Construction of Candida albicans Strains with ATP-Analog-Sensitive Protein Kinase A and Hog1.
Construction of Candida albicans Strains with ATP-Analog-Sensitive Protein Kinase A and Hog1.
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白色念珠菌是一种机会性的人类真菌病原体，也是粘膜微生物群的一员。为了在宿主中存活并引起疾病，白色念珠菌利用了几种毒力特征，包括对不同压力源的反应和适应能力，以及酵母和丝状形态之间的形态发生转换。虽然复杂的细胞回路控制着这些毒力属性，但以下两种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丝裂原激活的蛋白激酶（MAPK）级联和蛋白激酶A （PKA）途径。在这里，我们描述了白色念珠菌菌株的构建，在Hog1的ATP结合口袋和PKA、Tpk1和Tpk2的催化亚基中进行了取代，使它们的活性对大量ATP类似物的添加敏感。具体来说，ATP类似物1NM-PP1的抑制导致每个激酶基因对应的纯合缺失突变体的表型特征。这些菌株为快速特异性抑制PKA和Hog1激酶活性提供了工具集，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，真菌白色念珠菌依靠毒力特性引起疾病。它们包括从酵母菌形态转变为丝状形态的能力，以及在各种环境胁迫条件下生长的能力，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关键作用：Hog1和PKA。在这里，我们生成了对ATP类似物1NM-PP1的抑制敏感的每个激酶的版本。在类似物存在的情况下，激酶活性被迅速而特异性地抑制，便于分析这两种激酶在调节白色念珠菌形态发生和应激反应中的作用。总之，这些菌株代表了一个重要的工具集，进一步了解白色念珠菌的生物学和毒力。
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. IMPORTANCE 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.
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影响因子：
6.4
作者：
O'Meara TR;Duah K;Guo CX;Maxson ME;Gaudet RG;Koselny K;Wellington M;Powers ME;MacAlpine J;O'Meara MJ;Veri AO;Grinstein S;Noble SM;Krysan D;Gray-Owen SD;Cowen LE
通讯作者：
Cowen LE
肠道，坏和无害的：白色念珠菌是肠道中的共同和机会性病原体。
影响因子：
5.4
作者：
Kumamoto CA;Gresnigt MS;Hube B
通讯作者：
Hube B
通过表型、转录组和磷酸蛋白质组分析揭示白色念珠菌 PKA 催化亚基无效突变体中 cAMP/PKA 通路的全局调节作用
影响因子：
3.6
作者：
Cao, Chengjun;Wu, Mei;Huang, Guanghua
通讯作者：
Huang, Guanghua
影响因子：
30.8
作者：
通讯作者：
--
影响因子：
4.2
作者：
Kaba, Hani E. J.;Nimtz, Manfred;Bilitewski, Ursula
通讯作者：
Bilitewski, Ursula