Epigenetic Regulation of Sexual Mating and Biofilm formation in Candida albicans

白色念珠菌性交配和生物膜形成的表观遗传调控

• 批准号: 8783670
• 负责人: Christine Maria Scaduto
• 金额: $ 4.35万
• 依托单位: BROWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8783670
• 关键词: Address; Area; Bacterial Adhesins; Biology; Blood Circulation; Candida; Candida albicans; Cell Shape; Cells; Clinic; Communicable Diseases; Communities; DNA Binding; DNA Sequence Analysis; Data; Devices; Engineering; Epigenetic Process; Exhibits; Gastrointestinal tract structure; Genes; Genitourinary system; Goals; HIV; Health; Homologous Gene; Human; Immune; Immunoblotting; Individual; Infection; Laboratories; Leukocytes; Libraries; Life; Microbial Biofilms; Mitogen-Activated Protein Kinases; Molecular; Mucous Membrane; Mycoses; Operative Surgical Procedures; Oral cavity; Oral mucous membrane structure; Organism; Output; Partner in relationship; Pathway interactions; Patients; Pheromone; Prevalence; Proteins; Recombinants; Regulation; Reproduction; Research; Research Proposals; Saccharomyces cerevisiae; Sex Attractants; Signal Pathway; Signal Transduction; Skin; Source; Stimulus; Testing; Virulence; Work; cell type; cellular engineering; chromatin immunoprecipitation; combat; implantation; mortality; operation; oral infection; oropharyngeal thrush; overexpression; pathogen; research study; response; trait; transcription factor; transcriptome sequencing

DESCRIPTION (provided by applicant): The human fungal pathogen Candida albicans exhibits remarkable phenotypic plasticity during colonization and infection of multiple niches in the human host [2]. This is exemplified by its ability to switch between two phenotypic states, 'white' and 'opaque'. This epigenetic switch occurs between isogenic cells and is both heritable and reversible. White and opaque states differ in many key aspects of Candida biology, including their interaction with immune cells, their virulence, and their response to sexual pheromones. Thus, while white cells respond to pheromone by forming communities of adherent cells known as biofilms, opaque cells respond by undergoing sexual mating [20, 17]. In both cell types, a conserved MAPK pathway facilitates pheromone signaling [5,6], yet the underlying mechanism by which cells generate different responses to pheromone is unknown. Preliminary analysis indicates that opaque cells exhibit elevated expression of three components of the MAPK signaling pathway. Aim 1 will test the hypothesis that increased expression of specific MAPK components facilitates signal transduction and is necessary for efficient mating. To achieve this goal, strains locked in the white state will be engineered to express elevated levels of key MAPK components and mating efficiency quantified. Immunoblotting will also be used to compare expression levels of MAPK proteins between opaque cells, white cells, and engineered white cells, to determine if protein levels are reflectiv of overall mating efficiency. Aim 2 will define the mechanism of sexual (pheromone-induced) biofilm formation in white cells by examining the transcriptional targets of MAPK signaling. The transcription factor Cph1 is activated in both white and opaque cells [6], yet distinct sets of genes are induced in response to pheromone in both cell types [29]. This Aim will test the hypothesis that a white-specific transcription factor works cooperatively with Cph1 to promote biofilm formation. Chromatin immunoprecipitation and DNA sequencing analysis will be performed in white and opaque cells to identify the DNA binding targets for Cph1 in both cell types. In parallel with this approach, I will screen a transcription factor overexpression library (recently constructed in the Bennett laboratory) to define those factors that promote sexual biofilm formation. Together, completion of the experiments outlined in this proposal will define how an epigenetic switch can regulate phenotypic traits, including both sexual mating and biofilm formation. Given the prevalence of C. albicans biofilms within the oral cavity [18] and on surgical implantation devices [21], the research outlined in this proposal will have direct implications in combating debilitating fungal infections in the clinic.

描述（由申请人提供）：人类真菌病原体白色念珠菌在人类宿主的多个生态位定植和感染期间表现出显着的表型可塑性[2]。这可以通过其在“白色”和“不透明”两种表型状态之间切换的能力来例证。这种表观遗传开关发生在同基因细胞之间，并且是可遗传的和可逆的。白色和不透明状态在念珠菌生物学的许多关键方面有所不同，包括它们与免疫细胞的相互作用、它们的毒力以及它们对性信息素的反应。因此，虽然白细胞通过形成称为生物膜的贴壁细胞群落对信息素做出反应，但不透明细胞通过进行性交配做出反应[20, 17]。在这两种细胞类型中，保守的 MAPK 通路促进信息素信号传导 [5,6]，但细胞对信息素产生不同反应的潜在机制尚不清楚。初步分析表明，不透明细胞表现出 MAPK 信号通路三个组成部分的表达升高。目标 1 将检验以下假设：特定 MAPK 成分的表达增加有助于信号转导，并且是有效交配所必需的。为了实现这一目标，锁定在白色状态的菌株将被设计为表达升高水平的关键 MAPK 成分并量化交配效率。免疫印迹还将用于比较不透明细胞、白细胞和工程白细胞之间 MAPK 蛋白的表达水平，以确定蛋白水平是否反映整体交配效率。目标 2 将通过检查 MAPK 信号传导的转录靶标来定义白细胞中性（信息素诱导）生物膜形成的机制。转录因子 Cph1 在白色细胞和不透明细胞中均被激活 [6]，但两种细胞类型中都会诱导不同的基因组来响应信息素 [29]。该目标将测试白色特异性转录因子与 Cph1 协同作用促进生物膜形成的假设。将在白色和不透明细胞中进行染色质免疫沉淀和 DNA 测序分析，以确定两种细胞类型中 Cph1 的 DNA 结合靶标。与这种方法同时，我将筛选一个转录因子过表达库（最近在贝内特实验室构建）来定义那些促进性生物膜形成的因子。总之，完成本提案中概述的实验将定义表观遗传开关如何调节表型特征，包括性交配和生物膜形成。鉴于白色念珠菌生物膜在口腔内[18]和手术植入装置上[21]的普遍存在，本提案中概述的研究将对临床上对抗衰弱的真菌感染产生直接影响。