Document Type

Peer-Reviewed Article

Publication Date

12-2007

Journal Title or Book Title

Eukaryotic Cell

Volume

6

Issue

12

Version

Publisher's PDF

Publisher's Statement

ASM grants authors the right to post their accepted manuscripts in publicly accessible electronic repositories maintained by funding agencies, as well as appropriate institutional or subject-based open repositories established by a government or non-commercial entity.

Abstract

The Flo11/Muc1 flocculin has diverse phenotypic effects. Saccharomyces cerevisiae cells of strain background Σ1278b require Flo11p to form pseudohyphae, invade agar, adhere to plastic, and develop biofilms, but they do not flocculate. We show that S. cerevisiae var. diastaticusstrains, on the other hand, exhibit Flo11-dependent flocculation and biofilm formation but do not invade agar or form pseudohyphae. In order to study the nature of the Flo11p proteins produced by these two types of strains, we examined secreted Flo11p, encoded by a plasmid-borne gene, in which the glycosylphosphatidylinositol anchor sequences had been replaced by a histidine tag. A protein of approximately 196 kDa was secreted from both strains, which upon purification and concentration, aggregated into a form with a very high molecular mass. When secreted Flo11p was covalently attached to microscopic beads, it conferred the ability to specifically bind to S. cerevisiae var. diastaticus cells, which flocculate, but not to Σ1278b cells, which do not flocculate. This was true for the 196-kDa form as well as the high-molecular-weight form of Flo11p, regardless of the strain source. The coated beads bound to S. cerevisiae var. diastaticus cells expressing FLO11 and failed to bind to cells with a deletion of FLO11, demonstrating a homotypic adhesive mechanism. Flo11p was shown to be a mannoprotein. Bead-to-cell adhesion was inhibited by mannose, which also inhibits Flo11-dependent flocculation in vivo, further suggesting that this in vitro system is a useful model for the study of fungal adhesion.

The fungal adhesins are a family of cell surface proteins that mediate adherence to environmental substrates or to other cells (7, 45). Adhesins are critically important in the initial steps of fungal pathogenicity, when fungal cells must adhere to host tissue. For the common human pathogens Candida albicans and Candida glabrata, the involvement of multiple adhesins in the adherence of fungal cells to host tissue has been demonstrated (4, 5, 18, 26, 43).

Among the adhesins is the flocculin family of Saccharomyces cerevisiae cell wall proteins that mediate flocculation, which is asexual calcium-dependent cell-cell aggregation. The most recently described member of the yeast flocculin gene family, FLO11/MUC1 (24, 30), is the only flocculin expressed in the Σ1278b strain of S. cerevisiae (17), and it exhibits a wide variety of phenotypes. Some of these phenotypes are strain specific. Yeast cells of strain background Σ1278b have been shown to require FLO11 for invasive growth (23, 30), the development of pseudohyphae (24, 29), and the formation of biofilms on plastic (36), but they do not flocculate. On the other hand, the variant strain S. cerevisiae var. diastaticus, which is highly flocculent, has been shown to require FLO11 for flocculation (30). FLO11 is also required in Σ1278b strains for the formation of mats with hub and spoke structures on semisolid agar (36). The common laboratory strain background S288C does not express FLO11 due to a nonsense mutation in the transcriptional activator FLO8(28). In some industrial strains, FLO11 mediates formation of the specialized biofilms called flors that are necessary for the production of sherry wine (19, 48). The common feature of all these phenotypes is adhesion. Commensurate with the many different pathways that regulate its expression, FLO11 has been shown to have a promoter that is among the largest described for yeast, at over 3 kb (38). Much more is known about gene regulation of FLO11 (for reviews, see references 11, 25, and 32) than about the structure and function of the protein.

We further investigated the FLO11-dependent phenotypes of S. cerevisiae var. diastaticus and found that it also differs from Σ1278b in that the haploids do not invade agar and the diploids do not form pseudohyphae. In order to investigate these strain differences in the phenotypes of FLO11we expressed and purified the Flo11 proteins from S. cerevisiae var. diastaticus and from Σ1278b and examined their properties. An in vitro system was created for studying the adhesive characteristics of the expressed Flo11 by attaching the protein to microscopic beads and testing the adhesive properties of the beads.

Microscopic beads that can be coated covalently with proteins or ligands have been used to simplify several complex biological processes. For example, Gaur and Klotz used microscopic magnetic beads coated with extracellular matrix proteins to isolate a C. albicans adhesin gene, ALS5, by expression cloning in S. cerevisiae (13). Further work using such beads resulted in characterization of the adhesion properties of Ala1p and Als1p (12, 14, 15, 21, 22, 35). In this study, we have used this approach to study the in vitro properties of purified Flo11 proteins from two different strains.

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