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Molecular genetics and functional genomics of fungi

Abstract

The regulation of gene expression is vital for all organisms and is especially required by fungi for rapid adaptation to cellular stress conditions, as is the case when they infect a host or when they are submitted to antifungal drugs. These adaptive mechanisms are extremely complex and most of them have not been fully clarified. The objective of the present project is to continue to expand our research line in an attempt to elucidate the metabolic pathways or cellular processes that permit fungi to survive under adverse conditions. In addition, we intend to identify genes that may be involved in pathogenicity and that therefore may become targets for the development of new antifungal drugs. The model fungus Aspergillus nidulans and the dermatophyte Trichophyton rubrum will be used for this purpose. A determinant factor in the invasion and utilization of host tissue by fungi is the secretion of enzymes, which is frequently regulated by ambient pH. Consequently, the mechanisms of pathogenicity or even of resistance to inhibitors are likely to depend directly or indirectly on ambient pH monitoring. Thus, knowledge of the mechanisms that regulate the expression/secretion of enzymes involved in pathogenicity and of enzymes responsible for resistance to inhibitors will be of fundamental importance in the search for new therapeutic strategies, in the revelation of new drug targets, and therefore in the control of microorganisms that are harmful to man. To fulfill these objectives, three sub-projects, summarized below, will be developed: 1) mechanisms of resistance to inhibitory agents. Genes involved in resistance to inhibitors will be knocked out and the mutants will be used in experiments of differential expression. The information obtained will contribute to the understanding of the molecular mechanisms that impair antifungal therapy; 2) gene regulation in response to ambient pH. The objective of this sub-project is to knock out genes responsive to ambient pH and characterize these mutants by identifying the enzymes possibly involved in pathogenicity and determine their kinetic and structural properties. Genes differentialy expressed in these mutants will also be monitored. In addition, the utilization of bioinformatics and gel electrophoretic shift assay (GESA) in the analysis of promoters of genes responsive to pH will permit the identification of the genes that are being regulated by the same adaptive stimulus and that may participate in the same regulatory circuit; 3) experimental T. rubrum infection in animals. To evaluate whether genes involved in pH sensing and in resistance to antifungals are determinant in patogenicity, mutants of T. rubrum (selected or carrying knocked out genes) will be assayed for experimental infections in vivo. (AU)

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