Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
mSphere. 2023 Jun 22;8(3):e0009523. doi: 10.1128/msphere.00095-23. Epub 2023 Apr 11.
Candida albicans is an opportunistic human fungal pathogen and a member of the mucosal microbiota. To survive in the host and cause disease, C. albicans utilizes several virulence traits, including the ability to respond and adapt to diverse stressors, as well as the morphogenetic switch between yeast and filamentous morphologies. While complex cellular circuitry governs these virulence attributes, the following two kinase-mediated signaling pathways play particularly critical roles in controlling these processes: the Hog1 mitogen-activated protein kinase (MAPK) cascade and the protein kinase A (PKA) pathway. Here, we describe the construction of C. albicans strains harboring substitutions in the ATP-binding pockets of Hog1 and the catalytic subunits of PKA, Tpk1, and Tpk2 to render their activities sensitive to the addition of bulky ATP analogs. Specifically, inhibition by the ATP analog 1NM-PP1 resulted in phenotypes characteristic of the corresponding homozygous deletion mutants for each kinase gene. These strains represent a toolset for the rapid and specific inhibition of PKA and Hog1 kinase activity to further understand their roles in regulating C. albicans morphogenesis and stress responses. As an opportunistic pathogen in humans, the fungus Candida albicans relies on virulence traits to cause disease. They include the ability to transition from yeast to filamentous morphologies and the ability to grow in diverse environmental stress conditions, including nutrient limitation, as well as osmotic and heat shock. Previous work identified the following two kinases that play a critical role in regulating these responses: Hog1 and PKA. Here, we generated versions of each kinase that are sensitive to inhibition by a bulky ATP analog, 1NM-PP1. In the presence of the analog, kinase activity is inhibited rapidly and specifically, facilitating the analysis of both kinases in regulating C. albicans morphogenesis and stress responses. Together, these strains represent an important toolset to further our understanding of C. albicans biology and virulence.
白色念珠菌是一种机会性人类真菌病原体，也是黏膜微生物群的一员。为了在宿主中生存并引起疾病，白色念珠菌利用了几种毒力特性，包括对各种应激源的响应和适应能力，以及酵母和丝状形态之间的形态发生转换。虽然复杂的细胞电路控制着这些毒力属性，但以下两个激酶介导的信号通路在控制这些过程中起着特别关键的作用：Hog1 丝裂原激活蛋白激酶 (MAPK) 级联和蛋白激酶 A (PKA) 途径。在这里，我们描述了构建含有 Hog1 和 PKA 的催化亚基 Tpk1 和 Tpk2 的 ATP 结合口袋中的取代物的白色念珠菌菌株，以使它们的活性对大体积 ATP 类似物的添加敏感。具体而言，ATP 类似物 1NM-PP1 的抑制导致每个激酶基因的相应纯合缺失突变体的特征表型。这些菌株代表了用于快速和特异性抑制 PKA 和 Hog1 激酶活性的工具集，以进一步了解它们在调节白色念珠菌形态发生和应激反应中的作用。 作为人类的机会性病原体，真菌白色念珠菌依赖于毒力特性来引起疾病。它们包括从酵母到丝状形态的转变能力，以及在各种环境应激条件下生长的能力，包括营养限制以及渗透和热休克。先前的工作确定了以下两种激酶在调节这些反应中起着关键作用：Hog1 和 PKA。在这里，我们生成了每个激酶的版本，这些版本对大块 ATP 类似物 1NM-PP1 的抑制敏感。在类似物存在下，激酶活性被快速且特异性地抑制，从而促进了对两种激酶在调节白色念珠菌形态发生和应激反应中的分析。总之，这些菌株代表了进一步了解白色念珠菌生物学和毒力的重要工具集。
