Systematic in vitro and in vivo genetic analysis of C.albicans protein kinases

项目摘要 白色念珠菌既是正常人体微生物植物群的重要组成部分， 人类病原体虽然C.白色念珠菌主要局限于 相比之下，念珠菌病可累及胃肠道和泌尿生殖道， 系统，包括肾、脾、肝、心脏、眼、中枢神经系统和皮肤/粘膜。因此，C. 白色念珠菌是一种多功能的病原体，其适应于细菌内广泛的生理多样性环境。 人类宿主了解促进这种适应的系统对于制定详细的 C.白念珠菌的发病机制，并可以告知新的治疗方法的识别 接近念珠菌病。迄今为止，C.白念珠菌转录因子（TF）是最好的特点 这一系统的组成部分，部分是由于全面的TF缺失突变体集的可用性。在 相反，C.白色念珠菌生理学如蛋白激酶（PK）尚未被 系统地进行了相同程度的研究。为了解决这一知识差距，我们项目的长期目标是 系统地研究PKs在C.白色念珠菌病理生物学作为实现这一目标的第一步，我们 提出：1）产生和验证纯合和杂合C.白色念珠菌PK缺失 使用瞬时CRISPR/Cas9策略系统性地表征了突变体中的PK缺失突变体; 2）系统性地表征了突变体中的PK缺失突变体。 念珠菌病的两种鼠模型和两种毒力相关的体外过程：生物膜和菌丝形成; 3） 使用遗传学方法鉴定和表征调节生物膜和菌丝形成的PK-TF对网络 交互作用分析这些目标的成功实现将为人类提供有用的遗传资源， 研究社区，提供了第一个系统的功能表征C。体内和体内的白色念珠菌PK 体外，并为随后深入分析由C.白色念珠菌

PROJECT SUMMARY Candida albicans is both a commensal component of the normal human microbial flora and an important human pathogen. Although the commensal niches occupied by C. albicans are primarily limited to the gastrointestinal and genitourinary tracts, candidal disease, in contrast, can involve a wide range of organ systems including kidney, spleen, liver, heart, eye, the central nervous system, and skin/mucosae. As such, C. albicans is a versatile pathogen that adapts to a wide range of physiologically diverse environments within the human host. Understanding the systems that facilitate this adaptation is central to developing detailed mechanistic models of C. albicans pathogenesis and could inform the identification of new therapeutic approaches to candidiasis. To date, C. albicans transcription factors (TF) are the best characterized components of this system, due in part to the availability of comprehensive sets of TF deletion mutants. In contrast, upstream regulators of C. albicans physiology such as protein kinases (PK) have not been systematically studied to the same extent. To address this knowledge gap, the long-term goal of our project is to systematically characterize the role of PKs in C. albicans pathobiology. As a first step toward this goal, we propose to: 1) generate and validate a barcoded set of homozygous and heterozygous C. albicans PK deletion mutants using a transient CRISPR/Cas9 strategy; 2) Systematically characterize the PK deletion mutants in two murine model of candidiasis and two virulence relevant in vitro processes: biofilm and hyphae formation; 3) Identify and characterize the network of PK-TF pairs that regulate biofilm and hyphae formation using genetic interaction analysis. Successful completion of these aims will provide a useful genetic resource for the research community, provide the first systematic functional characterization of C. albicans PKs in vivo and in vitro, and set the stage for subsequent in-depth analyses of the genetic networks regulated by C. albicans PKs.