In this program project we will conduct basic studies on medically important fungi at the molecular and cellular level to: 1) enhance understanding of fungal pathogenesis; 2) develop biochemical markers of infection by fungi; 3) explore human phagocyte surface receptor interactions with ligands of Histoplasma capsulatum (Hc) and the subsequent signaling of intraphagocyte biochemical events; 4) further characterize the gene structure and organization of Pneumocystis carinii which we and others recently demonstrated to be a member of the fungi by small subunit ribosomal RNA comparative analysis. Proj. 1 will produce and characterize nucleic acid probes to analyze the gene(s) and other cognate proteins responsible for encoding a plasma membrane proton ion pump in Hc yeasts that maintains a reasonably constant internal pH and may enhance survival within the acidic milieu of host macrophage phagolysosomes. Proj. 2 will examine the fate of Histoplasma yeasts and microconidia in human monocyte/macrophages and alveolar macrophages. The ligands within yeasts and conidia that are recognized by the LFA-1,CR3,p150,95 receptors of macrophages will be identified and purified. Mechanisms of signal transduction initiated by yeast binding to these receptors will be compared to those induced by binding of C3bi-coated erythrocytes to CR3. Proj. 3 will study the role of elastinolytic proteinase in the virulence of invasive Aspergillus species by isolating and cloning the gene for Aspergillus elastase (AE). Cloned AE gene will be used to produce recombinant AE for use in diagnostic testing and virulence studies; the chromosomal location of the gene will be determined, and AE gene probes will be evaluated for diagnosis by in situ hybridization. Proj. 4 will pursue observations that pathogenic species of Aspergillus and Cryptococcus produce large amounts of the polyol, D-mannitol. Capillary gas-chromatography will be used to determine if this metabolite can be employed as a quantitative, diagnostic marker of invasive infection by these fungi in experimental animals and patients. Proj. 5 will extend our knowledge of the fungal nature of P. carinii by employing cloned DNA fragments to characterize gene structure and organization. Genes encoding ribosomal RNAs and the calmodulin gene will be primary cloning targets. Clones of rRNA genes, chromosome markers and repetitive elements will be used as hybridization probes to examine genomic variation among isolates of P. carinii from rat and human hosts.
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