Analysis of Candida albicans filamentation using SILAC

使用 SILAC 分析白色念珠菌丝状化

• 批准号: 8227948
• 负责人: Derek Paul Thomas
• 金额: $ 5.77万
• 依托单位: GRAND VALLEY STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-02-15 至 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8227948
• 关键词: Acquired Immunodeficiency Syndrome; Adherence; Affinity Chromatography; Amino Acids; Antibiotics; Benign; Biological; Biological Phenomena; Candida; Candida albicans; Candidiasis; Cell Culture Techniques; Cell physiology; Cells; Clinical; Complex; Data; Defect; Development; Developmental Process; Direct Costs; Disease; Economic Burden; Economics; Environment; Event; Genes; Health; Hospitals; Human; Hyphae; Immune; Infection; Knowledge; Left; Link; Mediating; Metals; Morbidity - disease rate; Mycoses; Nature; Nosocomial Infections; Organ Transplantation; Organism; Pathogenesis; Pathway interactions; Patients; Phosphoproteins; Phosphorylation; Process; Proteins; Proteome; Research; Sampling; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Stable Isotope Labeling; Steroid therapy; Techniques; Tissue Transplantation; Transcript; Virulence; Yeasts; analytical tool; base; cancer therapy; cell injury; fundamental research; mass spectrometer; mortality; mutant; pathogen; promoter; public health relevance; research study; response; stable isotope; tool; trait

DESCRIPTION (provided by applicant): The pathogenic potential of Candida albicans is intimately related with the way this organism senses and reacts to its surrounding environment, which, in the case of C. albicans, is the host. Candidiasis now represents the third most frequent nosocomial infection in hospitals both in the US and worldwide and C. albicans remains the most frequent causative agent of fungal infections. C. albicans is an increasingly common threat to human health as a consequence of AIDS, steroid therapy, organ and tissue transplantation, cancer therapy, broad spectrum antibiotics and other immune defects. Unfortunately these infections carry unacceptably high morbidity, mortality rates and important economic repercussions (estimated total direct cost of approximately 2 billion dollars in 1998 in US hospitals alone). C. albicans can grow as yeast cells, pseudohyphae or hyphae with its form being dictated by its surrounding conditions. The ability to form hyphal cells has been fundamentally linked to the disease potential of this organism. In fact, cells which cannot make the transition are avirulent. Hyphal formation is regulated by a complex network of signaling pathways. Research has centered on parts of a small number of specific signal transduction pathways that are known to mediate its environmental sensing and host-pathogen interactions. This has left substantial gaps in our knowledge including the originating signal and the nature of crosstalk within these very pathways. This is compounded by the possibility of other transduction pathways and the use of phosphorylation to modulate the activity of the proteins responsible for regulating filamentation. Although phosphorylation is important for a number of cellular and developmental processes, most studies have focused on a few specific proteins and there are limited proteome-wide data on signal transduction pathway proteins or phosphoproteins in general. This application will study the C. albicans phosphoproteome with an emphasis on signal transduction pathways. This project focuses on a central themes: i) that Candida filamentation is a virulence trait strongly associated with environmental sensing and that by studying the phosphoproteome we can identify environmental sensing molecules and pathways vital for this process. The basis of the proposed experimentation is to use Stable Isotope Labeling with Amino acids in Cell culture (SILAC) in conjunction with Immobilized Metal Affinity Chromatography (IMAC) enrichment of phosphoproteins followed by mass spectrometric analysis. The presence of the stable isotope causes a mass change allowing the direct quantitative comparison to an unlabeled sample on a mass spectrometer. PUBLIC HEALTH RELEVANCE: Candida albicans is the main causative agent of candidiasis, the most frequent fungal infection and the fourth leading cause of infections in US hospitals, with high mortality rates and significant economic burden. It frequently infects AIDS patients. Here we propose using a phosphoproteomic approach to investigate the sensing and signalling that mediate key virulence associated events, hopefully leading to preventative strategies against these frequent nosocomial infections.

描述（由申请人提供）：白色念珠菌的致病潜力与该生物体对其周围环境的感知和反应方式密切相关，在C.白色念珠菌是宿主。在美国和世界范围内，念珠菌病是第三大最常见的医院感染。白色念珠菌仍然是真菌感染的最常见病原体。C.由于艾滋病、类固醇治疗、器官和组织移植、癌症治疗、广谱抗生素和其它免疫缺陷，白色念珠菌对人类健康的威胁日益普遍。不幸的是，这些感染具有不可接受的高发病率、死亡率和严重的经济影响（仅在美国医院，1998年估计直接总成本约为20亿美元）。C.白色念珠菌可作为酵母细胞、假菌丝或菌丝生长，其形式取决于其周围条件。形成菌丝细胞的能力从根本上与这种生物体的疾病潜力有关。事实上，不能进行这种转变的细胞是无毒的。菌丝的形成受一个复杂的信号通路网络的调节。研究集中在少数特定的信号转导途径，已知介导其环境传感和宿主-病原体相互作用的部分。这在我们的知识中留下了大量的空白，包括这些路径内的原始信号和串扰的性质。这是复杂的其他转导途径的可能性和使用磷酸化来调节负责调节蛋白质的活性。尽管磷酸化对许多细胞和发育过程很重要，但大多数研究都集中在少数特定蛋白质上，并且一般而言，关于信号转导途径蛋白或磷蛋白的蛋白质组范围的数据有限。本应用程序将研究C.白念珠菌磷酸化蛋白质组与信号转导途径的重点。该项目的重点是一个中心主题：i）假丝酵母菌的表达是一种与环境感知密切相关的毒力性状，通过研究磷酸化蛋白质组，我们可以识别对这一过程至关重要的环境感知分子和途径。拟定实验的基础是使用细胞培养物中氨基酸的稳定同位素标记（SILAC）结合磷蛋白的固定化金属亲和色谱（IMAC）富集，然后进行质谱分析。稳定同位素的存在导致质量变化，允许在质谱仪上与未标记的样品进行直接定量比较。 公共卫生关系：白色念珠菌是念珠菌病的主要病原体，是美国医院最常见的真菌感染和第四大感染原因，具有高死亡率和显著的经济负担。它经常感染艾滋病患者。在这里，我们建议使用磷酸化蛋白质组学的方法来调查的传感和信号，介导的关键毒力相关的事件，希望导致这些频繁的医院感染的预防策略。