Worldwide, the dimorphic fungi cause several million infections each year. These fungi undergo a reversible transition between yeast (37oC) and mold (22oC). Growth as yeast promotes evasion of host immunity to cause disease, whereas growth as mold promotes survival in soil, genetic diversity through sexual reproduction, and transmission to new hosts. Despite the importance of thermal dimorphism, the question of how fungi regulate temperature adaptation is poorly understood and represents a major gap in knowledge. The long-term goal is to delineate the molecular mechanism(s) used by fungi to adapt to temperature. The research proposed investigates how a GATA transcription factor in Blastomyces dermatitidis, SREB (siderophore biosynthesis repressor in Blastomyces), governs the adaptation to temperature, and whether this regulation is linked with iron homeostasis. SREB null mutants fail to complete the temperature-dependent conversion to mold at 22oC and cannot properly regulate iron homeostasis. While most research has focused on the temperature change from 22oC to 37oC, the shift in the other direction - 37oC to 22oC - is underappreciated. Moreover, the downstream target genes and mechanisms used to respond to temperature (37oC or 22oC) remain ill defined. Analysis of SREB using gene expression microarrays revealed that deletion of this gene caused pleiotropic changes in transcription at 37oC and 22oC. Chromatin immunoprecipitation with quantitative real-time PCR (ChIP-qPCR) demonstrated SREB binds genes with disparate functions at 37oC and 22oC. Moreover, several candidate "non-iron" and "iron" genes under the control of SREB have been identified for functional testing. The hypothesis is SREB binds DNA at GATA motifs to regulate gene transcription, which in turn, controls the adaptation to temperature that is manifested by the transition to mold.
Aim 1 : Identify genes SREB binds in vivo on a genome-wide scale at 37oC and 22oC using ChIP with DNA sequencing (ChIP-seq). ChIP-seq is highly efficient and allows identification of SREB-bound genes without bias to specific motifs. When integrated with gene expression microarray and motif analyses, ChIP-seq will provide new, in-depth knowledge about how SREB impacts transcription at 37oC and 22oC.
Aim 2 : Functionally test SREB-bound genes we have "in-hand" (and those identified by ChIP-seq) for their impact on temperature adaptation (i.e., conversion to mold). Candidate genes "in-hand" will be tested by altering transcript abundance and analyzed for defects during the transition from 37oC to 22oC. Additional SREB-bound genes ("iron" and "non-iron") identified by ChIP-seq will be prioritized and tested in a similar fashion. The research is innovative because we are focusing on an understudied, but integral part of dimorphism, the transition to mold to understand how fungi adapt to temperature. The research is significant because the results will provide novel insight and serve as a foundation to decipher mechanisms used by fungi to adapt to temperature. Basic research on temperature adaptation has long-term potential to illuminate new therapeutic strategies for patients with fungal infections.
Collectively the dimorphic fungi are the most common agents of fungal disease worldwide, and cause pneumonia in persons with normal and impaired immune systems. The ability of these fungi to sense changes in temperature and switch between filamentous mold in soil and round, budding yeast in human tissue is critical for their biology and lifestyle. The proposed research, which dissects how dimorphic fungi adapt to temperature, will impact human health by providing novel insight on genes important for temperature adaptation;this, in turn, has the long-term potential to facilitate the discovery of new, innovative therapeutic strategies for treating patients with serious fungal infections. !