Cryptococcus gattii is an etiologic agent of cryptococcosis, a potentially fatal disease that affects humans and animals. The successful infection of mammalian hosts by cryptococcal cells relies on their ability to infect and survive in macrophages. Such phagocytic cells present a hostile environment to intracellular pathogens via the production of reactive nitrogen and oxygen species, as well as low pH and reduced nutrient bioavailability. To overcome the low-metal environment found during infection, fungal pathogens express high-affinity transporters, including members of the ZIP family. Previously, we determined that functional zinc uptake driven by Zip1 and Zip2 is necessary for full C. gattii virulence. Here, we characterized the ZIP3 gene of C. gattii, an ortholog of the Saccharomyces cerevisiae ATX2, which codes a manganese transporter localized to the membrane of the Golgi apparatus. Cryptococcal cells lacking Zip3 were tolerant to toxic concentrations of manganese and had imbalanced expression of intracellular metal transporters, such as the vacuolar Pmc1 and Vcx1, as well as the Golgi Pmr1. Moreover, null mutants of the ZIP3 gene displayed higher sensitivity to reactive oxygen species (ROS) and substantial alteration in the expression of ROS-detoxifying enzyme-coding genes. In line with these phenotypes, cryptococcal cells displayed decreased virulence in a non-vertebrate model of cryptococcosis. Furthermore, we found that the ZIP3 null mutant strain displayed decreased melanization and secretion of the major capsular component glucuronoxylomannan, as well as an altered extracellular vesicle dimensions profile. Collectively, our data suggest that Zip3 activity impacts the physiology, and consequently, several virulence traits of C. gattii.

Cryptococcus gattii是隐球菌病的病原体，隐球菌病是一种影响人类和动物的潜在致命疾病。隐球菌细胞成功感染哺乳动物宿主依赖于它们感染巨噬细胞并在巨噬细胞中存活的能力。这种吞噬细胞通过产生活性氮和氧物质以及低 pH 值和降低的营养生物利用度，为细胞内病原体提供了恶劣的环境。为了克服感染期间发现的低金属环境，真菌病原体表达高亲和力转运蛋白，包括 ZIP 家族的成员。以前，我们确定由 Zip1 和 Zip2 驱动的功能性锌吸收是完整的C. gattii 毒力所必需的。在这里，我们表征了C. gattii的ZIP3基因，酿酒酵母 ATX2的直向同源物，其编码定位于高尔基体膜的锰转运蛋白。缺乏 Zip3 的隐球菌细胞耐受有毒浓度的锰，并且细胞内金属转运蛋白的表达不平衡，例如液泡 Pmc1 和 Vcx1，以及高尔基体 Pmr1。此外，ZIP3基因的无效突变体对活性氧 (ROS) 表现出更高的敏感性，并且 ROS 解毒酶编码基因的表达发生了实质性改变。与这些表型一致，隐球菌细胞在隐球菌病的非脊椎动物模型中表现出降低的毒力。此外，我们发现ZIP3null 突变株显示主要荚膜成分葡糖醛酸木甘露聚糖的黑色化和分泌减少，以及细胞外囊泡尺寸分布改变。总的来说，我们的数据表明 Zip3 活性会影响C. gattii的生理学，并因此影响其几个毒力特征。