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 precedes 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 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 extracellular fungal pathogen Blastomyces dermatitidis. We determine the de novo structures of Hc H88 Cbp1 and the Paracoccidioides 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 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 Emergomyces 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 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 known Hc virulence 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.

中文翻译：

---

Cbp1，一种快速进化的真菌毒力因子，形成驱动巨噬细胞裂解的效应复合物

细胞内病原体分泌效应物来操纵它们的宿主细胞。荚膜组织胞浆菌( Hc ) 是人类的一种真菌细胞内病原体，在宿主中以酵母形式生长。Hc酵母被巨噬细胞吞噬，其中真菌细胞内复制先于巨噬细胞裂解。Hc酵母细胞分泌的最丰富的毒力因子是钙结合蛋白 1 (Cbp1)，它是巨噬细胞裂解所必需的。在这里，我们采用进化、结构和细胞生物学方法来理解 Cbp1 功能。我们发现 Cbp1 仅存在于 Ajellomycetaceae 家族的密切相关的双态真菌物种的基因组中，这些物种主要在其哺乳动物宿主中引导细胞内生活方式。组织胞浆菌、副球孢子菌和胚芽菌），但在细胞外真菌病原体皮炎芽生菌中不保守。我们确定了H c H88 Cbp1 和美洲副球孢子菌(Pb03) Cbp1 的从头结构，揭示了一种由螺旋二聚体排列组成的新型“双目”折叠，其中来自每个单体的两个螺旋有助于形成四螺旋束。与 Pb03 Cbp1 相比，我们表明当在Hc cbp1突变体中表达时，萌发菌Cbp1 直向同源物无法刺激巨噬细胞裂解。与此结果一致，我们发现野生型非洲芽孢菌酵母能够在原代巨噬细胞内生长，但不能裂解它们。最后，我们使用受感染巨噬细胞的亚细胞分离和间接免疫荧光来表明 Cbp1 在Hc感染期间定位于巨噬细胞胞质溶胶，这使得这是吞噬体人类真菌病原体将效应子引导到宿主细胞的胞质溶胶中的第一个实例。我们还表明 Cbp1 与 Yps-3 形成复合物，Yps-3 是另一种已知的Hc毒力因子，可进入细胞质。总之，这些数据意味着 Cbp1 是一种快速进化的真菌毒力因子，定位于细胞质以触发宿主细胞裂解。