Histoplasma capsulatum secretes cysteine-rich protein virulence factors to lyse host macrophages
Intracellular pathogens secrete effectors to manipulate their host cells. Histoplasma capsulatum (Hc) is a fungal intracellular pathogen of humans that grows in a yeast form in the host. Hc yeasts are phagocytosed by macrophages, where fungal intracellular replication is followed by macrophage lysis. The most abundant virulence factor secreted by Hc yeast cells is Calcium Binding Protein 1 (Cbp1), which is absolutely required for macrophage lysis. Here in Chapter 2, we take an evolutionary, structural, and cell biological approach to understand Cbp1 function. We find that Cbp1 is present only in the genomes of closely related dimorphic fungal species of the Ajellomycetaceae family that lead primarily intracellular lifestyles in their mammalian hosts (Histoplasma, Paracoccidioides, and Emergomyces), but not conserved in the closely related extracellular fungal pathogen Blastomyces dermatitidis. We determine the de novo structures of Cbp1 from Hc H88 Cbp1 and the Paracoccidioides americana (Pb03) Cbp1, revealing a novel “binocular” fold, consisting of a helical dimer arrangement where two helices from each monomer contribute to a four-helix bundle. In contrast to Pb03 Cbp1, we show that Emergomyces Cbp1 orthologs are unable to stimulate macrophage lysis when expressed in the Hc cbp1 mutant. Consistent with this result, we find that wild-type E. africanus yeast are able to grow within primary macrophages but are incapable of lysing them. Finally, we use subcellular fractionation of infected macrophages and indirect immunofluorescence to show that Cbp1 localizes to the macrophage cytosol during Hc infection, making this the first instance of a primary intracellular human fungal pathogen directing an effector into the cytosol of the host cell. We additionally show that Cbp1 forms a complex with Yps-3, another known Hc virulence factor. Taken together, these data imply that Cbp1 is a rapidly evolving fungal virulence factor that localizes to the cytosol to trigger host cell lysis. We also show that Cbp1 is not the only virulence factor that enters the macrophage cytosol. In Chapter 3, we identify other virulence factors including a Cerato-platanin domain containing protein (Hc Asp F13) and Yps-3, are also found in the macrophage cytosol and contribute to optimal lysis In Chapter 4, we identify a family of putative cysteine-knot containing proteins that are specifically expanded in Hc species using a new, unbiased algorithm known as KNOTTIN Finder. One putative cysteine-knot, Rockstar10, contributes to optimal lysis of macrophages during infection. Taken together, these data demonstrate that Hc relies on a strategy of secreting cysteine-rich proteins into the macrophage cytosol in order to potentiate the lysis of its host cell to further dissemination.