Analysis of Candida albicans filamentation using SILAC

描述(申请人提供)：白色念珠菌的致病潜力与这种生物对周围环境的感知和反应方式密切相关，在白色念珠菌的情况下，周围环境是宿主。念珠菌病现在是美国和世界各地医院感染的第三大常见病原体，而白色念珠菌仍然是最常见的真菌感染病原体。由于艾滋病、类固醇治疗、器官和组织移植、癌症治疗、广谱抗生素和其他免疫缺陷，白念珠菌对人类健康的威胁日益普遍。不幸的是，这些感染具有令人无法接受的高发病率、死亡率和重要的经济影响(仅在美国医院，估计1998年的直接总成本约为20亿美元)。白色念珠菌可以以酵母细胞、假菌丝或菌丝的形式生长，其形式取决于周围的条件。形成菌丝细胞的能力从根本上与这种有机体的致病潜力有关。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控。研究集中在少数特定信号转导通路的一部分，这些信号转导通路是已知的介导其环境感知和宿主-病原体相互作用的途径。这在我们的知识中留下了很大的空白，包括这些通路中的原始信号和串扰的性质。其他转导途径的可能性和使用磷酸化来调节负责调节细丝形成的蛋白质的活性，使这一点变得更加复杂。虽然磷酸化在许多细胞和发育过程中是重要的，但大多数研究都集中在少数几个特定的蛋白质上，关于信号转导途径蛋白或一般的磷蛋白的蛋白质组数据有限。这项应用将研究白色念珠菌的磷酸蛋白质组，重点是信号转导途径。该项目集中于一个中心主题：i)假丝酵母菌的丝状化是一种与环境感知密切相关的毒力特征，通过研究磷蛋白质组，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。拟议实验的基础是使用细胞培养中氨基酸稳定同位素标记(SILAC)和固定化金属亲和层析(IMAC)浓缩磷蛋白，然后进行质谱分析。稳定同位素的存在会引起质量变化，从而可以与质谱仪上的未标记样品进行直接定量比较。 与公共卫生相关：白色念珠菌是美国医院中最常见的真菌感染和第四大感染原因，是念珠菌病的主要病原体，死亡率高，经济负担巨大。它经常感染艾滋病患者。在这里，我们建议使用磷酸蛋白质组学的方法来研究介导关键毒力相关事件的感知和信号传递，希望能够导致预防这些频繁的医院感染的策略。

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.