Characterization of Histoplasma transcription factors using improved episomal DNA maintenance and controllable gene expression tools

Project Summary Histoplasma capsulatum causes pulmonary and systemic infections in both healthy and immunocompromised individuals, and is the most common cause of fungal respiratory infections in healthy hosts. It is a dimorphic fungal pathogen that can sense and respond to human body temperature by changing its growth program from a filamentous (mold) form to a parasitic yeast form. Infection occurs when the soil is disrupted, facilitating dispersion of hyphal fragments or spores that are inhaled by humans. Once introduced into the host, the pathogen converts to a budding-yeast form, which survives and replicates within host macrophages. In the laboratory, the switch between the infectious and parasitic states is modeled by changing the temperature: cells grow in the filamentous form at room temperature, whereas growth at 37ºC is sufficient to trigger growth in the yeast form and expression of virulence factors. In previous studies, we and others have identified four transcriptional regulators, Ryp1,2,3,4, which are required for yeast-phase growth and virulence gene expression. Ryps directly regulate expression of a set of yeast-phase specific genes that encode proteins with putative secretion signals and transmembrane helices; thus, form the core of the yeast-phase specific transcriptional network. In preliminary studies, we have found that two major players of the heat shock response, the heat shock factor Hsf1 and a chaperone Hsp90, also regulate the yeast-phase growth under in vitro conditions. However, these genes are essential and their function cannot be studied by conventional knockout strategies in Histoplasma. There is limited research done in Histoplasma, in part due to the limitation of the genetic tools available. In particular, there is no controllable gene expression systems that can be used to study gene function in vivo. In this project, we aim to develop controllable (Tet-inducible and Tet-repressible) gene expression systems and optimize episomal DNA maintenance. Using the Tet-inducible system, we will induce expression of knockdown cassettes for Ryp1,2,3,4, Hsf1 and Hsp90 under in vivo conditions and assess their role in maintenance of the parasitic growth in vivo. In addition, we will identify centromeric sequences and/or DNA elements that promote autonomous replication or segregation; and utilize them in episomal vectors to ensure proper segregation of the episomal DNA during cell division. We will generate knockdown and overexpression mutants of yeast- or hyphal- phase specific transcription factors using improved episomal vectors. Ultimately, results of the experiments proposed in this project will further advance our ability to study gene function in Histoplasma, and will expand our knowledge of the transcriptional network that governs the parasitic yeast-phase growth.