Histoplasma capsulatum, a dimorphic fungus, causes disease in humans that has a wide range of clinical manifestations. The long-term goal of this application is to develop an approach towards improving the outcome of histoplasmosis using biochemical and molecular techniques based on our earlier demonstration that virulent infection requires a phase transition at 37 degrees C in the host. We want to elucidate how genes coding for stress proteins and transition-specific (ts) proteins contribute to the morphologic transition and to the different degrees of pathogenicity in isolates from patients and soil. These studies will be of value in development of drugs that interfere with establishment of infection. There are three specific aims: 1. STudy genes involved in the early stages of the phase transition: a. Thermal adaptation - heat shock genes (hsg): clone and characterize the heat shock 83 gene and determine how regulation of its expression and maturation during phase transition at various temperatures in different isolates is related to thermotolerance and pathogenicity. Determine the physiological role the induction of hs proteins play in the different degrees of thermotolerance, morphogenesis, mitochondrial membrane integrity and virulence. b. Phase transition - clone and characterize ts gene that are expressed in the first 6 hrs of the mycelium to yeast differentiation process and determine the pattern of their expression in strains, including the transitional-defective PCMS strain, that show different levels of thermotolerance and pathogenicity. Within this group we expect to find genes that contain heat shock elements in their regulatory regions. 2. Determine the effect of disruption of genes involved in the phase transition and in virulence by: a. Establishing a transformation protocol - design a procedure that will yield an efficient transformation system in order to analyze the role in vitro-mutated genes play in morphogenesis and pathogenicity. b. In vitro mutagenesis to determine its effect on phase transition and adaption - analyze the effect mutagenesis has on the biochemical events that take place after the temperature shift. 3. Determine the role that hs and ts genes play in virulence by: a. Evaluating the in vitro-mutated strains in mice.
