Light-regulated genes affecting the pathogen potential of Cryptococcus neoformans

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DESCRIPTION (provided by applicant): The applicant is currently an experienced post-doctoral fellow transitioning into a role as an independent investigator. Immediate career objectives are to complete current research and obtain bridging resources to facilitate the establishment of an academic research position. The K22 award and career development plans would link within the long-term career objectives of developing an internationally-recognized laboratory investigating fungi that cause human disease or allergy. Light influences many properties in species of fungi, such as regulating sexual or asexual sporulation, pigment production, phototropism, and setting a circadian clock. The BWC1 and BWC2 genes, encoding a putative photoreceptor and interacting transcription factor, control inhibition of mating by light, tolerance to UV irradiation, and virulence in the human pathogenic fungus Cryptococcus neoformans. However, it is unclear what genes are regulated by light via Bwc1-Bwc2. Their identification, through this research, will provide directions towards controlling fungal infectious disease. Aim 1 is to identify genes regulated by light using a whole genome microarray analysis of transcription, comparing a wild type strain grown in constant darkness or treated with a pulse of light. Aim 2 is to demonstrate that the promoters of light-regulated genes physically interact with Bwc1-Bwc2 by gel mobility shift assays. Preliminary studies have revealed 30 genes that are light-regulated. One such gene encodes ferrochelatase, which catalyzes the final step of heme biosynthesis from protoporphyrin IX. Heme is an essential co-factor in many proteins, while porphyrins are highly photoreactive. Aim 3 will test the hypothesis that ferrochelatase activity is a major factor that mediates C. neoformans sensitivity to light and virulence. Strains with altered expression levels of ferrochelatase will be constructed and their phenotypes tested for mating, UV sensitivity and virulence in a macrophage cell line model. The relevance of the research is that it will enable the discovery of new genes that enable fungi to be virulent towards humans and to spread in nature. This information is expected to provide strategies to minimize exposure to fungal inocula to reduce initial infection or exposure to spores that cause allergies, and enable the design of strategies to use light as a therapy against fungal diseases.