The N-Linked Outer Chain Mannans and the Dfg5p and Dcw1p Endo-α-1,6-Mannanases Are Needed for Incorporation of Candida albicans Glycoproteins into the Cell Wall

Abstract

A biochemical pathway for the incorporation of cell wall protein into the cell wall of Neurospora crassa was recently proposed. In this pathway, the DFG-5 and DCW-1 endo-␣-1,6-mannanases function to covalently cross-link cell wall protein-associated N-linked galactomannans, which are structurally related to the yeast outer chain mannans, into the cell wall glucan-chitin matrix. In this report, we demonstrate that the mannosyltransferase enzyme Och1p, which is needed for the synthesis of the N-linked outer chain mannan, is essential for the incorporation of cell wall glycoproteins into the Candida albicans cell wall. Using endoglycosidases, we show that C. albicans cell wall proteins are cross-linked into the cell wall via their N-linked outer chain mannans. We further demonstrate that the Dfg5p and Dcw1p ␣-1,6-mannanases are needed for the incorporation of cell wall glycoproteins into the C. albicans cell wall. Our results support the hypothesis that the Dfg5p and Dcw1p ␣-1,6-mannanases incorporate cell wall glycoproteins into the C. albicans cell wall by cross-linking outer chain mannans into the cell wall glucan-chitin matrix.T he fungal cell wall plays a critical role in fungal survival, growth, and morphology. The fungal cell wall is generated by the cross-linking of glucans, chitin, and cell wall proteins in the cell wall space to create a three-dimensional matrix (1-6). In Candida albicans and Saccharomyces cerevisiae, the major glucans in the cell wall are ␤-1,3-glucan and ␤-1,6-glucan (1-8). The ␤-1,3-glucan and chitin elements are synthesized as linear polymers and extruded into the cell wall space during their synthesis by glucan synthases and chitin synthases. These enzymes use UDP-glucose and UDP-N-acetylglucosamine as substrates and add glucose and N-acetylglucosamine to the nonreducing ends of the nascent glucan and chitin polymers. As the glucan and chitin polymers are extruded into the cell wall space, they are cross-linked by groups of cell wall enzymes with glucanase and glucan transferase (transglycosylase) activities. ␤-1,6-Glucan has been implicated in crosslinking the other cell wall elements (1-3, 6). The enzymes involved in cross-linking the glucan and chitin polymers are encoded by multigene families which produce enzymes with overlapping specificities.Fungal cell walls have a characteristic array of integral cell wall proteins which are cross-linked into the glucan-chitin matrix. These include the glucanase/glucan transferase enzymes involved in cross-linking the wall polymers, adhesins, signal pathway receptors, and structural proteins (8)(9)(10)(11)(12)(13)(14). Some of these cell wall proteins have been shown to be critical for virulence and for the formation and functionality of the cell wall. These proteins contain signal peptide sequences at their N termini and travel through the canonical secretory pathway. As these proteins pass through the C. albicans endoplasmic reticulum (ER) and Golgi apparatus, they become heavily glycosylated with O-linked and N-linked oligosaccharides. Th...

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“…In this study we examined the functions of Dfg5 and Dcw1 in biofilm formation and Hog-1 signaling by using the double mutant strains, ES1 and ES195. Using the above C. albicans mutant strains we have previously confirmed that the Dfg5p and Dcw1p mannanases (crosslinking enzymes) function in cell wall biogenesis in C. albicans (Ao et al, 2015). We have also showed previously that the DFG5/DCW1 conditional mutants have a cell separation growth phenotype.…”

“…5 ul each of the undiluted, 1:10, 1:100, and 1:1000 dilution samples were spotted onto YNB pH7 plates containing one of the following cell wall stress agents: Calcofluor White (20 µg/ml), Caspofungin (0.25 µg/ml), Congo Red (1 µg/ml), 100 µg/ml SDS (100 µg/ml), or Sorbitol (1 M). Concentrations of stress agents were based on MIC values as described previously (Ao et al, 2015;Nikolaou et al, 2009;Heilmann et al, 2013). In addition, just for the 1M sorbitol experiments WT (SC5314), WT (BWP13), ES1 and ES195 were plated in YNB with 5mM methionine and 2 mM cysteine.…”

“…Based on the observation that the expression of HWP1, a wellknown cell wall gene expressed only in hyphae, was dependent upon the presence of Dfg5p, it has been thought to be involved in signal transduction mechanisms tied into the cell wall integrity pathway (Spreghini et al, 2003). We have showed that C. albicans Dfg5p and Dcw1p are involved in cell wall protein cross-linking within the cell wall (Ao et al, 2015). In a recent study in the yeast Saccharomyces cerevisiae it was observed that Hog-1 and Slt2 signaling pathways are affected in the DFG5 deletion background (Nasution et al, 2015).…”

Characterization of two homeodomain transcription factors with critical but distinct roles in virulence in the vascular pathogen Verticillium dahliae

“…Upadhya et al (2016) showed that deletion of the three chitin deacetylase genes of C. neoformans renders strains avirulent. VDAG_09238 was highly similar to dfg5 which encodes a glycosylphosphatidylinositol (GPI)-anchored cell wall a-1,6-mannanase required for the incorporation of glycoproteins into the cell wall (Ao et al, 2015;Maddi et al, 2012).…”

Unmasking fungal pathogens by studying MAPK-dependent cell wall regulation inCandida albicans

“…Mannosylation can impact the function, stability, and localization of proteins and can also lead to the cross linking of glycoproteins to polysaccharides in the cell wall. 12 The cell wall is a dynamic structure. It is rigid enough to provide support and protection, yet is malleable enough to allow for growth and differentiation.…”