Transcriptional Regulation of C. albicans Cell Fate and Host Interactions

项目摘要 白色念珠菌是人类微生物群中常见的寄生菌，也是一种重要的机会致病菌。 病原体这种真菌是变色龙般的能力，以替代细胞状态生长， 形态的形式，这种可塑性是至关重要的感染不同的壁龛在体内。这里我们 研究细胞命运决定的转录调控，驱动C. 白念珠菌包括表型转化、生物膜形成和表型转换。每一个项目 有助于该真菌在哺乳动物宿主中定殖和/或引起疾病的能力。先前 研究已经鉴定了调节这些发育程序的多种转录因子（TF）， 表明它们在高度协调的网络中共同作用以驱动基因表达。然而一个主要 关于细胞命运定义TF如何以协调的方式一起起作用而不是 个体实体。为了解决这一差距，我们强调初步数据显示，网络TF可以 进行液-液相分离（LLPS），并证明该过程能够形成 包含多个网络TF的复合物。我们进一步表明，相分离是由朊病毒样 关键的是，这些结构域的靶向突变可以消除 LLPS和TF功能。 为了建立在这些令人兴奋的观察基础上，目标1中概述的实验将确定 在体外和细胞中，PrLD的组成促进LLPS和多因子复合物的形成。 我们还讨论了LLPS的变化如何与C的功能相关。关键网络内的白色念珠菌TF， 包括控制表型转换和生物膜形成的那些。在目标2中，我们将确定其他 调节子C.使用覆盖C. albicans中所有TF的过表达文库，白色 基因组初步数据表明，通过这种方法可以发现多种新的调节剂， 新发现的转录因子将通过各种方法整合到现有的转录网络中 包括使用复杂单倍不足（CHI）分析。在目标3中，我们研究了阶段 分离C.白念珠菌转铁蛋白在真菌感染和致病过程中的作用 突变TF在小鼠感染模型中的作用。这些实验将通过条形码测序来促进 （条形码-SEQ）方法，其中可以平行评估多个菌株的竞争适应性。 总之，这些研究将导致对基本机制的新见解， 转录因子调控C.白色念珠菌，重点是如何LLPS使 功能性多因子TF复合物的形成。我们还将在这些网络中识别新的TF， 测试这些TF在传染性中的作用。鉴于TF在调节C.白色念珠菌细胞命运， 这些研究将确定针对这种重要的人类病原体进行治疗干预的新靶点。

Project Summary Candida albicans is a frequent commensal of the human microbiota and an important opportunistic pathogen. This fungus is chameleon-like in its ability to grow in alternative cellular states and different morphological forms, and this plasticity is critical for infection of diverse niches in the body. Here, we examine the transcriptional regulation of cell fate decisions that drive key developmental programs in C. albicans including filamentation, biofilm formation, and phenotypic switching. Each of these programs contributes to the ability of this fungus to colonize and/or cause disease in the mammalian host. Previous studies have identified multiple transcription factors (TFs) that regulate these developmental programs and showed that they act together in highly coordinated networks to drive gene expression. However, a major knowledge gap exists as to how cell fate-defining TFs act together in a coordinated manner rather than as individual entities. To address this gap, we highlight preliminary data revealing that network TFs can undergo liquid-liquid phase separation (LLPS) and demonstrate that this process enables the formation of complexes containing multiple network TFs. We further show that phase separation is driven by prion-like domains (PrLDs) present in each TF and, critically, that targeted mutation of these domains can abolish both LLPS and TF function. To build on these exciting observations, experiments outlined in Aim 1 will determine how the composition of PrLDs promotes LLPS and the formation of multifactorial complexes in vitro and in cells. We also address how changes in LLPS relate to the function of C. albicans TFs within key networks, including those controlling phenotypic switching and biofilm formation. In Aim 2, we will identify additional regulators of C. albicans cell fate using an overexpression library covering all TFs in the C. albicans genome. Preliminary data indicates that multiple novel regulators can be uncovered by this approach, and newly identified TFs will be integrated into existing transcriptional networks using a variety of approaches including the use of complex haploinsufficiency (CHI) analysis. In Aim 3, we examine how phase separation of C. albicans TFs impacts their function during commensalism and pathogenesis by testing mutant TFs in murine models of infection. These experiments will be facilitated by a barcode sequencing (barcode-SEQ) approach in which multiple strains can be evaluated in parallel for their competitive fitness. Together, these studies will lead to new insights into the fundamental mechanisms by which transcription factors regulate cell fate decisions in C. albicans, with an emphasis on how LLPS enables the formation of functional, multifactorial TF complexes. We will also identify novel TFs in these networks and test these TFs for their role in infectivity. Given the central role of TFs in regulating C. albicans cell fate, these studies will identify new targets for therapeutic intervention against this important human pathobiont.