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.

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A screening procedure was used to identify cell fusion (hyphal anastomosis) mutants in the Neurospora crassa single gene deletion library. Mutants with alterations in 24 cell fusion genes required for cell fusion between conidial anastomosis tubes (CATs) were identified and characterized. The cell fusion genes identified included 14 genes that are likely to function in signal transduction pathways needed for cell fusion to occur (mik-1, mek-1, mak-1, nrc-1, mek-2, mak-2, rac-1, pp2A, so/ham-1, ham-2, ham-3, ham-5, ham-9, and mob3). The screening experiments also identified four transcription factors that are required for cell fusion (adv-1, ada-3, rco-1, and snf5). Three genes encoding proteins likely to be involved in the process of vesicular trafficking were also identified as needed for cell fusion during the screening (amph-1, ham-10, pkr1). Three of the genes identified by the screening procedure, ham-6, ham-7, and ham-8, encode proteins that might function in mediating the plasma membrane fusion event. Three of the putative signal transduction proteins, three of the transcription factors, the three putative vesicular trafficking proteins, and the three proteins that might function in mediating cell fusion had not been identified previously as required for cell fusion.The process of cell-to-cell fusion plays a vital role in the life cycles of almost all multicellular organisms. Fertilization, the fusion of an egg and sperm, is a required cell-to-cell fusion event for sexually reproducing organisms. For vertebrates, cell fusion is a critical step in the development of muscle, placenta, and bone and plays an essential role in the formation of multinucleated giant cells in the immune system. Hyphal cell fusion plays an important role in the life cycle of Neurospora crassa and other filamentous fungi (1,6,12,24,38). During the life cycle of the filamentous fungus Neurospora crassa, cell fusions occur during at least three stages (12, 38). During sexual development, fertilization occurs as a protoperithecium (immature female mating structure) generates a trichogyne (long specialized hyphae) that chemotrophically grows toward a conidium (asexual spore) or hypha of the opposite mating type and undergoes cell fusion with it. During the germination of N. crassa conidia, the cells produce short, specialized thin hyphae called conidial anastomosis tubes (CATs), which mediate cell fusion between the germlings and generate an interconnected hyphal network (38,41). This process of cell fusion between conidia allows the cells to share resources and may be critical to the establishment of a colony under some environmental conditions. Cell fusions also occur during the growth of a vegetative N. crassa colony. A few millimeters behind the growing edge of the colony, specialized fusion hyphae are formed as branches from the vegetative hyphae. The fusion hyphae grow toward each other in a directed manner and undergo cell fusion to generate an interconnected hyphal network within the colony (18). Cytoplasm and organelles flow f...

Cell wall proteins from purified Candida albicans and Neurospora crassa cell walls were released using trifluoromethanesulfonic acid (TFMS) which cleaves the cell wall glucan/chitin matrix and deglycosylates the proteins. The cell wall proteins were then characterized by SDS PAGE and identified by proteomic analysis. The analyses for C. albicans identified 15 cell wall proteins and 6 secreted proteins. For N. crassa, the analyses identified 26 cell wall proteins and 9 secreted proteins. Most of the C. albicans cell wall proteins are found in the cell walls of both yeast and hyphae cells, but some cell type-specific cell wall proteins were observed. The analyses showed that the pattern of cell wall proteins present in N. crassa vegetative hyphae and conidia (asexual spores) are quite different. Almost all of the cell wall proteins identified in N. crassa have close homologs in the sequenced fungal genomes, suggesting that these proteins have important conserved functions within the cell wall.

Using mutational and proteomic approaches, we have demonstrated the importance of the glycosylphosphatidylinositol (GPI) anchor pathway for cell wall synthesis and integrity and for the overall morphology of the filamentous fungus Neurospora crassa. Mutants affected in the gpig-1, gpip-1, gpip-2, gpip-3, and gpit-1 genes, which encode components of the N. crassa GPI anchor biosynthetic pathway, have been characterized. GPI anchor mutants exhibit colonial morphologies, significantly reduced rates of growth, altered hyphal growth patterns, considerable cellular lysis, and an abnormal "cell-within-a-cell" phenotype. The mutants are deficient in the production of GPI-anchored proteins, verifying the requirement of each altered gene for the process of GPI-anchoring. The mutant cell walls are abnormally weak, contain reduced amounts of protein, and have an altered carbohydrate composition. The mutant cell walls lack a number of GPI-anchored proteins, putatively involved in cell wall biogenesis and remodeling. From these studies, we conclude that the GPI anchor pathway is critical for proper cell wall structure and function in N. crassa.In eukaryotic cells, a number of proteins are anchored to the outer leaflet of the plasma membrane via glycosylphosphatidylinositol (GPI) anchors. The presence of the GPI anchor is thought to play an important role in the trafficking of these proteins and providing them with an attachment to the plasma membrane, and in the case of fungi, to the cell wall as well (24,41). Proteins destined to receive a GPI anchor are directed into the lumen of the endoplasmic reticulum (ER) by a typical signal peptide. The carboxyl termini of these proteins have a sequence motif that is recognized by a protein complex located in the ER, known as the GPI transamidase. The GPI transamidase complex cleaves the substrate protein at a position within this motif, termed the omega site, and transfers the GPI anchor en bloc to the newly generated C terminus of the protein.The structures of the GPI anchor in the trypanosome, yeast, and mammalian systems have been determined. Although there are differences in the various substituents present on the GPI anchors produced by these organisms, all GPI anchors appear to share a common core structure (15,19,20). This core structure consists of a phosphatidylinositide (or inositolcontaining sphingolipid) with an attached oligosaccharide chain that is terminated with a phosphoethanolamine residue. The linkages between the sugar units within the carbohydrate chain are conserved, and the amino group of the phosphoethanolamine moiety is used to attach the GPI anchor to the C terminus of the target protein. The organization of this basic GPI anchor structure is as follows: protein-phosphoethanolamine-6Mannose␣1-2Mannose␣1-6Mannose␣1-4Glucos-amine␣1-6inositol-phospholipid.The process of GPI anchor production and attachment is mediated by the concerted actions of approximately 20 proteins, which are organized into biosynthetic complexes in the ER membrane. Seven primary steps ha...