SWITCHING AND CANDIDA PATHOGENESIS--A MOLECULAR ANALYSIS

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DESCRIPTION (Adapted from applicant's abstract): C. albicans switches spontaneously, reversibly and at high frequency between a number of general phenotypes distinguishable by colony morphology. Switching has a pleiotropic effect on cellular phenotype and occurs in commensal and pathogenic populations. The combinatorial changes of phenotype traits provide C. albicans an adaptive advantage for responding to changes in the host. Using the white-opaque phase transition in strain WO-1 as an experimental model, it was recently demonstrated that switching involves precise activation and deactivation of phase-specific genes, that in the case of the white phase-specific gene WH11, white-specific transcription is regulated by two transcription activation domains in its promoter, and these domains form phase-specific complexes with white, but not opaque cell protein extract. These results have led to the working hypothesis that white phase-specific genes are regulated by white phase-specific activators, and recent gel retardation studies suggest that opaque phase-specific genes may be regulated by white phase-specific repressors. The specific objectives of this proposal are: 1) to develop an accurate model for the circuitry involved in the regulation of phase-specific genes; 2) to identify the genetic locus and describe the general mechanism of the basic switch event; 3) to assess the roles played by individual phase-specific genes in the genesis of switch phenotypes and 4) to asses the role of individual phase-specific genes in virulence. To obtain a complete picture of the regulatory circuitry and hierarchy of regulatory events involved in the program of phase-specific gene regulation, we have developed several strategies for cloning additional phase-specific genes. The promoters of select white and opaque phase genes will be functionally characterized, using a newly developed Renilla reniformans luciferase bioluminescence reporter system, to identify cis-acting sequences and the mode of regulation (positive and negative), and gel retardation assays carried out with the cis-acting sequences and white or opaque cell extract to identify phase-specific complexes. To elucidate the basic switch event, a phase-specific trans-acting factor will be identified which controls expression of a phase-specific gene and itself is transcriptionally regulated. The cis-acting sequence will be used as a probe to screen an expression library for the trans-acting factor gene. If the regulated factor confers binding specificity but does not directly bind to the cis-acting regulatory sequence, the strategy for isolation will involve affinity purification of the factor, determination of protein primary sequence and the design of probes based on sequence to protein sequence to screen for the gene in question. The role of individual phase-specific genes in switching will be assessed by the generation of mis-expression mutants which express individual phase-specific in the wrong phase and disruptants of the phase-specific genes, and characterize the phenotypic consequences. Finally, to assess the role of switching and the expression of phase specific genes in pathogenesis, the same mis-expression and null mutants will be studied in two animal models, one model in which white cells are more virulent than opaque cells, and a mouse using model in which opaque cells are more virulent than white cells.