Cbp1, a rapidly evolving fungal virulence factor, forms an effector complex that drives macrophage lysis
Authors: Azimova D. , Herrera N. , Duvenage L. , Voorhies M. , English B.C. , Hoving J.C. , Rosenberg O. , Sil A. 
Published: September 2021

AbstractIntracellular 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.Hcyeasts are phagocytosed by macrophages, where fungal intracellular replication precedes macrophage lysis. The most abundant virulence factor secreted byHcyeast cells is Calcium Binding Protein 1 (Cbp1), which is absolutely required for macrophage lysis. Here 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, andEmergomyces), but not conserved in the extracellular fungal pathogenBlastomyces dermatitidis. We determine thede novostructures ofHc H88 Cbp1 and theParacoccidioides americana(Pb03) Cbp1, revealing a novel “binocular” fold consisting of a helical dimer arrangement wherein two helices from each monomer contribute to a four-helix bundle. In contrast to Pb03 Cbp1, we show thatEmergomycesCbp1 orthologs are unable to stimulate macrophage lysis when expressed in theHc cbp1mutant. Consistent with this result, we find that wild-typeEmergomyces africanusyeast 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 duringHcinfection, making this the first instance of a phagosomal 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 knownHcvirulence factor that accesses the cytosol. Taken together, these data imply that Cbp1 is a rapidly evolving fungal virulence factor that localizes to the cytosol to trigger host cell lysis.Author SummaryThe members of the Ajellomycetaceae fungal family are human pathogens that are responsible for a rising number of mycoses around the world. Calcium binding protein 1 (Cbp1) is a rapidly evolving virulence factor that is present in the genomes of the Ajellomycetaceae species that lead primarily intracellular lifestyles, includingHistoplasma, Paracoccidioides, andEmergomycesbut notBlastomyces, which remains largely extracellular during infection. BothParacoccidioidesandHistoplasmaCbp1 homologs are able to cause lysis of macrophages whereasEmergomyceshomologs cannot. This result is consistent withEmergomyces africanusnatural infection of macrophages, during which the yeast cells can replicate but cannot actively lyse the host cell. Despite divergence of the primary sequence ofHistoplasmaandParacoccidioidesCbp1 homologs, their protein structures are remarkably similar and reveal a novel fold. During infection, Cbp1 enters the cytosol of the host macrophage, making it the first known virulence factor from an intracellular human fungal pathogen that localizes to the cytosol of the host cell. We also show that Cbp1 forms a complex with another cytosolic virulence factor, Yps-3. Taken together, these studies significantly advance our understanding ofHistoplasmavirulence.