Histoplasma capsulatum (Hc) is a thermally dimorphic fungus and an intracellular pathogen of macrophages. Hc grows in the soil in a multicellular hyphal form. Once inhaled, Hc responds to mammalian body temperature by converting to a unicellular yeast form and initiating the expression of virulence genes important for macrophage colonization. We have extensive experience elucidating the gene networks that are transcriptionally induced in yeast cells. In our published work, we annotated the transcriptome of yeast-phase cells and discovered a family of small (≤ 200 AAs) predicted secreted proteins that exhibit a conserved C- terminal, 6-cysteine spacing pattern reminiscent of some insect toxins. The transcripts encoding these proteins showed highly differential expression in yeast cells compared to the remainder of the transcriptome, suggesting that they play an important role during infection. Further analysis revealed 26 Hc ORFs in this family, each containing a predicted cystine knot (or knottin) domain. In contrast, most fungal species contain 0-2 predicted knottin proteins in their genomes. Knottin domains are comprised of 3 interwoven disulfide bonds that form one of the smallest known stable globular domains, making these proteins extremely resistant to chemical, heat, and proteolytic stresses. Our preliminary data reveal the remarkable result that mutant strains lacking individual knottins show reduced virulence in the mouse model of Hc infection. All of these mutants are partially deficient in stimulating lysis of host macrophages, and some but not all display diminished growth within macrophages, indicating that knottins play key roles in Hc-host interactions. We will take advantage of our expertise in Hc- macrophage interactions and Hc molecular genetics to interrogate the role of individual and multiple knottins in Hc pathogenesis. We propose the following aims: First, using the mutant strains we have already generated, and taking advantage of CRISPR technology we have adapted to efficiently generate more mutant strains, we will further investigate the contribution of individual and multiple knottins to pathogenesis of Hc in macrophage and mouse models of infection. Second, our published work established that Hc activates apoptosis of infected macrophages by triggering an integrated stress response (ISR) in these cells. We will compare the transcriptional signature of macrophages to infection with wild-type vs mutant knottin strains to elucidate the contribution of individual knottins to the ISR and other aspects of the host molecular response to Hc. Additionally, since a subset of knottin mutants display reduced growth within macrophages, we will determine whether knottins affect the ability of Hc to block phagosome maturation, which is a key step in intracellular survival. Finally, to elucidate the molecular mechanism of knottin function, we will use standard pipelines in our laboratory to determine the subcellular localization and pr...