Mechanisms of fungal adhesion as revealed by strain variation in Flo11.

摘要

FLO11 is a yeast cell wall flocculin which mediates a variety of adhesive phenotypes and is an important determinant of cellular morphogenesis in Saccharomyces cerevisiae. It is found to be critical for several yeast developmental process including flocculation, pseudohyphae formation, agar invasion, microbial mats and biofilm formation. Display of these phenotypes has been shown to be strain specific and FLO11 dependent. To investigate the mechanisms by which this adhesin determines these differences in morphology it is meaningful to study the difference in FLO11 gene structure and function.;To investigate the effect of FLO11 mRNA expression levels on flocculation, quantitative RT-PCR analysis of FLO11 transcripts was performed. Interestingly, nonflocculant Σ1278b cells exhibit significantly higher FLO11 mRNA expression in stationary phase in comparison to highly flocculent S. cerevisiae var. diastaticus. Further experiments were performed to explore the influence of other factors such as cell wall hydrophobicity on FLO11-dependent cell adhesion. Results indicate that Flo11p doesn't make much contribution to cell surface hydrophobicity though strains vary in cell surface hydrophobicity.;Sequencing of the FLO11 gene in the highly flocculent strain S. cerevisiae var. diastaticus revealed differences between this strain and strains S288c and Σ1278b. One striking difference is the presence of a 15 amino acid insertion in the Σ1278b Flo11 which is not there in S. cerevisiae var. diastaticus or S288C. The differences in protein sequences were further investigated with emphasis on the Σ1278b adhesion domain to explore the role of this insertion by purifying the adhesion peptide from cells secreting the peptide which is expressed from a plasmid borne vector. Characterization of protein product was performed using an in vitro model system by coating microscopic beads with the secreted peptide. The coated beads exhibited preferential strain-specific binding to Σ1278b cells rather than S. cerevisiae var. diastaticus cells. To investigate whether the strain specific binding by the coated beads reflects strain specific binding in vivo, coflocculation assays were performed. Different strains were marked with different colored fluorescent proteins, then mixed and allowed to flocculate. I observed a striking pattern of floc formation, whereby the center of the flocs was occupied primarily by the strain exhibiting the strongest flocculation. The outer surface, however, was covered exclusively by the least flocculent strain. This pattern is similar to the one exhibited in the reconstruction of vertebrates embryos, leading me to propose a model that similar rules governs the construction of microbial communities and vertebrate embyros. These data have important implications also for the evolution of strains and species of fungi.