Transcriptional regulation of C. albicans biofilms by formation of phase-separated condensates

项目摘要/摘要 微生物形成生物膜的能力是感染人类宿主的关键毒力因素。 由于其复杂的结构组成，包括多层细胞群落和突出的胞外 在基质中，生物被膜往往对抗菌治疗顽固不化。机会性真菌病原菌念珠菌 白念珠菌是医院感染的主要原因之一，很大程度上是因为它能形成复合体。 生物膜。白色念珠菌可以定植于人体的每个主要器官，并在两种医用植入物上形成生物膜。 和粘膜表面。此外，在口咽念珠菌病(OPC)中，最常见的真菌感染是 人类、白色念珠菌生物膜在口腔组织和假牙上形成。从口腔，白色念珠菌可以 分散到新的感染部位，并在血液中侵袭性念珠菌病(IC)。IC的死亡率保持不变 尽管有临床干预，但约为40%，大多数病例被认为起源于共生生态位。解剖 因此，决定白色念珠菌在口腔壁龛定植的分子机制是至关重要的 更好地治疗慢性感染，并最大限度地减少疾病进展到IC。 由9个主要转录因子(TF)组成的定义明确的转录调控网络(TRN) 控制白色念珠菌的生物膜发育。这个网络中的多个TF绑定到自己的监管区域和 与其他8个因素相比，推测生物膜的调节依赖于物理相互作用 在这两个TF之间。有趣的是，序列分析表明，该电路中的7个转录因子含有类似蛋白的结构域 (PrLDs)。这些是本质上无序的区域，最近的研究表明，它们可以使蛋白质 经历一种称为相分离或液-液分离的现象。将蛋白质分离成一种 更集中和更稀薄的相与无膜细胞器的形成和在 阿尔茨海默氏症等疾病。然而，本申请中提出的实验是第一次检查 转铁蛋白如何形成相分离的冷凝物来调节白念珠菌生物膜的形成。 在我的初步数据中，我显示来自生物膜网络的五个TF已经被提纯，几个 这些蛋白质在体外很容易发生相分离，形成液状凝聚体。重要的是，在 在哺乳动物细胞报告系统中过度表达，我也发现TFS可以形成PrLD介导的 活细胞中的凝结物。此外，表达具有PrLD缺失的生物膜Tf的白色念珠菌菌株不能 形成成熟的生物膜。根据这一数据，我假设主生物膜TF经历了相分离 调节生物膜网络中的靶基因，这一过程的中断将阻止生物膜的形成 口腔。在目标1中，我将确定PrLD是否在体外介导多个生物膜TF的相分离。在目标2中， 我将确定含有PrLD的转录因子在促进OPC中白色念珠菌生物被膜形成和毒力方面的作用 模特。这些发现将揭示转铁蛋白复合体如何调节真核生物中的基因表达，并将导致 对抗真菌生物被膜形成和发病机制的治疗策略。

Project Summary/Abstract The ability of microorganisms to form biofilms is a critical virulence factor for infection of the human host. Due to their complex structural make up, including multi-layer cell communities and a prominent extracellular matrix, biofilms are often recalcitrant to antimicrobial treatment. The opportunistic fungal pathogen Candida albicans is one of the leading causes of nosocomial infections due, in large part, to its ability to form complex biofilms. C. albicans can colonize every major organ in the body and forms biofilms on both medical implants and mucosal surfaces. Furthermore, in oropharyngeal candidiasis (OPC), the most common fungal infection in humans, C. albicans biofilms are formed on oral tissues and dentures. From the oral cavity, C. albicans can disperse to seed new infection sites and invasive candidiasis (IC) in the bloodstream. Mortality rates for IC remain at ~40% despite clinical intervention, with most cases thought to originate from commensal niches. Dissecting the molecular mechanisms dictating C. albicans colonization of the oral niche is therefore crucial in order to better treat chronic infections and to also minimize disease progression to IC. A well-defined transcriptional regulatory network (TRN) consisting of 9 master transcription factors (TFs) controls biofilm development in C. albicans. Multiple TFs in this network bind to their own regulatory regions and to those of the other 8 factors, and it is postulated that biofilm regulation depends on physical interactions between these TFs. Interestingly, sequence analysis indicates that 7 TFs in the circuit contain prion-like domains (PrLDs). These are intrinsically disordered regions and recent studies indicate that they can enable proteins to undergo a phenomenon known as phase separation or liquid-liquid demixing. The separation of proteins into a more concentrated and a more dilute phase is implicated in the formation of membrane-less organelles and in diseases such as Alzheimer’s. The experiments proposed in this application, however, are the first to examine how TFs can form phase-separated condensates to regulate biofilm formation in C. albicans. In my preliminary data, I show that five TFs from the biofilm network have been purified and several of these proteins readily undergo phase separation to form liquid-like condensates in vitro. Importantly, upon overexpression in a mammalian cell reporter system, I have also found that TFs can form PrLD-mediated condensates in living cells. Moreover, C. albicans strains expressing biofilm TFs with PrLD deletions are unable to form mature biofilms. Based on this data, I hypothesize that master biofilm TFs undergo phase separation to regulate target genes in the biofilm network, and that disruption of this process will block biofilm formation in the oral cavity. In Aim 1, I will determine if PrLDs mediate phase separation of multiple biofilm TFs in vitro. In Aim 2, I will define the role of PrLD-containing TFs in promoting C. albicans biofilm formation and virulence in an OPC model. These findings will inform how TF complexes regulate gene expression in eukaryotes, and will lead to therapeutic strategies for combating fungal biofilm formation and pathogenesis.