) account for about 88% of all covalently linked CWPs. Approximately 90% of these GPI-CWPs, including Als1p and Als3p, are attached via beta1,6-glucan to beta1,3-glucan. The remaining GPI-CWPs are linked through beta1,6-glucan to chitin. The beta1,6-glucanase-resistant protein fraction is small and consists of Pir-related CWPs, which are attached to beta1,3-glucan through an alkali-labile linkage. Immunogold labelling and Western analysis, using an antiserum directed against Saccharomyces cerevisiae Pir2p/Hsp150, point to the localization of at least two differentially expressed Pir2 homologues in the cell wall of C. albicans. In mnn9Delta and pmt1Delta mutant strains, which are defective in N- and O-glycosylation of proteins respectively, we observed enhanced chitin levels together with an increased coupling of GPI-CWPs through beta1,6-glucan to chitin. In these cells, the level of Pir-CWPs was slightly upregulated. A slightly increased incorporation of Pir proteins was also observed in a beta1, 6-glucan-deficient hemizygous kre6Delta mutant. Taken together, these observations show that C. albicans follows the same basic rules as S. cerevisiae in constructing a cell wall and indicate that a cell wall salvage mechanism is activated when Candida cells are confronted with cell wall weakening.     

Altered extent of cross-linking of beta1,6-glucosylated mannoproteins to chitin in Saccharomyces cerevisiae mutants with reduced cell wall beta1,3-glucan content.

The yeast cell wall contains beta1,3-glucanase-extractable and beta1,3-glucanase-resistant mannoproteins. The beta1,3-glucanase-extractable proteins are retained in the cell wall by attachment to a beta1,6-glucan moiety, which in its turn is linked to beta1,3-glucan (J. C. Kapteyn, R. C. Montijn, E. Vink, J. De La Cruz, A. Llobell, J. E. Douwes, H. Shimoi, P. N. Lipke, and F. M. Klis, Glycobiology 6:337-345, 1996). The beta1,3-glucanase-resistant protein fraction could be largely released by exochitinase treatment and contained the same set of beta1,6-glucosylated proteins, including Cwp1p, as the B1,3-glucanase-extractable fraction. Chitin was linked to the proteins in the beta1,3-glucanase-resistant fraction through a beta1,6-glucan moiety. In wild-type cell walls, the beta1,3-glucanase-resistant protein fraction represented only 1 to 2% of the covalently linked cell wall proteins, whereas in cell walls of fks1 and gas1 deletion strains, which contain much less beta1,3-glucan but more chitin, beta1,3-glucanase-resistant proteins represented about 40% of the total. We propose that the increased cross-linking of cell wall proteins via beta1,6-glucan to chitin represents a cell wall repair mechanism in yeast, which is activated in response to cell wall weakening.     

Predicting the chemical composition and structure of Aspergillus nidulans hyphal wall surface by atomic force microscopy

In fungi, cell wall plays an important role in growth and development. Major macromolecular constituents of the aspergilli cell wall are glucan, chitin, and protein. We examined the chemical composition and structure of the Aspergillus nidulans hyphal wall surface by an atomic force microscope (AFM). To determine the composition of the cell wall surface, the adhesion forces of commercially available β-glucan, chitin, and various proteins were compared to those of corresponding fractions prepared from the hyphal wall. In both setups, the adhesion forces of β-glucan, chitin, and protein were 25–50, 1000–3000, and 125–300 nN, respectively. Adhesion force analysis demonstrated that the cell surface of the apical tip region might contain primarily chitin and β-glucan and relatively a little protein. This analysis also showed the chemical composition of the hyphal surface of the mid-region would be different from that of the apical region. Morphological images obtained by the tapping mode of AFM revealed that the hyphal tip surface has moderate roughness.     

Substantial decrease in cell wall α-1,3-glucan caused by disruption of the kexB gene encoding a subtilisin-like processing protease in Aspergillus oryzae

Disruption of the kexB encoding a subtilisin-like processing protease in Aspergillus oryzae (ΔkexB) leads to substantial morphological defects when the cells are grown on Czapek-Dox agar plates. We previously found that the disruption of kexB causes a constitutive activation of the cell wall integrity pathway. To understand how the disruption of the kexB affects cell wall organization and components, we analyzed the cell wall of ΔkexB grown on the plates. The results revealed that both total N-acetylglucosamine content, which constitutes chitin, and chitin synthase activities were increased. Whereas total glucose content, which constitutes β-1,3-glucan and α-1,3-glucan, was decreased; this decrease was attributed to a remarkable decrease in α-1,3-glucan. Additionally, the β-1,3-glucan in the alkali-insoluble fraction of the ΔkexB showed a high degree of polymerization. These results suggested that the loss of α-1,3-glucan in the ΔkexB was compensated by increases in the chitin content and the average degree of β-1,3-glucan polymerization.     

Chemical and ultrastructural studies on the cell walls of the yeastlike and mycelial forms of Histoplasma farciminosum

The cell wall of the yeast form of Histoplasma farciminosum contains 13.2% beta-1,3-glucan, 1.0% galactomannan, and 25.8% chitin, whereas the cell wall of mycelial form has 21.8, 4.5, and 40%, respectively, for the same polymers. Also, the cell wall of the yeast form contains alpha-1,3-glucan (13.5%) and an unidentified polymer (21.5%). Chitin, one of the structural polymers of both yeast and mycelial cell walls, is identified as thin isolated fibers (4 nm wide) or in thick bundles (50 nm wide) of fibers. beta-(1-3)-Glucan is also found as thin isolated fibers indistinguishable from isolated fibers of chitin. Fibers 14 nm wide and resembling alpha-(1-3)-glucan fibers of other fungi are found in the yeast form. The results reported here do not give support to the proposal for a different taxonomic classification.     

Dynamics of cell wall components of Magnaporthe grisea during infectious structure development

Oligosaccharides derived from cell wall of fungal pathogens induce host primary immune responses. To understand fungal strategies circumventing the host plant immune responses, cell wall polysaccharide localization was investigated using fluorescent labels during infectious structure differentiation in the rice blast fungus Magnaporthe grisea . α-1,3-glucan was labelled only on appressoria developing on plastic surfaces, whereas it was detected on both germ tubes and appressoria on plant surfaces. Chitin, chitosan and β-1,3-glucan were detected on germ tubes and appressoria regardless of the substrate. Major polysaccharides labelled at accessible surface of infectious hyphae were α-1,3-glucan and chitosan, but after enzymatic digestion of α-1,3-glucan, β-1,3-glucan and chitin became detectable. Immunoelectron microscopic analysis showed α-1,3-glucan and β-1,3-glucan intermixed in the cell wall of infectious hyphae; however, α-1,3-glucan tended to be distributed farther from the fungal cell membrane. The fungal cell wall became more tolerant to chitinase digestion upon accumulation of α-1,3-glucan. Accumulation of α-1,3-glucan was dependent on the Mps1 MAP kinase pathway, which was activated by a plant wax derivative, 1,16-hexadecanediol. Taken together, α-1,3-glucan spatially and functionally masks β-1,3-glucan and chitin in the cell wall of infectious hyphae. Thus, a dynamic change of composition of cell wall polysaccharides occurs during plant infection in M. grisea .     

Chemical and ultrastructural studies on the cell walls of the yeastlike and mycelial forms of Histoplasma capsulatum

Chemical and ultrastructural studies of the cell walls of the yeastlike (Y) and mycelial (M) forms ofHistoplasma capsulatum G-184B revealed that the Y form contained about 46.5% ofα-glucan, 31.0% ofβ-glucan, 7.7% of galactomannan and 11.5% of chitin, whereas the M form cell wall contained about 18.8% ofβ-glucan, 24.7% of galactomannan, 25.8% of chitin, and essentially noα-glucan. Theα-glucan of the Y form contained mainly anα-(1 → 3)-linkage. Theβ-glucans of both forms may have mainly aβ-(1 → 3)-linkage. Chitin microfibrils were located mainly in the inner portion of the cell walls of the Y and M forms, whereas theα-glucan fibers were observed only in the outer portion of the Y form cell wall.     

Characterization of Aspergillus nidulans mutants deficient in cell wall chitin or glucan.

By screening for the osmotically remediable phenotype, mutations in two genes (orlA and orlB) affecting the cell wall chitin content of Aspergillus nidulans were identified. Strains carrying temperature-sensitive alleles of these genes produce conidia which swell excessively and lyse when germinated at restrictive temperatures. Growth under these conditions is remedied by osmotic stabilizers and by N-acetylglucosamine (GlcNAc). Remediation by GlcNAc suggests that the mutations affect early steps in the synthesis of chitin. Temperature and medium shift experiments indicate that the phenotype is the result of decreased synthesis rather than increased chitin degradation and that osmotic stabilizers act to stabilize a defective wall rather than to stabilize the gene product. Two genes, orlC and orlD, which affect cell wall beta-1,3-glucan content were also identified. Walls from strains carrying mutations in these genes exhibit normal amounts of alpha-1,3-glucan and chitin but reduced amounts of beta-1,3-glucan. As for the chitin-deficient mutants, orlC and orlD mutants spontaneously lyse on conventional media but are remedied by osmotic stabilizers. These results indicate that both chitin and beta-1,3-glucan are likely to contribute to the structural rigidity of the cell wall.     

A refined method for the determination of Saccharomyces cerevisiae cell wall composition and beta-1,6-glucan fine structure.

In yeast and other fungi, cell division, cell shape, and growth depend on the coordinated synthesis and degradation of cell wall polymers. We have developed a reliable and efficient micro method to determine Saccharomyces cerevisiae cell wall composition that distinguishes between beta1,3- and beta1,6-glucan. The method is based on the sequential treatment of cell walls with specific hydrolytic enzymes followed by dialysis. The low molecular weight (MW) products thus separated account for each particular cell wall polymer. The method can be applied to as little as 50-100 mg (wet wt) of radioactively labeled cells. A combination of chitinase and recombinant beta-1,3-glucanase is initially used, releasing all of the chitin and 60-65% of the beta1,3-glucan from the cell walls. Next, recombinant endo-beta-1,6-glucanase from Trichoderma harzianum is utilized to release all the beta-1,6-glucan present in the wall. The chromatographic pattern of endoglucanase digested beta-1,6-glucan provides a characteristic "fingerprint" of beta-1,6-glucan and the fine structure of the oligosaccharides in this pattern was determined by 1H NMR and electrospray ionization mass spectroscopy. The final enzymatic step uses laminarinase and beta-glucosidase to release the remaining beta-1,3-glucan. The cell wall mannan remains as a high MW fraction at the end of the fractionation procedure. Good sensitivity and correlation with cell wall composition determined by traditional methods were observed for wild-type and several cell wall mutants.     

Individual chitin synthase enzymes synthesize microfibrils of differing structure at specific locations in the Candida albicans cell wall

The shape and integrity of fungal cells is dependent on the skeletal polysaccharides in their cell walls of which beta(1,3)-glucan and chitin are of principle importance. The human pathogenic fungus Candida albicans has four genes, CHS1, CHS2, CHS3 and CHS8, which encode chitin synthase isoenzymes with different biochemical properties and physiological functions. Analysis of the morphology of chitin in cell wall ghosts revealed two distinct forms of chitin microfibrils: short microcrystalline rodlets that comprised the bulk of the cell wall; and a network of longer interlaced microfibrils in the bud scars and primary septa. Analysis of chitin ghosts of chs mutant strains by shadow-cast transmission electron microscopy showed that the long-chitin microfibrils were absent in chs8 mutants and the short-chitin rodlets were absent in chs3 mutants. The inferred site of chitin microfibril synthesis of these Chs enzymes was corroborated by their localization determined in Chsp-YFP-expressing strains. These results suggest that Chs8p synthesizes the long-chitin microfibrils, and Chs3p synthesizes the short-chitin rodlets at the same cellular location. Therefore the architecture of the chitin skeleton of C. albicans is shaped by the action of more than one chitin synthase at the site of cell wall synthesis.     

Inhibition of fungal cell wall synthesizing enzymes by trans-cinnamaldehyde

This study examined the inhibitory effects of trans-cinnamaldehyde (CA), an aromatic aldehyde derived from Cinnamomi Cortex, on Saccharomyces cerevisiae cell wall synthesizing enzymes in vitro. This compound was found to be a noncompetitive inhibitor of beta-(1,3)-glucan synthase and a mixed inhibitor of chitin synthase 1 with 50% inhibitory concentrations (IC50) of 0.84 and 1.44 mM, respectively. Chitin synthases 2 and 3 were less sensitive than chitin synthase 1 to CA. CA can be useful as a model compound of cell wall inhibitors for the development of effective antifungal agents.     

The stress response protein Hsp12p increases the flexibility of the yeast Saccharomyces cerevisiae cell wall

The yeast S. cerevisiae cell wall comprising a 10 nm thick layer of polysaccharides, predominantly beta(1,3)-glucan and proteins, is the interface between the cell and the neighbouring environment. As such it is not a static entity but rather one that is dynamically remodelled in response to changes in the environmental conditions. We have recently proposed from studies using yeast cells lacking the gene encoding Hsp12p (Deltahsp12 yeast) and from incorporation of Hsp12p into agarose, used as a model system for the beta-glucan layer of the cell wall, that the hydrophilic stress response cell wall protein Hsp12p acts as a cell wall plasticizer. In this report we have used force spectroscopy to confirm that Deltahsp12 yeast are indeed less flexible than the wild type strain. The spring constant of the cell wall of Deltahsp12 yeast, kcw was determined to be 72+/-3 mN m-1 as compared to 17+/-5 mN m-1 obtained for the wild type strain. A similar result was found on the basis of a quantitative analysis of the electrophoretic mobilities measured for the two yeast strains. Those indicated that the hydrodynamic permeability quantified through the softness parameter of the external layer of Deltahsp12 cells was smaller than the one of wild type cells. We proposed from surface infrared spectroscopy measurements that yeast compensate for the lack of Hsp12p by reducing the carbohydrate/proteins ratio of the cell wall or increasing the cell wall chitin content.     

Two homologous genes,DCW1 (YKL046c) and DFG5, are essential for cell growth and encode glycosylphosphatidylinositol (GPI)-anchored membrane proteins required for cell wall biogenesis in Saccharomyces cerevisiae

The cell wall of Saccharomyces cerevisiae consists of glucan, chitin and various kinds of mannoproteins. Major parts of mannoproteins are synthesized as glycosylphosphatidylinositol (GPI)-anchored proteins and are then transferred to cell wall beta-1,6-glucan. A glycosyltransferase has been hypothesized to catalyse this transfer reaction. A database search revealed that the products of YKL046c and DFG5 are homologous to bacterial mannosidase. These genes are homologous to each other and have primary structures characteristic of GPI-anchored proteins. Although single disruptants of ykl046c and dfg5 were viable, ykl046cDelta was hypersensitive to a cell wall-digesting enzyme (zymolyase), suggesting that this gene is involved in cell wall biosynthesis. We therefore designated this gene as DCW1 (defective cell wall). A double disruptant of dcw1 and dfg5 was synthetically lethal, indicating that the functions of these gene products are redundant, and at least one of them is required for cell growth. Cells deficient in both Dcw1p and Dfg5p were round and large, had cell walls that contained an increased amount of chitin and secreted a major cell wall protein, Cwp1p, into the medium. Biochemical analyses showed that epitope-tagged Dcw1p is an N-glycosylated, GPI-anchored membrane protein and is localized in the membrane fraction including the cell surface. These results suggest that both Dcw1p and Dfg5p are GPI-anchored membrane proteins and are required for normal biosynthesis of the cell wall.     

Mechanical double layer model for Saccharomyces Cerevisiae cell wall

The elastic modulus of the Baker’s yeast (Saccharomyces cerevisiae) cell wall reported in studies using atomic force microscopy (AFM) is two orders of magnitude lower than that obtained using whole cell compression by micromanipulation. Using finite element modelling, it is shown that Hertz-Sneddon analysis cannot be applied to AFM indentation data for single layer core–shell structures. In addition, the Reissner solution for shallow homogeneous spheres is not appropriate for thick walls such as those of yeast cells. In order to explain yeast compression measurements at different length scales, a double layer wall model is presented considering a soft external layer composed of mannoproteins, and a stiff inner layer of β-glucan fibres and chitin. Under this model, previous AFM studies using sharp indenters provide reasonable estimates of the external layer elastic modulus, while micromanipulation provides the total stiffness of the cell wall. Data from both measurements are combined to estimate the mechanical properties of the inner stiff layer.     

Cell wall structure of secreted laccase-silenced strain in Lentinula edodes

Laccase1 (Lcc1) is abundantly secreted from vegetative mycelia into culture medium by Lentinula edodes. Down-regulation of lcc1 in L. edodes results in abnormal hyphal structure and thinner cell wall in mycelia. In this study, we observed the effects of Lcc1 on the hyphal morphology and cell wall structure of L. edodes. A thick cell wall and fibrous layer were clearly observed in the lcc1-silenced strain ivrL1#32, when purified Lcc1 (0.1 mU/mL) was added to the culture medium. The ratio of cell wall polysaccharide contents was compared between the ivrL1#32 strain and the wild-type (WT) strain SR-1, revealing that levels of the alkali soluble β-1,3-1,6-glucan were significantly lower in the lcc1-silenced strain than in the WT strain. Chronological analysis revealed that chitin content in the cell wall did not increase over time, but that the alkali soluble β-1,3-1,6-glucan content increased after Lcc1 secretion in the WT. Taken together, these data suggest that the increased level of β-1,3-1,6-glucan induced by Lcc1 in the mycelial cell wall contributes to increased cell wall thickness and strength.     

The structure and synthesis of the fungal cell wall

The fungal cell wall is a dynamic structure that protects the cell from changes in osmotic pressure and other environmental stresses, while allowing the fungal cell to interact with its environment. The structure and biosynthesis of a fungal cell wall is unique to the fungi, and is therefore an excellent target for the development of anti-fungal drugs. The structure of the fungal cell wall and the drugs that target its biosynthesis are reviewed. Based on studies in a number of fungi, the cell wall has been shown to be primarily composed of chitin, glucans, mannans and glycoproteins. The biosynthesis of the various components of the fungal cell wall and the importance of the components in the formation of a functional cell wall, as revealed through mutational analyses, are discussed. There is strong evidence that the chitin, glucans and glycoproteins are covalently cross-linked together and that the cross-linking is a dynamic process that occurs extracellularly.     

Ultrastructural and cytochemical aspects of the interaction between the ectomycorrhizal fungus Laccaria laccata and the saprotrophic fungi, Trichoderma harzianum and T. virens

Different interactions between soil fungi competing in the rhizosphere with each other are necessary to understand their influence on plant growth and health. The interactions between the ectomycorrhizal (ECM) fungus Laccaria laccata and soil saprotrophic fungi (T. harzianum, T. virens) were studied by transmission electron microscopy, and by gold cytochemistry to assess the potential role of cell wall lytic enzymes in mycoparasitism. Anti-β-1,3-glucan antibody, WGA/ovomucoid-gold complex and PATAg test were used to localize β-1,3-glucan, chitin and polysaccharides. Cytoplasm disorganisation of the saprotrophic fungi occurred concurrently with dissolution of β-1,3-glucan in walls of hyphae and conidia of the saprotrophic fungi. Then digestion of polysaccharides and chitin of colonised fungal structures occurred. The studies suggest sequential contribution of cell wall lytic enzymes and importance of disturbing the host's cell integrity during mycoparasitism. We conclude that the ECM fungus can parasitise on the saprotrophic fungi not only in dual culture on artificial medium but also in the rhizosphere of Scots pine.     

The contribution of the O-glycosylated protein Pir2p/Hsp150 to the construction of the yeast cell wall in wild-type cells and beta 1,6-glucan-deficient mutants

The cell wall of yeast contains a major structural unit, consisting of a cell wall protein (CWP) attached via a glycosylphosphatidylinositol (GPI)-derived structure to beta 1,6-glucan, which is linked in turn to beta 1, 3-glucan. When isolated cells walls were digested with beta 1,6-glucanase, 16% of all CWPs remained insoluble, suggesting an alternative linkage between CWPs and structural cell wall components that does not involve beta 1,6-glucan. The beta 1,6-glucanase-resistant protein fraction contained the recently identified GPI-lacking, O-glycosylated Pir-CWPs, including Pir2p/Hsp150. Evidence is presented that Pir2p/Hsp150 is attached to beta 1,3-glucan through an alkali-sensitive linkage, without beta 1,6-glucan as an interconnecting moiety. In beta 1,6-glucan-deficient mutants, the beta 1,6-glucanase-resistant protein fraction increased from 16% to over 80%. This was accompanied by increased incorporation of Pir2p/Hsp150. It is argued that this is part of a more general compensatory mechanism in response to cell wall weakening caused by low levels of beta 1,6-glucan.