N6-methyladenosine in Candida white-opaque switching and oral infection

项目总结/摘要 白色念珠菌白色不透明转换是一个表观遗传过程，调节其交配能力，毒力， 以及在人类宿主中不同生态位的殖民化。这些壁龛之一是口腔，念珠菌 这种真菌会引起称为口咽念珠菌病（OPC）的疼痛感染。虽然已知 环境线索和转录调节因子控制白色不透明开关，一种机制， 了解特定环境如何触发切换到一种状态或另一种状态，以及这如何有助于 殖民化和感染是缺乏的。该提案将研究RNA修饰N6- 甲基腺苷（m6 A）介导C.白色念珠菌初步研究 从我的合作者那里，我发现缺乏IME 4（m6 A甲基转移酶）的细胞， 温度诱导的不透明到白色转换，并具有增加的匹配效率。是有着积极 背景念珠菌白色不透明开关和m6 A生物学，我是唯一有资格调查 m6 A调节介导诱导表型变化的环境信号假说 介导C.白色念珠菌目的1是在白色不透明细胞中鉴定m6 A修饰的靶标。 通过使用eCLIP来定位m6 A修饰的单核苷酸位点和MRB 1的结合位点， 阅读器，以及野生型和ime 4突变体白色和不透明细胞中的基因表达分析。目标2将 确定环境如何通过产生基因表达来调节m6 A的转换和表达 以及在环境条件如高温或CO2下细胞的代谢组学特征， 白色不透明的开关或显示。目的3探讨m6 A在口腔念珠菌定植中的作用。白色 通过使用细胞培养和小鼠OPC模型。白色不透明转换调节口腔定植，我们 假设IME 4在这个过程中发挥了作用。在这个目标，我也将研究如何辐射影响念珠菌 缺乏IME 4的细胞以及它如何影响鼠口腔生态位的定植。在目标4中，我将研究函数 m6 A与口腔微生物组的相互作用。我发现的许多m6 A修饰基因都涉及 在物种间的相互作用，这表明IME 4和m6 A调节宿主内的这些相互作用。这 一项研究计划将是第一个联合收割机结合的领域epitranscriptomics和念珠菌生物学研究如何m6 A 有助于临床相关真菌物种的生活方式和致病性，有可能铺平道路， 新的抗真菌药物治疗OPC的方法。我已经提出了一个全面的培训计划， 该提案的K99阶段，包括获得生物信息学，代谢组学，动物模型方面的新技能 口腔感染，微生物组分析，杰出导师咨询委员会的指导， 合作者，以及发展关键的专业和领导技能。总的来说，这个计划将有助于我 在R 00阶段过渡到独立，并为我作为独立研究员的职业生涯做好准备。

Project Summary / Abstract Candida albicans white-opaque switching is an epigenetic process that regulates its mating ability, virulence, and colonization of different niches in the human host. One of these niches is the oral cavity, where Candida species can cause painful infections called oropharyngeal candidiasis (OPC). While there are known environmental cues and transcriptional regulators that control the white-opaque switch, a mechanistic understanding of how specific environments trigger switching to one state or another and how this contributes to colonization and infection is lacking. This proposal will investigate how the RNA modification N6- methyladenosine (m6A) acts to mediate white-opaque switching and mating in C. albicans. Preliminary studies from my collaborator and I have found that cells lacking IME4, the m6A methyltransferase, are impaired in temperature-induced opaque to white switching and have an increased mating efficiency. As someone with a background in Candida white-opaque switching and m6A biology, I am uniquely qualified to investigate the hypothesis that m6A regulation mediates environmental signals that induce phenotypic changes that mediate infection by C. albicans. Aim 1 is to identify targets of m6A modification in white-opaque switching by using eCLIP to map single nucleotide sites of m6A modification and binding sites of MRB1, the m6A reader, as well as gene expression analysis in wild-type and ime4 mutant white and opaque cells. Aim 2 will determine how the environment regulates m6A in switching and filamentation by generating gene expression and metabolomic profiles of cells under environmental conditions such as high temperature or CO2 that induce white-opaque switching or filamentation. Aim 3 will investigate the role of m6A in oral colonization of C. albicans by using cell culture and mouse models of OPC. White-opaque switching regulates oral colonization, and we hypothesize that IME4 plays a role in this process. In this aim, I will also study how radiation affects Candida cells lacking IME4 and how it affects colonization of the murine oral niche. In aim 4 I will investigate the function of m6A in interactions with the oral microbiome. Many of the m6A modified genes I have identified are involved in interspecies interactions, suggesting that IME4 and m6A regulate these interactions within the host. This research plan will be the first to combine the fields of epitranscriptomics and Candida biology to study how m6A contributes to the lifestyle and pathogenicity of a clinically relevant fungal species, with the potential to pave the way for new anti-fungal drug treatments for OPC. I have proposed a comprehensive training program for the K99 phase of this proposal including acquisition of new skills in bioinformatics, metabolomics, animal models of oral infection, microbiome analyses, guidance from an advisory committee of prominent mentors and collaborators, and development of key professional and leadership skills. Collectively, this plan will facilitate my transition to independence during the R00 phase and prepare me for a career as an independent researcher.