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

通过形成相分离冷凝物对白色念珠菌生物膜的转录调节

• 批准号: 10172833
• 负责人: Mae Staples
• 金额: $ 3.71万
• 依托单位: BROWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-01 至 2022-01-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10172833
• 关键词: Affect; Alzheimer' s Disease; Amino Acid Sequence; Area; Binding; Biological; Biological Process; Blood Circulation; Candida; Candida albicans; Candidiasis; Cells; Cellular biology; Chromatin; Chronic; Clinical; Complex; Data; Dentures; Development; Disease; Disease Progression; Escherichia coli; Eukaryota; Extracellular Matrix; Focal Infection; Gene Expression; Genes; Genetic Transcription; Goals; Human; Human body; In Vitro; Infection; Intervention; Intracellular Space; Label; Length; Liquid substance; Malignant Neoplasms; Mammalian Cell; Mediating; Medical Device; Membrane; Microbial Biofilms; Microscopy; Modeling; Molecular; Monitor; Mucous Membrane; Mus; Mutation; Mycoses; Nosocomial Infections; Nuclear Pore; Nucleic Acid Regulatory Sequences; Oral; Oral cavity; Organ; Organelles; Pathogenesis; Pathology; Patients; Phase; Physical condensation; Play; Prion Diseases; Process; Protein Region; Proteins; Regulation; Reporter; Research Proposals; Risk; Role; Seeds; Sequence Analysis; Site; Structure; Surface; System; Testing; Therapeutic; Transcriptional Regulation; Virulence; Virulence Factors; antimicrobial; base; cell community; chronic infection; druggable target; experimental study; gut colonization; in vivo; infection management; macromolecule; medical implant; microbial; microorganism; mortality; nervous system disorder; novel; novel therapeutic intervention; oral infection; oral tissue; oropharyngeal thrush; overexpression; pathogen; pathogenic fungus; prion-like; protein aggregation; protein complex; stress granule; transcription factor

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.

项目总结/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子。 由于其复杂的结构组成，包括多层细胞群落和突出的细胞外 基质、生物膜通常对抗微生物处理是不利的。机会致病真菌念珠菌 白色念珠菌是医院感染的主要原因之一，这在很大程度上是由于其能够形成复杂的 生物膜C.白色念珠菌可以在身体的每一个主要器官定植，并在两种医疗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， humans，C.在口腔组织和假牙上形成白色念珠菌生物膜。从口腔分离，C.白色念珠菌可以 在血流中传播以播种新的感染部位和侵袭性念珠菌病（IC）。IC的死亡率仍然是 尽管进行了临床干预，但仍有约40%的病例，大多数病例被认为起源于骨龛。解剖 C.因此，口腔生态位的白色念珠菌定殖至关重要， 更好地治疗慢性感染，并最大限度地减少疾病进展为IC。 由9个主转录因子（TF）组成的明确的转录调控网络（TRN） 控制C.白色念珠菌。该网络中的多个TF结合到它们自己的调节区域， 其他8个因素，它是假定，生物膜调节依赖于物理相互作用 在这些TF之间。有趣的是，序列分析表明，电路中的7个TF含有朊病毒样结构域 （PrLD）。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 经历称为相分离或液-液分层的现象。将蛋白质分离成 更浓的相和更稀的相与无膜细胞器的形成有关， 老年痴呆症等疾病。然而，在本申请中提出的实验是第一次检查 TF如何形成相分离的冷凝物来调节C.白色念珠菌。 在我的初步数据中，我显示了来自生物膜网络的五个TF已经被纯化， 这些蛋白质在体外容易发生相分离以形成液体状缩合物。重要的是，在 通过在哺乳动物细胞报告系统中过度表达，我还发现TF可以形成PrLD介导的 在活细胞中凝结此外，C.表达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。基于这些数据，我假设主生物膜TF经历相分离， 调节生物膜网络中的靶基因，并且该过程的破坏将阻断生物膜中的生物膜形成。 口腔在目标1中，我将确定PrLD是否在体外介导多种生物膜TF的相分离。在目标2中， 我将定义含PrLD的TF在促进C. OPC中的白色念珠菌生物膜形成和毒力 模型这些发现将告知TF复合物如何调节真核生物中的基因表达，并将导致 用于对抗真菌生物膜形成和发病机制的治疗策略。