Characterization of Neurospora crassa Cell Wall Biogenesis
The fungal cell wall is a vital organelle for its growth, morphology, and survival. For pathogenic fungi, the cell wall is a good target for anti-fungal agents because it is critical for fungal viability, as well as for virulence and pathogenicity. While a great deal of cell biology is shared between the fungi and their hosts, the cell wall is a uniquely fungal structure and so antifungal directed against cell wall biogenesis do not affect host functions. The fungal cell wall is composed of glucans, chitin, and cell wall glycoproteins. All three components are cross-linked together by cell wall cross-linking enzymes to generate a functional cell wall matrix. Although the composition of fungal cell wall is well-known, the mechanisms of how the cell wall components are cross-linked together has not been fully elucidated. This thesis contributes to our current understanding of how cell wall proteins are cross-linked to the cell wall matrix, and it may provide some potential possibilities for the development of anti-fungal agents. In Candida Albicans and Saccharomyces cerevisiae, two mechanisms for the incorporation of cell wall proteins into the cell wall matrix have been demonstrated in the past. Glycosylphosphatidylinositol (GPI)-anchored proteins have been shown to be cross-linked to cell wall via glycosidic bonds between cell wall glucans and the oligosaccharide portion of the GPI anchor. Proteins with internal repeats (PIRs) have been shown to be cross-linked to the cell wall via a linkage between a glutamine residue in the repeat and cell wall β-1,3-glucan. In chapter 2 of this thesis, we demonstrate a third mechanism for cross-linking cell wall proteins into the cell wall matrix in C. albicans. We show that the N-linked outer chain mannan structure present on cell wall proteins is required for the incorporation of cell wall proteins into the wall. This was demonstrated by showing the C. albicans och1 mutants, which are unable to elaborate the outer chain mannan associated with N-linked oligosaccharides, are defective in the incorporation of cell wall proteins into the cell wall. The involvement of the outer chain mannan in cell wall proteins incorporation was further demonstrated by showing that digestion of C. albicans purified cell walls with endoglycosidases with specificities for N-linked oligosaccharides releases GPI-anchored and non-anchored cell wall proteins. In addition to showing the attachment of cell wall protein into the wall occurs thru N-linked oligosaccharides, we also show that the Dfg5p, and Dcw1p -1,6-mannanases are required for cell wall protein incorporation into cell wall matrix in C. albicans. These mannanases have been previously shown to be involved in cross-linking Neurospora crassa cell wall proteins into the cell wall matrix through their N-linked oligosaccharides. My thesis research demonstrates that cell wall -1,6-mannanases, Dfg5p, and Dcw1p, are involved in C. albicans cell wall glycoproteins incorporation into cell wall matrix using the N-linked outer chain mannans. In Neurospora crassa, the N-linked outer chain mannan and the cell wall -1,6-mannanases (Dfg5p and Dcw1p) are required for cell wall proteins incorporation into the cell wall matrix. In chapter 3 of this thesis, another family of cell wall cross-linking enzymes were shown to be required for cell wall proteins incorporation as well. The GH 72 family of β-1,3-glucanases (GEL-1, GEL-2, GEL-3, and GEL-5) are found in cell wall proteome, and different combinations of these enzymes are expressed in different cell types. My thesis work demonstrated that these enzymes have overlapping activities, and are required for cell N. crassa cell wall protein incorporation into the cell wall matrix. The current available data in N. crassa supports the hypothesis that the -1,6-mannanses and -1,3-glucanases work in concert to cross-link the glycoproteins to cell wall glucan using the N-linked galactomannan present on the target proteins. In chapter 4 of my thesis I report on the results of a proteomic analysis of the N. crassa conidia cell wall. These results were compared with the proteome of the hyphal cell wall. We found that approximately half of the conidial cell wall proteins were specific to the conidia and not present in the vegetative hyphal cell wall, demonstrating that the cell wall is altered during asexual development. Promoter::RFP and promoter::GFP constructs of the promoters for 15 of the conidia-specific cell wall genes gave conidia-specific expression, demonstrating cell type-specific expression for the conidia cell wall proteins identified in our proteomic analysis. Our analysis also provided experimental evidence demonstrating that cell wall cross-linking enzymes are encoded by multigene families, and different members from these multigene families are expressed at different points in the N. crassa life cycle. We also showed that two conidia-specific cell wall glycosyl hydrolases, the CGL-1 β-1,3-glucanase (NCU07523) and the NAG-1 exochitinase (NCU10852), are required for proper conidia separation.