MOLECULAR AND CELL BIOLOGY OF FUNGAL PATHOGENS

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.