Division of Infectious Diseases, Boston Children's Hospital/Harvard Medical School, Boston, Massachusetts, United States of America.
Division of Oral Biosciences, School of Dental Science, Trinity College Dublin, Ireland.
PLoS Pathog. 2022 Jun 10;18(6):e1010089. doi: 10.1371/journal.ppat.1010089. eCollection 2022 Jun.
Whether to commit limited cellular resources toward growth and proliferation, or toward survival and stress responses, is an essential determination made by Target of Rapamycin Complex 1 (TORC1) for a eukaryotic cell in response to favorable or adverse conditions. Loss of TORC1 function is lethal. The TORC1 inhibitor rapamycin that targets the highly conserved Tor kinase domain kills fungal pathogens like Candida albicans, but is also severely toxic to human cells. The least conserved region of fungal and human Tor kinases are the N-terminal HEAT domains. We examined the role of the 8 most N-terminal HEAT repeats of C. albicans Tor1. We compared nutritional- and stress responses of cells that express a message for N-terminally truncated Tor1 from repressible tetO, with cells expressing wild type TOR1 from tetO or from the native promoter. Some but not all stress responses were significantly impaired by loss of Tor1 N-terminal HEAT repeats, including those to oxidative-, cell wall-, and heat stress; in contrast, plasma membrane stress and antifungal agents that disrupt plasma membrane function were tolerated by cells lacking this Tor1 region. Translation was inappropriately upregulated during oxidative stress in cells lacking N-terminal Tor1 HEAT repeats despite simultaneously elevated Gcn2 activity, while activation of the oxidative stress response MAP kinase Hog1 was weak. Conversely, these cells were unable to take advantage of favorable nutritional conditions by accelerating their growth. Consuming oxygen more slowly than cells containing wild type TOR1 alleles during growth in glucose, cells lacking N-terminal Tor1 HEAT repeats additionally were incapable of utilizing non-fermentable carbon sources. They were also hypersensitive to inhibitors of specific complexes within the respiratory electron transport chain, suggesting that inefficient ATP generation and a resulting dearth of nucleotide sugar building blocks for cell wall polysaccharides causes cell wall integrity defects in these mutants. Genome-wide expression analysis of cells lacking N-terminal HEAT repeats showed dysregulation of carbon metabolism, cell wall biosynthetic enzymes, translational machinery biosynthesis, oxidative stress responses, and hyphal- as well as white-opaque cell type-associated genes. Targeting fungal-specific Tor1 N-terminal HEAT repeats with small molecules might selectively abrogate fungal viability, especially when during infection multiple stresses are imposed by the host immune system.
真核细胞在有利或不利条件下，通过雷帕霉素靶蛋白复合物 1（TORC1）来决定是将有限的细胞资源用于生长和增殖，还是用于生存和应激反应。TORC1 功能的丧失是致命的。雷帕霉素是一种靶向高度保守的 Tor 激酶结构域的 TORC1 抑制剂，可杀死白色念珠菌等真菌病原体，但对人类细胞也有严重毒性。真菌和人类 Tor 激酶中最不保守的区域是 N 端 HEAT 结构域。我们研究了白色念珠菌 Tor1 的 8 个最 N 端 HEAT 重复序列的作用。我们比较了表达受 TetO 抑制的截短 Tor1 信使的细胞与表达 TetO 或天然启动子来源的野生型 TOR1 的细胞的营养和应激反应。部分应激反应（但不是所有应激反应）因 Tor1 N 端 HEAT 重复缺失而受到显著影响，包括氧化应激、细胞壁应激和热应激；相比之下，对破坏质膜功能的质膜应激和抗真菌药物，缺乏该 Tor1 区域的细胞是可以耐受的。尽管 Gcn2 活性同时升高，但在缺乏 N 端 Tor1 HEAT 重复的细胞中，氧化应激期间翻译被不恰当地上调，而氧化应激反应 MAP 激酶 Hog1 的激活很弱。相反，这些细胞无法通过加速生长来利用有利的营养条件。在葡萄糖生长过程中，与含有野生型 TOR1 等位基因的细胞相比，缺乏 N 端 Tor1 HEAT 重复的细胞消耗氧气更慢，此外，它们还无法利用非发酵性碳源。它们对呼吸电子传递链中特定复合物的抑制剂也很敏感，这表明这些突变体中无效的 ATP 生成和用于细胞壁多糖的核苷酸糖构建块的缺乏导致细胞壁完整性缺陷。缺乏 N 端 HEAT 重复的细胞的全基因组表达分析显示，碳代谢、细胞壁生物合成酶、翻译机制生物合成、氧化应激反应以及菌丝和白- opaque 细胞类型相关基因的调控失调。用小分子靶向真菌特异性 Tor1 N 端 HEAT 重复可能会选择性地消除真菌的活力，特别是在感染期间，宿主免疫系统会施加多种应激。
