The GPI-modified proteins Pga59 and Pga62 of Candida albicans are required for cell wall integrity

Abstract

The fungal cell wall is essential in maintaining cellular integrity and plays key roles in the interplay between fungal pathogens and their hosts. The PGA59 and PGA62 genes encode two short and related glycosylphosphatidylinositol-anchored cell wall proteins and their expression has been previously shown to be strongly upregulated when the human pathogen Candida albicans grows as biofilms. Using GFP fusion proteins, we have shown that Pga59 and Pga62 are cell-walllocated, N-and O-glycosylated proteins. The characterization of C. albicans pga59D/pga59D, pga62D/pga62D and pga59D/pga59D pga62D/pga62D mutants suggested a minor role of these two proteins in hyphal morphogenesis and that they are not critical to biofilm formation. Importantly, the sensitivity to different cell-wall-perturbing agents was altered in these mutants. In particular, simultaneous inactivation of PGA59 and PGA62 resulted in high sensitivity to Calcofluor white, Congo red and nikkomicin Z and in resistance to caspofungin. Furthermore, cell wall composition and observation by transmission electron microscopy indicated an altered cell wall structure in the mutant strains. Collectively, these data suggest that the cell wall proteins Pga59 and Pga62 contribute to cell wall stability and structure. INTRODUCTIONThe cell wall is an essential component of fungal cells, preserving cellular integrity and playing a central role in the interaction of fungi with their environment. This is particularly the case for pathogenic fungi such as the opportunistic yeast pathogen Candida albicans, where the cell wall has been shown to play central roles in adhesion, virulence, biofilm formation, infection and immunomodulation (Albrecht et al., 2006;Douglas, 2003;Netea et al., 2006;Richard et al., 2002b;Sundstrom, 2002). Because of the essential role of the cell wall in cellular integrity and fungal specificity of some enzymes involved in its biogenesis, it is a recognized target for the development of novel antifungals (e.g. echinocandins that target the b-1,3-glucan synthase) (Latge, 2007).The organization of the fungal cell wall has been mainly characterized in the yeasts Saccharomyces cerevisiae and C. albicans and in the filamentous fungus Aspergillus fumigatus (Klis et al., 2006;Latge, 2007;Lesage & Bussey, 2006;Ruiz-Herrera et al., 2006). The yeast cell wall has a bilayered structure. The inner part is composed of a network of b-1,3-glucan molecules linked by hydrogen bonds. These chains can be bound covalently to b-1,6-glucan molecules and to chitin chains. The outer part of the cell wall is composed mainly of mannoproteins (Klis et al., 2006Lesage & Bussey, 2006). Most proteins in the cell wall of ascomycetous yeasts are glycosylphosphatidylinositolanchored proteins (GPI-modified proteins) that become covalently linked to b-1,6-glucan through a remnant of their GPI anchor. As the b-1,6-glucan moiety can be linked to b-1,3-glucan or chitin, the cell wall GPI-modified proteins are strongly linked to the cell wall Klis et al., 2001;Richard & Plaine, 2007).GPI-...

Unexpected Role for a Serine/Threonine-Rich Domain in the Candida albicans Iff Protein Family

“…4A). The fluorophore displayed a similar strong peripheral staining for these four proteins, as already described for GPI-anchored proteins (47,50). Only Iff8 showed a weak peripheral signal, suggesting that the V5 epitope was not accessible to the antibody from the surface.…”

“…Some GPI-modified proteins have been successfully tagged and localized. Examples include Pga59 tagged with green fluorescent protein (GFP) (50), Eap1 tagged with hemagglutinin (43), and Dfg5 tagged with the V5 epitope (59). However, to date only a quarter of the putative GPIanchored proteins have been detected at the surface of the cells based on liquid chromatography-tandem mass spectrometry, leaving around 75 proteins with an unknown localization (9,14,46,49,58).…”

“…Are there specific partner proteins? Several putative GPI-anchored proteins that display tandem repeats containing cysteine residues have been reported (Cx 4 Cx 18-22 YTTYCPL [50]); these proteins might be good candidates for such a disulfide bond and thus deserve specific attention in future studies. Whether this interaction is only important for localization or has some functional importance has not yet been determined, but this question would need the identification of at least 2 partners.…”

Insight into the Antiadhesive Effect of Yeast Wall Protein 1 of Candida albicans

“…The 533-amino-acid (aa) polypeptide encoded by YWP1 has the following features: (i) an N-terminal signal peptide of 21 aa that directs the nascent polypeptide into the secretory pathway but is then cleaved and lost; (ii) a tribasic sequence (RRR) followed closely by a dibasic sequence (KR), both of which are cleaved to create an N-terminal propeptide of about 100 aa (at least one of these cleavages is predicted to occur intracellularly, prior to secretion); (iii) a segment of about 360 aa that is especially rich in threonine, with a third of the residues in this segment being either threonine or serine (this segment contains three tandem copies of a 42-to 51-aa motif that is also found in several other wall proteins [23,39,54]); and (iv) a hydrophobic C terminus and adjacent signal that result in replacement of the C-terminal 22 aa with a GPI moiety anchored in the plasma membrane. The GPI anchor is subsequently cleaved, and the 378-aa core of Ywp1 becomes attached covalently to the glucan network of the cell wall through a lipid-free remnant of the GPI anchor.…”

Structure and Function of Glycosylated Tandem Repeats from Candida albicans Als Adhesins

“…Tandem repeats are a common feature in the adhesins, and they usually occur near the middle of the sequences. These repeats are commonly conserved in length and sequence within an adhesin family, and many such repeats are predicted to be ␤-sheet-rich domains (8,16,28,47,49,51). More repeats are associated with greater aggregation in the S. cerevisiae flocculins, as they are in the C. albicans Als proteins (16,47,48).…”