The incidence of fungal infections is dramatically increasing and resistance to currently available antifungal agents is on the rise. The molecular mechanisms for fungal virulence are emerging gene-by-gene. What is needed is a rapid means to evaluate the function of multiple genes in the fungal genome to identify new antifungal targets. A powerful tool used for analysis of gene function in a wide variey of species has been antisense repression. The PI proposes to develop antisense repression as a screening method to identify genes essential for virulence of Cryptococcus neoformans. Specifically, the PI will focus on genes induced by growth of the fungus at 37 degreesdegrees C, since the ability to grow at mammalian physiologic temperatures is absolutely required for human pathogens. The PI has used a cDNA subtraction approach to enrich for genes upregulated at 37 degreesdegrees C, and these will serve as a substrate for the refinement of antisense in C. neoformans. Once developed, this technology could be used to screen genes differentially expressed under other conditions associated with fungal pathogenesis such as physiologic pH and CO2 levels. In addition, this methodology could be transferred to certain medically-relevant pathogens where transformation efficiencies and rates of homologous recombination are not optimal. Once genes essential for growth at 37 degreesdegrees C are identified, their suitability as antifungal targets needs to be determined. Therefore, the PI will use some of the techniques developed in the genomic screening procedure and in viral and mammalian systems to evaluate the potential of using antisense oligonucleotides (ODNs) in antifungal therapy. Specifically, the PI will define regions in the nucleotide sequence of CNA1, the gene that encodes calcineurin, a protein phosphatase already known to be essential for growth at 37 degreesdegrees C and virulence. The PI will then evaluate a series of ODNs designed to bind to the fungal-specific DNA sequences in vivo. This will be accomplished using the model system Saccharomyces cerevisiae. The PI will test the ODNs against mammalian and cryptococcal calcineurin being expressed in a yeast strain deficient in the expression of its own calcineurin gene. In the presence of the ODNs, the PI will screen for phenotypes associated with a calcineurin mutant.
