Abstract

The pathogenesis of Histoplasma capsulatum is linked to the yeast phase and the factors produced by cells in this state. We have used a proteomics approach to identify the extracellular proteins produced by this pathogenic phase as these factors may affect host macrophages and immune defense systems. The major components identified from Histoplasma yeast culture filtrates include cell-wall related glycosidases and enzymes involved in defense against oxidative stress. Approximately one-fourth of the extracellular proteins represent novel factors with unknown functions. Enriched expression of genes encoding extracellular proteins in the pathogenic phase as compared to non-pathogenic mycelia was used to prioritize factors for functional studies. Using an RNAi sentinel system that co-targets the gene of interest and GFP fluorescence of an expressed gfp transgene, we have generated strains lacking individual extracellular factors. Culture filtrates harvested from these strains confirm depletion of the respective factors and these strains are being employed in cultured macrophage and animal models of histoplasmosis to assess their contribution to Histoplasma pathogenesis.਀㰀瀀㸀 We are also employing Agrobacterium-mediated transformation to generate random T-DNA insertions in Histoplasma with which to identify mutants in virulence-related genes. To isolate mutations in specific genes, we optimized methods to pool insertion mutants and screen them by PCR for disruptions in a targeted gene of interest. Successive subdivision of pools containing the insertion mutant permits isolation of the desired strain. This methodology enables isolation of gene knock-outs without reliance on homologous recombination. For forward genetics, random T-DNA insertion mutants are being screened in cultured phagocytes for yeast strains with reduced virulence. We developed an efficient assay to measure replication of Histoplasma yeast in infected macrophages through the cytopathic effects of yeast on phagocytic host cells. This assay has been adapted to a 96-well plate format allowing for higher throughput screens to be performed. With this virulence screen, we have isolated three Histoplasma mutants with reduced replication in phagocytes.਀㰀⼀昀漀渀琀㸀㰀⼀瀀㸀㰀戀爀㸀㰀戀㸀䬀攀礀眀漀爀搀㨀 㰀⼀戀㸀☀渀戀猀瀀㬀䠀椀猀琀漀瀀氀愀猀洀愀Ⰰ 洀甀琀愀琀椀漀渀Ⰰ 刀一䄀椀Ⰰ 猀攀挀爀攀琀椀漀渀Ⰰ 琀爀愀渀猀昀漀爀洀愀琀椀漀渀㰀⼀琀搀㸀㰀⼀琀爀㸀㰀⼀琀愀戀氀攀㸀㰀⼀琀爀㸀㰀⼀琀搀㸀㰀⼀琀愀戀氀攀㸀㰀⼀戀漀搀礀㸀㰀⼀栀琀洀氀㸀