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区系中常见的共生菌，也是一种重要的机会。 病原体。这种真菌的能力就像变色龙一样，能够在不同的细胞状态下生长 这种可塑性对于感染体内不同的壁龛是至关重要的。在这里，我们 研究细胞命运决定的转录调控，这些决定驱动C。 白念珠菌包括丝状化、生物膜形成和表型转换。这些计划中的每一个 有助于这种真菌在哺乳动物宿主中定居和/或引起疾病的能力。上一首 研究已经确定了调节这些发育程序的多个转录因子(TF)和 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，一个主要的 关于定义细胞命运的函数如何以协调的方式共同作用而不是像 单个实体。为了解决这一差距，我们重点介绍了初步数据，这些数据揭示了网络功能可以 进行液-液相分离(LLP)，并证明该过程能够形成 包含多个网络TF的络合物。我们进一步证明了相分离是由类普里子驱动的 存在于每个转铁蛋白中结构域(PrLD)，关键是，这些结构域的定向突变可以消除 LLP和Tf都起作用。 为了建立在这些令人兴奋的观察结果的基础上，目标1中概述的实验将确定 PrLD的组成在体外和细胞内促进LLP和多因素复合体的形成。 我们还讨论了LLP的变化如何与关键网络中白念珠菌转录因子的功能相关， 包括那些控制表型转换和生物膜形成的基因。在目标2中，我们将确定其他 利用覆盖白念珠菌中所有转录因子的过表达文库调控白念珠菌细胞命运 基因组。初步数据表明，这种方法可以发现多个新的调节子，并且 新确定的转录因子将使用各种方法整合到现有的转录网络中 包括使用复合单倍体不足(CHI)分析。在目标3中，我们考察了阶段 白念珠菌转录因子的分离对其共生功能及致病机制的影响 小鼠感染模型中突变的转录因子。这些实验将通过条形码测序来促进 (条形码-序列)方法，可以并行评估多个菌株的竞争适合度。 总之，这些研究将导致对以下基本机制的新见解： 转录因子调节白念珠菌的细胞命运决定，重点是LLP如何使 形成功能性、多因子的转铁蛋白络合物。我们还将在这些网络中识别新的TF，并 测试这些因子在传染性中的作用。鉴于转录因子在调控白念珠菌细胞命运中的核心作用， 这些研究将确定针对这种重要的人类疾病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.