Functional genomics of cell wall biogenesis in fungi

In pathogenic fungi, glycosylphosphatidyl inositol cell wall proteins (GPI-CWPs) mediate adhesion of the fungus to a host during infection---a step that is necessary for the fungus to be able to initiate and establish infection. Genes involved in the biosynthetic pathway that incorporate GPI-CWPs to the wall thus represent excellent targets for antifungal drug design. A mechanistically-based genomic screen was developed and applied to search for genes involved in fungal cell wall biogenesis. Specifically, the screen identifies genes required for the cross-linking of glycoproteins (GPI-CWPs) to cell wall polysaccharides via GPI anchors. GPI-CWPs are the most abundant class of proteins at the wall and play both structural and biosynthetic functions.;The rationale for the screening assay was that cells unable to properly cross-link GPI-CWPs to the wall would either retain a GPI-CWP reporter protein within the cell (hypo-excretors) or excrete the reporter protein into the growth medium (hyper-excretors), relative to wild type cells. This idea was validated in cell wall mutants known to hyper-excrete GPI-CWPs. To screen for the hypo- and hyper-excretion phenotypes, a GPI-CWP-marker protein was made by fusing the green fluorescent protein (GFP) to the carboxyl end of the GPI-CWP alpha-agglutinin. The marker protein was expressed in 178 gene deletants missing genes previously implicated in cell wall biogenesis and in corresponding wild type parental strains. Strains expressing the reporter protein were examined for deficiencies in anchoring the marker protein to the wall. The screen used conditions that facilitated sample processing at large-scale and enhanced marker protein yields while minimizing noise in marker protein expression. These conditions required expression of the reporter protein from the constitutive S. cerevisiae GPD1 promoter at lower temperature than the standard 30°C in the presence of 1 M sorbitol, and growth of the cell cultures to similar cell concentrations in medium buffered to pH 6.5. Sample analysis at large scale was possible by growing the cells as 3 ml cultures in glass test tubes from which OD measurements were taken directly. Microliter volumes of cell-free supernatants were evaluated for GFP levels using a 96-well plate reader fluorimeter. Interesting mutants were subjected to secondary screens using anti-GFP antibodies and by collecting complete fluorescence spectra. The strains were also analyzed for cell surface fluorescence and levels of cytosolic and total protein in the culture medium.;Deletions in MCD4, GPI13, TDH3 and GDA1 caused hyper-excretion of the reporter protein. MCD4 and GPI13 are essential genes that play a role in GPI-anchor biosynthesis, GDA1 is involved in GPI-CWP O-glycosylation and TDH3 is a glycolytic enzyme that resides at the cell wall and has been implicated in mediating adhesion of fungal cells to host surfaces. The genomic screen was performed without the use of pepstatin A, which was found to inhibit GFP degradation by extracellular protease activity and thus to enhance the sensitivity of the screening assay without affecting cell growth and viability. Identified mutants thus hyper-excrete large enough amounts of the GPI-CWP reporter protein to allow detection of the hyper-excretion phenotype even in the presence of GFP proteolysis. Deletion of the transglycosidases Dcwp1 and Dfg5p, suspected of playing a direct role in cross-linking GPI-CWPs to the cell wall, hyper-excreted the reporter protein when grown in the presence of pepstatin A. Gene deletants subjected to secondary screens exhibited 1.5-2.0 fold less fluorescence at the wall than wild type cells except for MCD4/mcd4 and GPI13/gpi13 cells which hyper-express the marker protein at the wall 2-4-fold relative to wild type cells. This increase may be in compensation for the high failure rate of adding the GPI-anchor to GPI-CWPs precursors. All strains examined showed less than wild type levels of the cytosolic marker, 3-phosphoglycerate kinase (PGK) and wild type levels of total protein in cell-free supernatants.