Roles of Candida albicans Dfg5p and Dcw1p Cell Surface Proteins in Growth and Hypha Formation

Abstract

The Candida albicans cell wall participates in both growth and morphological transitions between yeast and hyphae. Our studies here focus on Dfg5p and Dcw1p, two similar proteins with features of glycosylphosphatidylinositol-linked cell surface proteins. Mutants lacking Dfg5p are defective in alkaline pH-induced hypha formation; mutants lacking Dcw1p have no detected hypha formation defect. Both homozygote-triplication tests and conditional expression strategies indicate that dfg5 and dcw1 mutations are synthetically lethal. Therefore, Dfg5p and Dcw1p share a function required for growth. Epitope-tagged Dfg5p, created through an insertional mutagenesis strategy, is found in cell membrane and cell wall extract fractions, and endoglycosidase H digestion shows that Dfg5p undergoes N-linked mannosylation. Surprisingly, Dfg5p is required for expression of the hypha-specific gene HWP1 in alkaline media. Because Dfg5p is a cell surface protein, it is poised to generate or transmit an external signal required for the program of hypha-specific gene expression.

Role of Pir1 in the construction of the Candida albicans cell wall

“…This epitope is a 14 aa peptide (Gly-Lys-Pro-Ile-Pro-Asn-Pro-Leu-Leu-GlyLeu-Asp-Ser-Thr) that has been described as being convenient for tagging C. albicans extracellular proteins (Spreghini et al, 2003). The V5 epitope was introduced into the C-terminal end of CaPir1 as described in Methods.…”

Site-Directed and Random Insertional Mutagenesis in Medically Important Fungi

“…The technology has been adapted for diploid organisms using the parasexual cycle, haploid insufficiency, and homologous recombination (Carr et al, 2010;Davis et al, 2002;Firon et al, 2003;Juarez-Reyes et al, 2011;Spreghini et al, 2003;Uhl et al, 2003). The use of transposons has superseded auxotrophic and STM approaches.…”

“…This library is widely used by the Candida community Norice et al, 2007;Park et al, 2009). Spreghini et al (2003) exploited transposon mutagenesis to add an epitope to the putative cell wall protein, Dfg5p. Since conventional epitope tagging of amino and carboxyl termini was not an option, they wanted to identify an internal site which, when disrupted with a tag, did not compromise function.…”

Budding off: bringing functional genomics toCandida albicans

“…This method was used to construct 217 homozygous deletion strains [133], and identified a key regulator of alkaline-dependent filamentation [134]. This technique was subsequently used in targeted gene disruptions [135,136] and additional deletion libraries [137][138][139][140]. A separate approach relied on the generation of heterozygous mutants by insertional mutagenesis with a URA3-marked transposon into the genome [141].…”