Large scale synthetic genetic analysis in Candida albicans

描述(申请人提供)：白色念珠菌是人类最常见的真菌病原体，也是艾滋病毒/艾滋病患者发病的重要原因。事实上，几乎所有感染艾滋病毒的人都会在一生中的某个时候患上口腔咽部(OPC)或食道念珠菌病(EC)。白念珠菌致病的能力与 圆形酵母型和丝状菌丝型之间的形态发生转变。因此，这一过程一直是密集研究的主题，并已识别出许多影响酵母向细丝转变的基因。人们对这些基因和调控通路如何相互作用来协调这一复杂的生物过程还知之甚少。这在一定程度上是因为，在我们的工作开始之前，还没有在白色念珠菌中开发出大规模遗传相互作用筛选的策略。我们基于复杂单倍体不足的概念开发了一种大规模白念珠菌遗传互作筛选的方法，并将其应用于形态发生中的RAM网络(Ace2p和形态发生的调节)的研究。通过这种方式，我们发现在形态发生过程中，RAM途径与cAMP/蛋白激酶A(PKA)途径相互作用，调节一组共同基因的转录。在本申请中，我们建议确定RAM和PKA通路的活动协调的机制(目标1)。我们还将使用我们的突变体集合来研究RAM途径在血清诱导的形态发生过程中如何独立于其特征良好的转录因子ACE2发挥作用(AIM 2)。最后，我们将开发一个条码转录因子缺失盒的文库，并将它们应用于定向复杂的单倍体不足筛选方法，以更完整地定义RAM和PKA途径的遗传相互作用网络(目标3)。这些筛选将在体外进行，并使用体内念珠菌病的小鼠模型，因此，将代表第一个体内遗传相互作用的筛选。 公共卫生相关性：几乎每个艾滋病毒/艾滋病携带者在其一生中都会患上口咽或食道念珠菌病。白色念珠菌是人类最常见的真菌病原体，其引起粘膜疾病的能力与其经历从圆形酵母形态到丝状菌丝形态变化的能力有关。本申请中提出的项目是基于使用一种新的遗传方法来加深对控制这一过程的细胞调控网络的理解。了解这一过程的机制及其与发病机制的关系可能会导致治疗或预防这些感染的新方法。

DESCRIPTION (provided by applicant): Candida albicans is the most prevalent human fungal pathogen and is an important cause of morbidity in patients living with HIV/AIDS. Indeed, nearly all HIV-infected individuals will develop either oral pharyngeal (OPC) or esophageal candidiasis (EC) at some point in their lives. The ability of C. albicans to cause disease is associated with a morphogenetic transition between round yeast form and filamentous hyphal forms. Consequently, this process has been the subject of intense study and many genes have been identified that affect the yeast-to-filament transition. Less understood is how these genes and regulatory pathways interact to orchestrate this complex biological process. This is due, in part, to the fact that strategies for large-scale genetic interaction screening had not been developed in C. albicans prior to the initiation of our work. We have developed an approach to large scale genetic interaction screening in C. albicans based on the concept of complex haploinsufficiency and applied it to the study of the RAM network (Regulation of Ace2p and Morphogenesis) in morphogenesis. In this way, we found that the RAM pathway interacts with the cAMP/protein kinase A (PKA) pathway to regulate the transcription of a common set of genes during morphogenesis. In this application, we propose to determine the mechanism through which the activities of the RAM and PKA pathways are coordinated (Aim 1). We will also use our collection of mutants to investigate how the RAM pathway functions independently of its well characterized transcription factor Ace2 during serum- induced morphogenesis (Aim 2). Finally, we will develop a library of bar-coded transcription factor deletion cassettes and apply them to a directed complex haploinsufficiency screening approach to more completely define the genetic interaction networks for the RAM and PKA pathways (Aim 3). These screens will be carried out in vitro and using in vivo mouse models of candidiasis and, as such, will represent the first in vivo genetic interaction screen. PUBLIC HEALTH RELEVANCE: Nearly every person living with HIV/AIDS will develop oropharyngeal or esophageal candidiasis during their lifetime. The ability of Candida albicans, the most common human fungal pathogen, to cause mucosal disease is associated with its ability to undergo a morphological change from round yeast form to filamentous hyphal forms. The project proposed in this application is based on the use of a novel genetic approach to develop a deeper understanding the cellular regulatory networks that control this process. Understanding the mechanisms of this process and its relation to pathogenesis could lead to novel approaches to treating or preventing these infections.