Functional Mapping of Transcription Factor Grf10 That Regulates Adenine-Responsive and Filamentation Genes in Candida albicans.
Functional Mapping of Transcription Factor Grf10 That Regulates Adenine-Responsive and Filamentation Genes in Candida albicans.
复制标题
调节白色念珠菌腺嘌呤反应和丝状基因的转录因子 Grf10 的功能定位。
DOI：
10.1128/msphere.00467-18
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发表时间：
2018
期刊：
影响因子：
4.8
通讯作者：
Rolfes,RondaJ
中科院分区：
文献类型：
--
作者：
Wangsanut,Tanaporn;Tobin,JoshuaM;Rolfes,RondaJ
Grf10是一种含有同源结构域的转录因子，在人类真菌白色念珠菌中调节腺苷酸和单碳代谢和形态发生。在这里，我们通过单杂交和突变分析确定了参与转录因子活性的功能域和关键残基。我们通过单杂交分析将激活域定位到Grf10蛋白的c端一半，并利用生物信息学分析确定了基序；其中一个特征激活域（AD1）响应温度。LexA-Grf10融合蛋白以腺嘌呤依赖的方式激活了exaop - his1报告蛋白，这种激活不依赖于Bas1，表明腺嘌呤限制信号直接传递给Grf10。LexA-Grf10的过表达导致成丝，这需要一个功能同源结构域，与Grf10控制关键成丝基因的表达一致；LexA-Grf10过表达诱导的丝化与腺嘌呤水平和Bas1无关。在LexA-Grf10和Grf10的保守相互作用区（IR）内进行丙氨酸替换以研究其在转录中的作用。在LexA-Grf10中，D302A突变组成性地激活转录，E305A突变受腺嘌呤调控。当这些突变被引入到原生基因位点时，D302A突变不能补充ADE表型，并且在诱导菌丝的条件下不能促进成丝；E305A突变体在ADE表型上表现为原生基因，在诱导菌丝方面存在部分缺陷。这些结果证明了不同表型的等位基因特异性反应，与Grf10与多个伴侣蛋白相互作用能力的扰动一致。白色念珠菌是一种主要的人类真菌病原体，在哺乳动物宿主体内存活并感染多种身体部位，代谢适应和形态发生是必不可少的。白色念珠菌利用转录因子严密控制代谢基因和形态发生基因的转录。Grf10是一种关键的同源结构域转录因子，在腺嘌呤限制下控制嘌呤和单碳代谢，Grf10是酵母到菌丝形态转换所必需的，这是一种已知的毒力因子。在这里，我们进行了单杂交和突变分析，以确定Grf10的功能域。我们的研究结果表明，Grf10分别调控代谢和形态发生基因，并且它包含一个保守的蛋白结构域，用于蛋白质伴侣相互作用，允许Grf10控制多种不同途径的转录。我们的发现有助于理解转录因子在白色念珠菌中控制多种致病性状的作用和机制。
Grf10, a homeodomain-containing transcription factor, regulates adenylate and one-carbon metabolism and morphogenesis in the human fungal pathogen Candida albicans. Here, we identified functional domains and key residues involved in transcription factor activity using one-hybrid and mutational analyses. We localized activation domains to the C-terminal half of the Grf10 protein by one-hybrid analysis and identified motifs using bioinformatic analyses; one of the characterized activation domains (AD1) responded to temperature. The LexA-Grf10 fusion protein activated thelexAop-HIS1reporter in an adenine-dependent fashion, and this activation was independent of Bas1, showing that the adenine limitation signal is transmitted directly to Grf10. Overexpression of LexA-Grf10 led to filamentation, and this required a functioning homeodomain, consistent with Grf10 controlling the expression of key filamentation genes; filamentation induced by LexA-Grf10 overexpression was independent of adenine levels and Bas1. Alanine substitutions were made within the conserved interaction regions (IR) of LexA-Grf10 and Grf10 to investigate roles in transcription. In LexA-Grf10, the D302A mutation activated transcription constitutively, and the E305A mutation was regulated by adenine. When these mutations were introduced into the native gene locus, the D302A mutation was unable to complement the ADE phenotype and did not promote filamentation under hypha-inducing conditions; the E305A mutant behaved as the native gene with respect to the ADE phenotype and was partially defective in inducing hyphae. These results demonstrate allele-specific responses with respect to the different phenotypes, consistent with perturbations in the ability of Grf10 to interact with multiple partner proteins.IMPORTANCEMetabolic adaptation and morphogenesis are essential for Candida albicans, a major human fungal pathogen, to survive and infect diverse body sites in the mammalian host. C. albicans utilizes transcription factors to tightly control the transcription of metabolic genes and morphogenesis genes. Grf10, a critical homeodomain transcription factor, controls purine and one-carbon metabolism in response to adenine limitation, and Grf10 is necessary for the yeast-to-hypha morphological switching, a known virulence factor. Here, we carried out one-hybrid and mutational analyses to identify functional domains of Grf10. Our results show that Grf10 separately regulates metabolic and morphogenesis genes, and it contains a conserved protein domain for protein partner interaction, allowing Grf10 to control the transcription of multiple distinct pathways. Our findings contribute significantly to understanding the role and mechanism of transcription factors that control multiple pathogenic traits in C. albicans.