Molecular Basis for Invasiveness in A Pathogenic Fungus

Dranginis, Anne

Abstract

The yeast Candida albicans is the most frequently isolated fungal pathogen in humans. It has recently been shown that virulence of this yeast requires the ability to switch from a single cell form of growth to a filamentous form. Although it is very difficult to directly analyze Candida genetically, much progress has been made in understanding the dimorphic switch by using a related dimorphic yeast, Saccharomyces cerevisiae, as a guide. Diploid strains of Saccharomyces switch from a single cell form to a filamentous form, while haploid strains invade agar. Both these processes are regulated by a MAP kinase cascade in Saccharomyces that results in activation of the transcriptional activator Ste12. A homologous MAP kinase cascade exists in Candida and several of its components have been cloned by complementation in Saccharomyces. We have recently identified a Saccharomyces gene, FLO11, that encodes a cell surface molecule that is critically required for cell cell adhesion, invasiveness, and pseudohyphal formation. FLO11 is the only target of Ste12 that is required for invasion by Saccharomyces. It is the aim of this proposal to clone the C. albicans homolog of FLO11 by complementation of the invasion defect of the FLO11 mutation in Saccharomyces. A FLO11 mutant Saccharomyces strain will be transformed with a plasmid genomic library from C. albicans. Transformants will be selected and assayed for invasiveness. Transformants will also be observed for complementation of a colony morphology phenotype which is also conferred on Saccharomyces by FLO11 mutation. Invasive transformants will be mated with FLO11 mutant haploids to produce diploid strains, and the diploids will be tested for complementation of pseudohyphae formation. Plasmids from invasive transformants will be transformed into E. coli. Restriction maps of the plasmid inserts will be constructed and used to group plasmids containing identical Candida sequences. The inserted Candida gene from one member of each group will then be sequenced and the sequence will be compared with those currently in the database. This study will lead to the identification of a Candida functional homolog of FLO11, which will eventually be used to create a gene knockout in Candida where its effect on filamentation and pathogenicity may be studied. A cell surface molecule that is critically required for filamentation and invasion such as FLO11 may be a promising target for anti-fungal therapies.