Histoplasma capsulatum is a pathogenic dimorphic fungus which causes a pulmonary mycosis of world-wide medical importance. In its yeast form, Histoplasma capsulatum apparently resides in the acidic environment of the macrophage phago-lysosome. One possible strategy for survival in such a hostile environment is the production of a proton pump like that which exists in the plasma membranes of the fungi Neurospora crassa and Saccharomyces cerevisiae, and the protozoan Leishmania donovoni, where there appears to be a life-stage difference in the pump's expression.
The aim of this proposal is to apply molecular cloning technology to produce molecular probes with which to analyze the gene or genes (and their cognate proteins) responsible for encoding the Histoplasma plasma membrane ion pumps. Our long term goal is to understand the structure, function and regulation of this enzyme or enzymes and, through this understanding, to provide new avenues to the control of this important pathogen. We have succeeded in demonstrating that Histoplasma capsulatum genomic DNA carries at least one, and as many as three, regions of homology to oligonucleotide probes designed to recognize this family of genes; we have also succeeded in the construction of a lambda EMBL3 genomic library of the Histoplasma capsulatum genome sufficiently large to ensure the isolation of these hybridizing sequences. We have the necessary nucleic acid probes with which to clone the H. capsulatum gene(s), and propose to use these genes to achieve the following specific aims: 1. Determine the DNA sequences of the genes. 2. Predict the amino acid sequences of the products of these genes. 3. Use this sequence information to produce nucleic acid and antibody probes for the analysis of expression of cation pump genes. 4. Determine the constitution of the two Histoplasma capsulatum life stages with respect to the amounts of pump-specific RNA and protein produced. 5. Determine the cation specificity of the pump(s) by expression in heterologous systems; and 6. Compare the abundance of the gene products to specific ion pumping and rates of ATP hydrolysis.
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