Quantitative genetic approaches to Candida albicans pathogenesis

白色念珠菌发病机制的定量遗传学方法

• 批准号: 10390393
• 负责人: Matthew Z Anderson
• 金额: $ 45.31万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-12 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10390393
• 关键词: Adherence; Adhesions; Agar; Benign; Biological; Biology; Blood Circulation; Candida; Candida albicans; Candidate Disease Gene; Candidiasis; Cell physiology; Cells; Clinical; Detection; Development; Disease; Disease Outcome; Endocytosis; Epithelial; Epithelial Cells; Equilibrium; Esophagus; Filament; Gastrointestinal tract structure; Gene Expression; Genes; Genetic; Genetic Determinism; Genetic Polymorphism; Genetic Screening; Genetic Transcription; Genetic Variation; HIV; Human; Hyphae; Immune response; Immunocompetent; Immunocompromised Host; In Vitro; Incubated; Individual; Integration Host Factors; Investigation; Lead; Life; Link; Liquid substance; Measures; Mucous Membrane; Mus; Nutritional; Oral; Oral candidiasis; Oral cavity; Oral mucous membrane structure; Oropharyngeal; Partner in relationship; Pathogenesis; Pathogenicity; Pathway interactions; Patients; Penetration; Phagocytes; Phenotype; Ploidies; Process; Production; Property; Quality of life; Quantitative Genetics; Quantitative Trait Loci; Recombinants; Role; Signal Transduction; Skin; Solid; Surface; Symbiosis; System; Systemic disease; Systemic infection; Testing; Tissues; Variant; Virulence; Work; Yeasts; cell growth regulation; cell injury; clinically relevant; differential expression; experimental study; fitness; fungus; genetic approach; genome-wide; human pathogen; in vivo; in vivo evaluation; insight; interest; mouse model; mutant; novel; opportunistic pathogen; oral cavity epithelium; oral infection; oropharyngeal thrush; pathogenic fungus; programs; response; therapeutic target; tissue culture; trait; transcriptome sequencing; uptake; urogenital tract; virtual; wasting

ABSTRACT Candida albicans is a common fungal commensal and opportunistic pathogen occupying the skin, oral cavity, gastrointestinal tract, and genitourinary tract of its human host. Overgrowth of C. albicans within these host niches can lead to mucosal and systemic disease and typically relies on pathogenic processes such as the yeast-hyphal transition. Hyphae are capable of breaching host mucosal barriers through active penetration and endocytic uptake that both lead to host cell destruction and transmigration of C. albicans to deeper tissues. Thus, host epithelial cells serve a vital role in detecting C. albicans overgrowth and signaling to professional phagocytes to remove invasive fungal cells. Interestingly, natural C. albicans isolates obtained from clinical setting display a range of filamentation phenotypes, including strains unable to produce hyphae. Here, we will investigate the genetic determinants underlying natural occurring phenotypic variation in C. albicans that regulates the ability to filament and/or colonize the oral cavity through the first use of quantitative genetic approaches in C. albicans. A genetically diverse set of sequenced clinical C. albicans isolates forms the basis of this study as they display significant variation in filamentation processes in vitro and the ability to cause disease in vivo. First, gene expression across the sequenced isolates will facilitate construction of co- expression network modules that associate with in vitro filamentation phenotypes. Expression of key transcriptional regulators within each predicted module will be tested in multiple strain backgrounds for altered in vitro filamentation across a variety of solid and liquid substrates. Automated phenotyping of filamentation, adhesion, and invasion of agar substrates will be built to facilitate scoring mutant phenotypes (Aim 1). In Aim 2, quantitative trait loci (QTL) mapping will be developed utilizing the parasexual program, an alternative mating and ploidy reduction system in C. albicans, to identify the genes responsible for differences in filamentation and epithelial damage between C. albicans strains incubated with OKF6/TERT-2 oral epithelial cells. Identified genes that modulate filamentation and epithelial damage in tissue culture systems will be subsequently tested for in vivo colonization and filamentation phenotypes of a murine model of oropharyngeal candidiasis. Preliminary experiments suggest that filamentation and damage of oral epithelial cells are separable phenotype, which will be explored further through dual-species RNA sequencing of strains separated by filamentation (+/-) and cell damage (+/-) phenotypes incubated with OKF6/TERT-2 cells (Aim 3). This Aim will also determine the role of recently identified host genes with differential expression between damaging and colonizing strains in host cell signaling and transcriptional responses that lead to either filamentation or host cell damage during interaction with oral epithelial cells. Together, these studies will determine the pathways and processes modulated by genetic diversity in C. albicans that lead to divergent filamentation and epithelial damage phenotypes that contribute to commensalism v. disease.

摘要 白色念珠菌是一种常见的真菌共生和机会致病菌，占据皮肤、口腔 寄主的腔、胃肠道和泌尿生殖道。白念珠菌在这些区域内过度生长 宿主的生态位可导致粘膜和全身疾病，通常依赖于以下致病过程 酵母菌向菌丝的转变。菌丝能够通过主动穿透突破宿主的粘膜屏障 和内吞摄取，两者都导致宿主细胞破坏和白念珠菌向更深的组织迁移。 因此，宿主上皮细胞在检测白色念珠菌过度生长和向专业人士发出信号方面起着至关重要的作用。 吞噬细胞来清除入侵的真菌细胞。有趣的是，从临床上获得的天然白色念珠菌分离株 设置显示一系列丝化表型，包括不能产生菌丝的菌株。在这里，我们将 研究自然发生的白念珠菌表型变异背后的遗传决定因素 通过第一次使用数量遗传学来调节成丝和/或定植口腔的能力 白色念珠菌的致病途径。一组基因多样性的临床白色念珠菌分离株的测序形成了基础 因为它们在体外成丝过程中显示出显著的差异，并能够导致 活体内的疾病。首先，跨测序分离株的基因表达将有助于构建共- 与体外丝状化表型相关的表达网络模块。关键字的表示 每个预测模块中的转录调控因子将在多个菌株背景下进行测试，以确定是否发生了改变 在各种固体和液体底物上的体外丝状化。丝状化的自动表型分析， 将建立琼脂基质的粘附性和侵袭性，以便于对突变表型进行评分(目标1)。在目标2中， 数量性状基因座(QTL)定位将利用另一种交配方式--准性交程序进行开发 和白念珠菌倍体减少系统，以确定导致细丝形成差异的基因 与OKF6/TERT-2口腔上皮细胞孵育的白念珠菌株之间的上皮损伤。已确定 随后将对组织培养系统中调节丝状化和上皮损伤的基因进行测试 用于口咽部念珠菌病小鼠模型的体内定植和丝状化表型。 初步实验表明，口腔上皮细胞的丝状化和损伤是可以分离的。 表型，这将通过双物种RNA测序进一步探索 与OKF6/TERT-2细胞孵育的丝状化(+/-)和细胞损伤(+/-)表型(目标3)。这一目标将 也确定最近发现的宿主基因的作用，这些宿主基因在破坏性和 在宿主细胞信号和转录反应中定植导致丝状或宿主的菌株 口腔上皮细胞相互作用过程中的细胞损伤。总而言之，这些研究将确定 以及由白念珠菌遗传多样性调节的导致不同丝状和上皮细胞分化的过程 损害有助于共生对抗疾病的表型。