Crh1p and Crh2p are required for the cross-linking of chitin to beta(1-6)glucan in the Saccharomyces cerevisiae cell wall

In budding yeast, chitin is found in three locations: at the primary septum, largely in free form, at the mother-bud neck, partially linked to beta(1-3)glucan, and in the lateral wall, attached in part to beta(1-6)glucan. By using a recently developed strategy for the study of cell wall cross-links, we have found that chitin linked to beta(1-6)glucan is diminished in mutants of the CRH1 or the CRH2/UTR2 gene and completely absent in a double mutant. This indicates that Crh1p and Crh2p, homologues of glycosyltransferases, ferry chitin chains from chitin synthase III to beta(1-6)glucan. Deletion of CRH1 and/or CRH2 aggravated the defects of fks1Delta and gas1Delta mutants, which are impaired in cell wall synthesis. A temperature shift from 30 degrees C to 38 degrees C increased the proportion of chitin attached to beta(1-6)glucan. The expression of CRH1, but not that of CRH2, was also higher at 38 degrees C in a manner dependent on the cell integrity pathway. Furthermore, the localization of both Crh1p and Crh2p at the cell cortex, the area where the chitin-beta(1-6)glucan complex is found, was greatly enhanced at 38 degrees C. Crh1p and Crh2p are the first proteins directly implicated in the formation of cross-links between cell wall components in fungi.     

Immobilization of the Glycosylphosphatidylinositol-anchored Gas1 Protein into the Chitin Ring and Septum Is Required for Proper Morphogenesis in Yeast

Gas1p is a glucan-elongase that plays a crucial role in yeast morphogenesis. It is predominantly anchored to the plasma membrane through a glycosylphosphatidylinositol, but a fraction was also found covalently bound to the cell wall. We have used fusions with the green fluorescent protein or red fluorescent protein (RFP) to determine its localization. Gas1p was present in microdomains of the plasma membrane, at the mother-bud neck and in the bud scars. By exploiting the instability of RFP-Gas1p, we identified mobile and immobile pools of Gas1p. Moreover, in chs3Δ cells the chitin ring and the cross-linked Gas1p were missing, but this unveiled an additional unexpected localization of Gas1p along the septum line in cells at cytokinesis. Localization of Gas1p was also perturbed in a chs2Δ mutant where a remedial septum is produced. Phenotypic analysis of cells expressing a fusion of Gas1p to a transmembrane domain unmasked new roles of the cell wall-bound Gas1p in the maintenance of the bud neck size and in cell separation. We present evidence that Crh1p and Crh2p are required for tethering Gas1p to the chitin ring and bud scar. These results reveal a new mechanism of protein immobilization at specific sites of the cell envelope.     

Comparison of cell wall localization among Pir family proteins and functional dissection of the region required for cell wall binding and bud scar recruitment of Pir1p

We examined the localization of the Pir protein family (Pir1 to Pir4), which is covalently linked to the cell wall in an unknown manner. In contrast to the other Pir proteins, a fusion of Pir1p and monomeric red fluorescent protein distributed in clusters in pir1Delta cells throughout the period of cultivation, indicating that Pir1p is localized in bud scars. Further microscopic analysis revealed that Pir1p is expressed inside the chitin rings of the bud scars. Stepwise deletion of the eight units of the repetitive sequence of Pir1p revealed that one unit is enough for the protein to bind bud scars and that the extent of binding of Pir1p to the cell wall depends on the number of these repetitive units. The localization of a chimeric Pir1p in which the repetitive sequence of Pir1p was replaced with that of Pir4p revealed the functional role of the different protein regions, specifically, that the repetitive sequence is required for binding to the cell wall and that the C-terminal sequence is needed for recruitment to bud scars. This is the first report that bud scars contain proteins like Pir1p as internal components.     

Two Novel Techniques for Determination of Polysaccharide Cross-Links Show that Crh1p and Crh2p Attach Chitin to both β(1-6)- and β(1-3)Glucan in the Saccharomyces cerevisiae Cell Wall

Previous work, using solubilization of yeast cell walls by carboxymethylation, before or after digestion with β(1-3)- or β(1-6)glucanase, followed by size chromatography, showed that the transglycosylases Crh1p and Crh2p/Utr2p were redundantly required for the attachment of chitin to β(1-6)glucan. With this technique, crh1Δ crh2Δ mutants still appeared to contain a substantial percentage of chitin linked to β(1-3)glucan. Two novel procedures have now been developed for the analysis of polysaccharide cross-links in the cell wall. One is based on the affinity of curdlan, a β(1-3)glucan, for β(1-3)glucan chains in carboxymethylated cell walls. The other consists of in situ deacetylation of cell wall chitin, generating chitosan, which can be extracted with acetic acid, either directly (free chitosan) or after digestion with different glucanases (bound chitosan). Both methodologies indicated that all of the chitin in crh1Δ crh2Δ strains is free. Reexamination of the previously used procedure revealed that the β(1-3)glucanase preparation used (zymolyase) is contaminated with a small amount of endochitinase, which caused erroneous results with the double mutant. After removing the chitinase from the zymolyase, all three procedures gave coincident results. Therefore, Crh1p and Crh2p catalyze the transfer of chitin to both β(1-3)- and β(1-6)glucan, and the biosynthetic mechanism for all chitin cross-links in the cell wall has been established.The fungal cell wall protects the cell against internal turgor pressure and external mechanical injury. To fulfill these functions, it must be endowed with a resilient structure. Presumably, the cell wall strength is largely due to the cross-links that bind together its components, mainly polysaccharides, giving rise to a tightly knit mesh (6, 11-13). Interestingly, the cross-links must be created outside the plasma membrane, because most of the polysaccharides are extruded as they are synthesized at the membrane; therefore, they do not exist inside the cell. This posits a thermodynamic problem, because there are no obvious sources of energy in the periplasmic space. About 20 years ago we proposed that the free energy may come from existing bonds in the polysaccharide chains and that the new cross-links may be originated by transglycosylation, thus creating a new linkage for each one that is broken (5).Ascertaining the mechanism of cross-link formation seemed a worthwhile endeavor, both because of the theoretical implications and because the cell wall is a proven target for antifungal compounds; therefore, more knowledge about its synthesis can be of practical interest. For this type of investigation to proceed, it was necessary to devise some method for the quantitative analysis of cell wall cross-links. We developed such a procedure for the evaluation of the proportion of cell wall chitin that is free or bound to β(1-3)- or β(1-6)glucan (4). In this methodology, chitin was specifically labeled in vivo with [¹⁴C]glucosamine; cell walls were isolated, and their proteins were eliminated by alkali treatment. The insoluble residue was solubilized by carboxymethylation and analyzed by size fractionation chromatography. By treating the cell walls with different glucanases before carboxymethylation and comparing the chromatographic profiles, we were able to determine the amount of chitin bound to the different glucans, as well as the fraction that was free (4). Armed with this procedure, we could now analyze the cell wall of different mutants that appeared to be candidates for cross-links defects. In this way we found that the two putative transglycosylases Crh1p and Crh2p were redundantly required for the formation of the chitin-β(1-6)glucan linkage. A double mutant crh1Δ crh2Δ had no chitin attached to β(1-6)glucan, although it still contained apparently normal amounts of chitin-β(1-3)glucan complex (7). Further work supported the notion that Crh1p and Crh2p function as transglycosylases, transferring portions of chitin chains to glucan (8). This confirmed our earlier hypothesis.With the initial intention of finding easier and faster methods, I devised two novel procedures for cell wall analysis. One is based on the affinity between β(1-3)glucan chains, the other on the conversion of chitin in situ into its deacetylated product, chitosan, followed by extraction of the chitosan with acetic acid before or after treatment with specific glucanases. With a wild-type strain, both procedures gave similar results to those of the carboxymethylation-chromatography technique. However, in the double mutant crh1Δ crh2Δ all of the chitin appeared to be free with both new methods. Further investigation showed that the older procedure led to erroneous results for the double mutant, because of the presence of a small amount of chitinase in the β(1-3)glucanase preparation used. After reconciling the results, I conclude that Crh1p and Crh2p are necessary for the formation of cross-links between chitin and either β(1-6) or β(1-3)glucan.     

Swm1p, a subunit of the APC/cyclosome, is required to maintain cell wall integrity during growth at high temperature in Saccharomyces cerevisiae

Swm1p, a subunit of the APC cyclosome, was originally identified for its role in the later stages of the sporulation process and is required for spore wall assembly. In addition, this protein is required to maintain cell wall integrity in vegetative cells during growth at high temperature. Electron microscopy analyses of mutant cells grown at the restrictive temperature in the absence of osmotic support show that the cell wall is clearly abnormal, with large number of discontinuities that may be responsible for the observed lysis. The mutant cells show a 7-fold reduction in glucan synthase activity during growth at 38 degrees C and a 3.5-fold increase in the chitin content of the cell wall. The chitin is deposited in a delocalized manner all over the cell wall, where it accumulates in patches in abnormal regions. The excess chitin is mainly synthesized by the action of chitin synthase III (Chs3p), since it disappears in the swm1 chs3 double-mutant.     

Ultrastructural localization of anionic sites on the surface of yeast, hyphal and germ-tube forming cells of Candida albicans

The cell wall of Candida albicans contains chitin, beta-glucans and phosphorylated mannoproteins, and possesses a fuzzy coat which is thought to play a role in pathogenicity, phagocytosis, and adherence of this dimorphic yeast. Using scanning electron microscopy and the gold method, mannoproteins were detected on the whole surface of blastoconidia including the bud scars, but chitin was absent even after alpha-mannosidase treatment of the cells. The presence of surface beta-(1----6)glucan (but not beta(1----3)glucan) was observed only after extensive alpha-mannosidase and alkaline phosphatase treatments of blastoconidia. Using transmission and scanning electron microscopy, the locations of anionic sites were revealed by polycationic colloidal gold-chitosan complexes on the surface of blastoconidia, germ tubes and hyphae. Anionic sites were dispersed evenly over the surface of blastoconidia bearing bud scars. Depending upon the growth conditions, anionic sites could be detected on emerging buds and young cells. However, bud scars were always free of marking. When germ-tube formation was induced, anionic sites were present at different densities on all cell surfaces, the highest density being observed on cells with bud scars. Anionic sites were detected at a remarkably high density on all hyphal surfaces. An apical concentration of anionic sites was observed on germ tubes and hyphae. The distribution of anionic sites was not modified by endoglucosaminidase treatment of blastoconidia, germ tubes and hyphae. The anionic sites were associated with the fuzzy coat. As the hyphal form is regarded as possessing the greatest invasiveness, it is suggested that anionic sites play an important role in establishing tissue colonization by this human pathogen.     

Rho1p mutations specific for regulation of beta(1-->3)glucan synthesis and the order of assembly of the yeast cell wall

In the yeast Saccharomyces cerevisiae, the GTP-binding protein Rho1 is required for beta(1-->3)glucan synthase activity, for activation of protein kinase C and the cell integrity pathway and for progression in G1, cell polarization and exocytosis. A genetic screen for cells that become permeabilized at non-permissive temperature was used to isolate in vitro-generated mutants of Rho1p. After undergoing a battery of tests, several of them appeared to be specifically defective in the beta(1-->3) glucan synthesis function of Rho1p. At the non-permissive temperature (37 degrees C), the mutants developed defects in the cell wall, especially at the tip of new buds. In the yeast cell wall, beta(1-->6)glucan is linked to both beta(1-->3)glucan and mannoprotein, as well as occasionally to chitin. We have used the rho1 mutants to study the order of assembly of the cell wall components. The incorporation of [(14)C]-glucose into beta(1-->3)glucan at 37 degrees C was decreased or abolished in the mutants. Concomitantly, a partial defect in the incorporation of label into cell wall mannoproteins and beta(1-->6)glucan was observed. In contrast, YW3458, an inhibitor of glycosylphosphatidylinositol anchor formation, prevented mannoprotein incorporation, whereas the beta(1-->3)-beta(1-->6)glucan complex was synthesized at almost normal levels. As beta(1-->3)glucan can be synthesized in vitro or in vivo independently, we conclude that the order of addition in vivo is beta(1-->3)glucan, beta(1-->6)glucan, mannoprotein. Previous observations indicate that chitin is the last component to be incorporated into the complex.     

Targeting of Chitin Synthase 3 to Polarized Growth Sites in Yeast Requires Chs5p and Myo2p

Chitin is an essential structural component of the yeast cell wall whose deposition is regulated throughout the yeast life cycle. The temporal and spatial regulation of chitin synthesis was investigated during vegetative growth and mating of Saccharomyces cerevisiae by localization of the putative catalytic subunit of chitin synthase III, Chs3p, and its regulator, Chs5p. Immunolocalization of epitope-tagged Chs3p revealed a novel localization pattern that is cell cycledependent. Chs3p is polarized as a diffuse ring at the incipient bud site and at the neck between the mother and bud in small-budded cells; it is not found at the neck in large-budded cells containing a single nucleus. In large-budded cells undergoing cytokinesis, it reappears as a ring at the neck. In cells responding to mating pheromone, Chs3p is found throughout the projection. The appearance of Chs3p at cortical sites correlates with times that chitin synthesis is expected to occur. In addition to its localization at the incipient bud site and neck, Chs3p is also found in cytoplasmic patches in cells at different stages of the cell cycle. Epitope-tagged Chs5p also localizes to cytoplasmic patches; these patches contain Kex2p, a late Golgi-associated enzyme. Unlike Chs3p, Chs5p does not accumulate at the incipient bud site or neck. Nearly all Chs3p patches contain Chs5p, whereas some Chs5p patches lack detectable Chs3p. In the absence of Chs5p, Chs3p localizes in cytoplasmic patches, but it is no longer found at the neck or the incipient bud site, indicating that Chs5p is required for the polarization of Chs3p. Furthermore, Chs5p localization is not affected either by temperature shift or by the myo2-66 mutation, however, Chs3p polarization is affected by temperature shift and myo2-66. We suggest a model in which Chs3p polarization to cortical sites in yeast is dependent on both Chs5p and the actin cytoskeleton/Myo2p.     

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.     

Synthase III-dependent chitin is bound to different acceptors depending on location on the cell wall of budding yeast

In yeast, chitin is laid down at three locations: a ring at the mother-bud neck, the primary septum and, after cytokinesis, the cell wall of the daughter cell. Some of the chitin is free and the remainder attached to beta(1-3)glucan or beta(1-6)glucan. We recently reported that the chitin ring contributes to the prevention of growth at the mother-bud neck and hypothesized that this inhibition is achieved by a preferential binding of chitin to beta(1-3)glucan at that site. Here, we devised a novel strategy for the analysis of chitin cross-links in [14C]glucosamine-labeled cell walls, involving solubilization in water of alkali-treated walls by carboxymethylation. Intact cell walls or their digestion products with beta(1-3)glucanase or beta(1-6)glucanase were carboxymethylated and fractionated on size columns, and the percentage of chitin bound to different polysaccharides was calculated. Chitin dispersed in the wall was labeled in maturing unbudded cells and that of the ring in early budding cells. The former was mostly attached to beta(1-6)glucan and the latter to beta(1-3)glucan. This confirmed our hypothesis and indicated that the cell has mechanisms to attach chitin, a water-insoluble substance, synthesized here through chitin synthase III, to different acceptors, depending on location. In contrast, most of the chitin synthase II-dependent chitin of the primary septum was free, with the remainder linked to beta(1-3)glucan.     

Increase in chitin as an essential response to defects in assembly of cell wall polymers in the ggp1delta mutant of Saccharomyces cerevisiae.

The GGP1/GAS1 gene codes for a glycosylphosphatidylinositol-anchored plasma membrane glycoprotein of Saccharomyces cerevisiae. The ggp1delta mutant shows morphogenetic defects which suggest changes in the cell wall matrix. In this work, we have investigated cell wall glucan levels and the increase of chitin in ggp1delta mutant cells. In these cells, the level of alkali-insoluble 1,6-beta-D-glucan was found to be 50% of that of wild-type cells and was responsible for the observed decrease in the total alkali-insoluble glucan. Moreover, the ratio of alkali-soluble to alkali-insoluble glucan almost doubled, suggesting a change in glucan solubility. The increase of chitin in ggp1delta cells was found to be essential since the chs3delta ggp1delta mutations determined a severe reduction in the growth rate and in cell viability. Electron microscopy analysis showed the loss of the typical structure of yeast cell walls. Furthermore, in the chs3delta ggp1delta cells, the level of alkali-insoluble glucan was 57% of that of wild-type cells and the alkali-soluble/alkali-insoluble glucan ratio was doubled. We tested the effect of inhibition of chitin synthesis also by a different approach. The ggp1delta cells were treated with nikkomycin Z, a well-known inhibitor of chitin synthesis, and showed a hypersensitivity to this drug. In addition, studies of genetic interactions with genes related to the construction of the cell wall indicate a synthetic lethal effect of the ggp1delta kre6delta and the ggp1delta pkc1delta combined mutations. Our data point to an involvement of the GGP1 gene product in the cross-links between cell wall glucans (1,3-beta-D-glucans with 1,6-beta-D-glucans and with chitin). Chitin is essential to compensate for the defects due to the lack of Ggp1p. Moreover, the activities of Ggp1p and Chs3p are essential to the formation of the organized structure of the cell wall in vegetative cells.     

Sbe2p and sbe22p, two homologous Golgi proteins involved in yeast cell wall formation

The cell wall of fungal cells is important for cell integrity and cell morphogenesis and protects against harmful environmental conditions. The yeast cell wall is a complex structure consisting mainly of mannoproteins, glucan, and chitin. The molecular mechanisms by which the cell wall components are synthesized and transported to the cell surface are poorly understood. We have identified and characterized two homologous yeast proteins, Sbe2p and Sbe22p, through their suppression of a chs5 spa2 mutant strain defective in chitin synthesis and cell morphogenesis. Although sbe2 and sbe22 null mutants are viable, sbe2 sbe22 cells display several phenotypes indicative of defects in cell integrity and cell wall structure. First, sbe2 sbe22 cells display a sorbitol-remediable lysis defect at 37 degrees C and are hypersensitive to SDS and calcofluor. Second, electron microscopic analysis reveals that sbe2 sbe22 cells have an aberrant cell wall structure with a reduced mannoprotein layer. Finally, immunofluorescence experiments reveal that in small-budded cells, sbe2 sbe22 mutants mislocalize Chs3p, a protein involved in chitin synthesis. In addition, sbe2 sbe22 diploids have a bud-site selection defect, displaying a random budding pattern. A Sbe2p-GFP fusion protein localizes to cytoplasmic patches, and Sbe2p cofractionates with Golgi proteins. Deletion of CHS5, which encodes a Golgi protein involved in the transport of Chs3p to the cell periphery, is lethal in combination with disruption of SBE2 and SBE22. Thus, we suggest a model in which Sbe2p and Sbe22p are involved in the transport of cell wall components from the Golgi apparatus to the cell surface periphery in a pathway independent of Chs5p.     

Polyoxin D inhibits colloidal gold-wheat germ agglutinin labelling of chitin in dimorphic forms of Candida albicans

Yeasts and mycelia of the pathogen Candida albicans grown in the presence of polyoxin D, a competitive inhibitor of chitin synthase, formed chains of swollen bulbous cells as observed by fluorescence microscopy. Wheat germ agglutinin (WGA) complexed to colloidal gold (Au) was used as a specific label at the ultrastructural level to visualize chitin in walls of control and polyoxin-treated cells. In control cells, Au-WGA labelling was preferentially localized in the innermost wall layers and was predominant at bud scars and septa. After 4.5 h in 4 mM-polyoxin D, budding in yeasts and lateral wall growth in mycelia continued, but primary septa failed to form and no Au-WGA labelling was detected in the walls. These results demonstrated that the morphological alterations caused by polyoxin D were due to the absence of chitin, a wall component important for formation of primary septa and for maintenance of structural integrity during morphogenesis.     

Presence of a large β(1-3)glucan linked to chitin at the Saccharomyces cerevisiae mother-bud neck suggests involvement in localized growth control

Previous results suggested that the chitin ring present at the yeast mother-bud neck, which is linked specifically to the nonreducing ends of β(1-3)glucan, may help to suppress cell wall growth at the neck by competing with β(1-6)glucan and thereby with mannoproteins for their attachment to the same sites. Here we explored whether the linkage of chitin to β(1-3)glucan may also prevent the remodeling of this polysaccharide that would be necessary for cell wall growth. By a novel mild procedure, β(1-3)glucan was isolated from cell walls, solubilized by carboxymethylation, and fractionated by size exclusion chromatography, giving rise to a very high-molecular-weight peak and to highly polydisperse material. The latter material, soluble in alkali, may correspond to glucan being remodeled, whereas the large-size fraction would be the final cross-linked structural product. In fact, the β(1-3)glucan of buds, where growth occurs, is solubilized by alkali. A gas1 mutant with an expected defect in glucan elongation showed a large increase in the polydisperse fraction. By a procedure involving sodium hydroxide treatment, carboxymethylation, fractionation by affinity chromatography on wheat germ agglutinin-agarose, and fractionation by size chromatography on Sephacryl columns, it was shown that the β(1-3)glucan attached to chitin consists mostly of high-molecular-weight material. Therefore, it appears that linkage to chitin results in a polysaccharide that cannot be further remodeled and does not contribute to growth at the neck. In the course of these experiments, the new finding was made that part of the chitin forms a noncovalent complex with β(1-3)glucan.     

Chitin and β(1–3) glucan synthases in the protoplastic entomophthorales

(1–3) glucan and chitin synthases were studied in spontaneously produced protoplasts and in the mycelium (hyphal body) of the entomopathogenic Entomophthorale species Entomophaga aulicae, Conidiobolus obscurus and Entomophthora muscae. The absence of wall in protoplasts was correlated to an absence of chitin synthase and to a very low (1–3) glucan synthase activity, whereas these two polysaccharide synthases were present and active in the walled hyphal bodies. Physicochemical properties of chitin and (1–3) glucan synthases such as localization, optimum pH and temperature, activation by disaccharides and proteases were similar to those found in other fungi unable to spontaneously produce protoplasts and could not be related to the ability for protoplastic Entomophthorale species to produce and proliferate under a protoplast form. The absence or the low chitin and glucan synthase activites in Entomophthorale protoplasts was not due to an absence of proteolytic activation of the enzyme. However, all protoplast fractions contained inhibitory substances of glucan and chitin synthase activities. These inhibitors were stable and specific of the protoplast stage. They were not glucanase nor chitinase. These results suggest that the absence of wall synthesis in Entomophthorale protoplasts is due to a continuous inhibition of (1–3) glucan and chitin synthase activities by intracellular compounds and also for glucan synthase by protoplast medium constituents such as NaCl and fetal calf serum.Abbreviations BSA bovine serum albumin - DFP diisopropylfluorophosphate - EDTA ethylenediamine tetraaoetic acid - FCS fetal calf serum - GlcNAc N-acetylglucosamine - TCA trichloroacetic acid - 2 k pellet 2,000 g wall fraction - 140 k pellet 140,000 g particulate fraction - 140 k supernatant 140,000 g soluble fraction     

Mannan as a major component of the bud scars of Saccharomyces cerevisiae

Summary Bud scars were prepared from the cell walls of Saccharomyces cerevisiae by means of an exo--d-(13)-glucanase. On acid hydrolysis, these structures were shown to consist of mannose (85%), glucose (4%) and glucosamine (2.7%). In addition the isolated bud scars were incubated either with mercurylabelled Concanavalin A or with Concanavalin A coated colloidal gold. They were then examined by electron microscopy and were found to be heavily marked, indicating the presence of mannan. It was thus shown that mannan is a true component of bud scars, which until now had been thought to consist solely of chitin and glucan.     

Loss of cell wall alpha(1-3) glucan affects Cryptococcus neoformans from ultrastructure to virulence

Yeast cell walls are critical for maintaining cell integrity, particularly in the face of challenges such as growth in mammalian hosts. The pathogenic fungus Cryptococcus neoformans additionally anchors its polysaccharide capsule to the cell surface via alpha(1-3) glucan in the wall. Cryptococcal cells disrupted in their alpha glucan synthase gene were sensitive to stresses, including temperature, and showed difficulty dividing. These cells lacked surface capsule, although they continued to shed capsule material into the environment. Electron microscopy showed that the alpha glucan that is usually localized to the outer portion of the cell wall was absent, the outer region of the wall was highly disorganized, and the inner region was hypertrophic. Analysis of cell wall composition demonstrated complete loss of alpha glucan accompanied by a compensatory increase in chitin/chitosan and a redistribution of beta glucan between cell wall fractions. The mutants were unable to grow ina mouse model of infection, but caused death in nematodes. These studies integrate morphological and biochemical investigations of the role of alpha glucan in the cryptococcal cell wall.     

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.