Cell cycle-independent phospho-regulation of Fkh2 during hyphal growth regulates Candida albicans pathogenesis.
Cell cycle-independent phospho-regulation of Fkh2 during hyphal growth regulates Candida albicans pathogenesis.
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DOI：
10.1371/journal.ppat.1004630
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发表时间：
2015-01
期刊：
影响因子：
6.7
通讯作者：
Sudbery PE
中科院分区：
文献类型：
--
作者：
Greig JA;Sudbery IM;Richardson JP;Naglik JR;Wang Y;Sudbery PE
人类机会致病真菌白念珠菌在从致病性到致病性的转变中经历了形态和转录的适应。尽管先前的基因敲除研究已经确定了许多参与这种转变的因素，但仍不清楚这些因素如何调节以协调开关。通过翻译后磷酸化研究形态发生控制已经产生了对这一过程的重要监管见解，特别是关注细胞周期蛋白依赖性激酶Cdc28的协调控制。在这里，我们已经确定了Fkh2转录因子作为Cdc28和细胞壁生物合成激酶Cbk1的调节靶点，在细胞周期进程中的作用不同于其保守的功能。在静止期酵母细胞中，2D凝胶电泳显示存在Fkh2磷酸化同种型的多样性库。对于菌丝诱导的短窗口，远在细胞周期开始之前，磷酸化谱在恢复到酵母谱之前被转化。当静止期细胞重新接种到支持酵母生长的新鲜培养基中时，不会发生这种转化。质谱和突变分析鉴定了Cdc28和Cbk1磷酸化的残基。用非磷酸化丙氨酸取代这些残基改变了酵母的磷酸化特征，并消除了菌丝特征的转化。磷酸化位点突变体的转录谱显示，菌丝磷酸化谱是参与发病机制、宿主相互作用和生物膜形成的基因表达所必需的。我们证实了这些基因表达的变化导致了致病过程中的相应缺陷。此外，我们发现Fkh2与染色质修饰剂Pob3以磷酸化依赖的方式相互作用，从而提供了Fkh2磷酸化调节其特异性的可能机制。因此，我们发现了一种新的细胞周期独立的磷酸调控事件，颠覆了细胞周期机制的关键组成部分，在从细菌性疾病到致病性的转变中发挥作用。真菌白色念珠菌是人类微生物群中的寄生虫，负责浅表感染，如口腔和阴道鹅口疮。然而，它可以变得高度致命，在严重免疫功能低下的患者中引起危及生命的全身性念珠菌血症，包括那些服用免疫抑制药物用于移植的患者，艾滋病和中性粒细胞减少症患者，以及正在接受化疗或处于极端年龄的个体。随着全球人口老龄化的加剧，C。白色念珠菌和其他真菌病原体将变得更加普遍，需要更好地了解它们的发病机理以开发有效的治疗方法。真菌致病性需要一组转录因子协调基因表达模式的变化。在这里，我们已经发现，转录因子，Fkh2，被修改的磷酸化激酶Cdc28和Cbk 1的控制下，在激活毒力因子表达的条件。Fkh2参与包括细胞增殖在内的多种细胞过程，但这种磷酸化赋予其促进宿主中组织侵入、生物膜形成和发病所需基因表达的专门功能。这项研究强调了蛋白磷酸化在调节发病机制中的作用，并进一步加深了我们对这种重要的机会致病真菌病原体的致病开关的理解。
The opportunistic human fungal pathogen, Candida albicans, undergoes morphological and transcriptional adaptation in the switch from commensalism to pathogenicity. Although previous gene-knockout studies have identified many factors involved in this transformation, it remains unclear how these factors are regulated to coordinate the switch. Investigating morphogenetic control by post-translational phosphorylation has generated important regulatory insights into this process, especially focusing on coordinated control by the cyclin-dependent kinase Cdc28. Here we have identified the Fkh2 transcription factor as a regulatory target of both Cdc28 and the cell wall biosynthesis kinase Cbk1, in a role distinct from its conserved function in cell cycle progression. In stationary phase yeast cells 2D gel electrophoresis shows that there is a diverse pool of Fkh2 phospho-isoforms. For a short window on hyphal induction, far before START in the cell cycle, the phosphorylation profile is transformed before reverting to the yeast profile. This transformation does not occur when stationary phase cells are reinoculated into fresh medium supporting yeast growth. Mass spectrometry and mutational analyses identified residues phosphorylated by Cdc28 and Cbk1. Substitution of these residues with non-phosphorylatable alanine altered the yeast phosphorylation profile and abrogated the characteristic transformation to the hyphal profile. Transcript profiling of the phosphorylation site mutant revealed that the hyphal phosphorylation profile is required for the expression of genes involved in pathogenesis, host interaction and biofilm formation. We confirmed that these changes in gene expression resulted in corresponding defects in pathogenic processes. Furthermore, we identified that Fkh2 interacts with the chromatin modifier Pob3 in a phosphorylation-dependent manner, thereby providing a possible mechanism by which the phosphorylation of Fkh2 regulates its specificity. Thus, we have discovered a novel cell cycle-independent phospho-regulatory event that subverts a key component of the cell cycle machinery to a role in the switch from commensalism to pathogenicity. The fungus Candida albicans is a commensal in the human microbiota, responsible for superficial infections such as oral and vaginal thrush. However, it can become highly virulent, causing life-threatening systemic candidemia in severely immunocompromised patients, including those taking immunosuppressive drugs for transplantation, sufferers of AIDS and neutropenia, and individuals undergoing chemotherapy or at extremes of age. With a rapidly increasing ageing population worldwide, C. albicans and other fungal pathogens will become more prevalent, demanding a greater understanding of their pathogenesis for the development of effective therapeutics. Fungal pathogenicity requires a coordinated change in the pattern of gene expression orchestrated by a set of transcription factors. Here we have discovered that a transcription factor, Fkh2, is modified by phosphorylation under the control of the kinases Cdc28 and Cbk1 in response to conditions that activate virulence factor expression. Fkh2 is involved in a wide variety of cellular processes including cell proliferation, but this phosphorylation endows it with a specialized function in promoting the expression of genes required for tissue invasion, biofilm formation, and pathogenesis in the host. This study highlights the role of protein phosphorylation in regulating pathogenesis and furthers our understanding of the pathogenic switch in this important opportunistic fungal pathogen.