Propeptide genesis by Kex2-dependent cleavage of yeast wall protein 1 (Ywp1) of Candida albicans.

Candida albicans is a prevalent fungal resident and opportunistic pathogen of humans, exhibiting a variety of ovoid and filamentous morphologies. Anchored within the cell wall of the ovoid yeast form of C. albicans is an abundant glycoprotein termed yeast wall protein 1 (Ywp1). Ywp1 has an antiadhesive effect that may facilitate yeast cell dispersal; it also contributes to the masking of the glucan matrix of the yeast cell wall, potentially providing shielding from recognition by the human immune system. Mature Ywp1 consists of an O-glycosylated core of 378 amino acids associated with an N-glycosylated propeptide that originates from an N-terminal segment of Ywp1. A tribasic (-RRR-) sequence in the immature Ywp1 polypeptide is separated by 8 amino acids from a dibasic (-KR-) sequence that is a canonical site for cleavage by the intracellular endopeptidase Kex2, and cleavage occurs at both of these sites to generate an 11 kilodalton (kDa) propeptide that remains strongly associated with the mature core of Ywp1. Previous studies demonstrated an absence of the 11 kDa propeptide in strains lacking Kex2, but the presence of lesser amounts of a 12 kDa propeptide ostensibly (and paradoxically) arising from cleavage at the dibasic site. Subsequent studies of wild type strains, however, suggested that post-secretion cleavages were carried out in vitro by acid proteases in unbuffered cultures to generate the 12 kDa propeptide. Here, intact and Gfp-tagged Ywp1 are utilized to show that neither of the two multibasic sites is normally cleaved in the absence of Kex2, but that uncleaved Ywp1 is still N-glycosylated and subsequently anchored to the cell wall. This furthers our understanding of the multistep cleavage of this highly conserved sequence, as well as the possible contributions of the cleaved propeptide to the maturation and functioning of Ywp1.

Accessibility and contribution to glucan masking of natural and genetically tagged versions of yeast wall protein 1 of Candida albicans.

Yeast wall protein 1 (Ywp1) is an abundant glycoprotein of the cell wall of the yeast form of Candida albicans, the most prevalent fungal pathogen of humans. Antibodies that bind to the polypeptide backbone of isolated Ywp1 show little binding to intact yeast cells, presumably because the Ywp1 epitopes are masked by the polysaccharides of the mannoproteins that form the outer layer of the cell wall. Rare cells do exhibit much greater anti-Ywp1 binding, however, and one of these was isolated and characterized. No differences were seen in its Ywp1, but it exhibited greater adhesiveness, sensitivity to wall perturbing agents, and exposure of its underlying ß-1,3-glucan layer to external antibodies. The molecular basis for this greater epitope accessibility has not been determined, but has facilitated exploration of how these properties change as a function of cell growth and morphology. In addition, previously engineered strains with reduced quantities of Ywp1 in their cell walls were also found to have greater ß-1,3-glucan exposure, indicating that Ywp1 itself contributes to the masking of wall epitopes, which may be important for understanding the anti-adhesive effect of Ywp1. Ectopic production of Ywp1 by hyphae, which reduces the adhesivity of these filamentous forms of C. albicans, was similarly found to reduce exposure of the ß-1,3-glucan in their walls. To monitor Ywp1 in the cell wall irrespective of its accessibility, green fluorescent protein (Gfp) was genetically inserted into wall-anchored Ywp1 using a bifunctional cassette that also allowed production from a single transfection of a soluble, anchor-free version. The wall-anchored Ywp1-Gfp-Ywp1 accumulated in the wall of the yeast forms but not hyphae, and appeared to have properties similar to native Ywp1, including its adhesion-inhibiting effect. Some pseudohyphal walls also detectably accumulated this probe. Strains of C. albicans with tandem hemagglutinin (HA) epitopes inserted into wall-anchored Ywp1 were previously created by others, and were further explored here. As above, rare cells with much greater accessibility of the HA epitopes were isolated, and also found to exhibit greater exposure of Ywp1 and ß-1,3-glucan. The placement of the HA cassette inhibited the normal N-glycosylation and propeptide cleavage of Ywp1, but the wall-anchored Ywp1-HA-Ywp1 still accumulated in the cell wall of yeast forms. Bifunctional transformation cassettes were used to additionally tag these molecules with Gfp, generating soluble Ywp1-HA-Gfp and wall-anchored Ywp1-HA-Gfp-Ywp1 molecules. The former revealed unexpected electrophoretic properties caused by the HA insertion, while the latter further highlighted differences between the presence of a tagged Ywp1 molecule (as revealed by Gfp fluorescence) and its accessibility in the cell wall to externally applied antibodies specific for HA, Gfp and Ywp1, with accessibility being greatest in the rapidly expanding walls of budding daughter cells. These strains and results increase our understanding of cell wall properties and how C. albicans masks itself from recognition by the human immune system.

Insight into the antiadhesive effect of yeast wall protein 1 of Candida albicans.

Ywp1 is a prominent glycosylphosphatidylinositol (GPI)-anchored glycoprotein of the cell wall of Candida albicans; it is present in the yeast form of this opportunistic fungal pathogen but absent from filamentous forms and chlamydospores. Yeast cells that lack Ywp1 are more adhesive and form thicker biofilms, implying an antiadhesive activity for Ywp1, with a possible role in yeast dispersal. The antiadhesive effect of Ywp1 is transplantable from yeast to hyphae, as hyphae that are forced to express YWP1 lose adhesion in an in vitro assay. Deletion of the GPI anchor results in loss of Ywp1 to the surrounding medium and reduction of the antiadhesive effect, implying an importance of time-dependent residency in the cell wall. Anchor-negative versions of Ywp1 possessing or lacking a C-terminal green fluorescent protein (GFP) tag were created in C. albicans and harvested from culture supernatants; in addition to serving as quantifiable markers for Ywp1 secretion, they revealed that the cleaved 11-kDa propeptide of Ywp1 remains strongly but noncovalently associated with the Ywp1 core. This association is resistant to highly acidic and basic solutions, 8 M urea, and 1% SDS (below 45°C). Above 50°C, SDS dissociates the isolated complex, but even higher temperatures are required to dissociate the propeptide from native Ywp1 that is anchored in a cell wall. This property has permitted detection, for the first time, of orthologs of Ywp1 in other members of the Candida clade. The cleaved propeptide, which carries the sole N-glycan of Ywp1, must participate in the antiadhesive effect of Ywp1.

Yeast wall protein 1 of Candida albicans.

Yeast wall protein 1 (Ywp1, also called Pga24) of Candida albicans is predicted to be a 533 aa polypeptide with an N-terminal secretion signal, a C-terminal glycosylphosphatidylinositol anchor signal and a central region rich in serine and threonine. In yeast cultures, Ywp1p appeared to be linked covalently to glucans of the wall matrix, but, as cultures approached stationary phase, Ywp1p accumulated in the medium and was extractable from cells with disulfide-reducing agents. An 11 kDa propeptide of Ywp1p was also present in these soluble fractions; it possessed the sole N-glycan of Ywp1p and served as a useful marker for Ywp1p. DNA vaccines encoding all or part of Ywp1p generated analytically useful antisera in mice, but did not increase survival times for disseminated candidiasis. Replacement of the coding sequence of YWP1 with the fluorescent reporter GFP revealed that expression of YWP1 is greatest during yeast exponential-phase growth, but downregulated in stationary phase and upon filamentation. Expression was upregulated when the extracellular phosphate concentration was low. Disruption by homologous recombination of both YWP1 alleles resulted in no obvious change in growth, morphology or virulence, but the Ywp1p-deficient blastoconidia exhibited increased adhesiveness and biofilm formation, suggesting that Ywp1p may promote dispersal of yeast forms of C. albicans.