Defining the role of the TLO gene family in Candida albicans parasexual processes

定义 TLO 基因家族在白色念珠菌副性过程中的作用

• 批准号: 10536048
• 负责人: Andrew Logan Woodruff
• 金额: $ 3.48万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-16 至 2024-07-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10536048
• 关键词: Adopted; Affect; Biological Assay; Candida albicans; Cells; ChIP-seq; Chromatin; Chromosomes; Clustered Regularly Interspaced Short Palindromic Repeats; Complement; Complex; DNA Binding; Data; Diploidy; Disease; Enzymes; Epigenetic Process; Epitopes; Equilibrium; Eukaryota; Family member; Feedback; Filament; Gene Dosage; Gene Duplication; Gene Family; Gene Targeting; Genes; Genetic; Genetic Transcription; Genotype; Head; Homologous Gene; Human; Immunocompromised Host; Individual; Infection; Lead; Life; Link; MED15; Mating Types; Mediator of activation protein; Molecular; Morphology; Mucous Membrane; Oral cavity; Organism; Partner in relationship; Pathogenesis; Phenotype; Pheromone; Play; Process; Regulation; Regulon; Role; Skin; Sterility; Symbiosis; System; Tail; Testing; Transcriptional Regulation; Virulence; Work; Yeasts; base; chromatin modification; chromosome loss; clinically relevant; fitness; fungus; gene function; human pathogen; insight; macrophage; member; mutant; novel; opportunistic pathogen; overexpression; paralogous gene; pathogenic fungus; programs; promoter; reference genome; response; restoration; telomere; trait; transcriptome sequencing; urogenital tract

PROJECT SUMMARY In eukaryotes, cell states transitions often require major transcriptional shifts that are subsequently reinforced through feedback loops that maintain stable expression of the newly acquired cellular phenotype. Candida albicans, a diploid commensal yeast and significant opportunistic pathogen, has been found to adopt a number of morphologically-distinct cell states that modulate its interaction with the human host. The phenotypic switch between the white and opaque cell states governs entry into the parasexual mating system by opaque cells and regulates the balance between virulence and commensalism in different host niches. Previous work has shown that switching between these states is regulated, in part, by the Mediator complex, a major transcriptional regulatory complex in eukaryotes. However, mutants within the Tail module subunits of Mediator that are necessary for formation of the opaque state affect incorporation of other subunits into the complex. More specifically, deletion of MED3 from the Tail module also causes the loss of Med2 from Mediator. In C. albicans, MED2 is encoded by the telomere-associated (TLO) expanded gene family, which can function as interchangeable Med2 subunits of Mediator. Thus, it is unclear if C. albicans phenotypic switching is affected by inactivation of MED3 or TLO genes in Mediator. Individual TLO genes have diverse functions based on overexpression studies with multiple genes often regulating a single trait. Therefore, we hypothesize that the TLO gene family as a whole is necessary for the white-opaque switch and progression through the parasexual cycle, with individual TLO paralogs possessing unique regulatory impacts on parasexual processes. Aim 1 will test necessity of the TLO gene family on interconversion between the white and opaque cell states by utilizing a recently built TLO-null strains in comparison to the med3/ and wild-type strains. Furthermore, we will evaluate the phenotypic impact of complete TLO loss on the pheromone response and mating efficiency of opaque cells, as MED3 mutants ectopically induced into the opaque state also suffered from reduced mating efficiency. To determine if specific TLO paralogs are responsible for alterations in cell state transition and mating, Aim 2 will produce a panel of strains containing a single epitope-tagged TLO paralog with their native promoters reintroduced into the null background. These strains will be tested for restoration of wild-type phenotypes for traits identified in Aim 1. These TLO reintroduction strains will also be used to identify paralog-specific bound DNA targets by chromatin immunoprecipitation sequencing (ChIP-Seq) and affected regulons via RNA sequencing (RNA-Seq). This work will determine the role of the TLO gene family and individual paralogs in the parasexual cycle of C. albicans. Furthermore, it will begin to determine the extent of functional diversification among members of a large paralogous gene family, which is largely untested beyond gene duplicates, and provide greater understanding into how genes may be retained in tightly packed evolutionary spaces.

项目摘要 在真核生物中，细胞状态转换通常需要随后发生的主要转录转变。 通过维持新获得的细胞表型的稳定表达的反馈回路来加强。 白色念珠菌是一种二倍体假丝酵母菌，是重要的条件致病菌， 调节其与人类宿主相互作用的形态学上不同的细胞状态的数量。表型 在白色和不透明细胞状态之间的转换控制着进入准性交配系统， 在不同的寄主生态位中调节毒力和寄生性之间的平衡。以前的工作 已经表明，这些状态之间的转换是受调节的，部分，由中介复合体，一个主要的 真核生物中的转录调控复合物。然而，在Mediator的Tail模块亚基内的突变体 形成不透明状态所必需的那些影响其它亚基结合到复合物中。更 具体而言，从尾模块中删除MED 3也会导致Mediator中丢失Med 2。In C.白色念珠菌， MED 2由端粒相关（TLO）扩展基因家族编码，其可以作为 Mediator的可互换Med 2亚基。因此，尚不清楚C。白念珠菌表型转换受以下因素影响 Mediator中MED 3或TLO基因的失活。个体TLO基因具有不同的功能， 多个基因的过表达研究通常调节单个性状。因此，我们假设， TLO基因家族作为一个整体对于白色不透明的转换和通过准有性的进展是必要的。 周期，个别TLO旁系同源物具有独特的调节影响准性过程。目标1将 通过利用一种新的方法测试TLO基因家族在白色和不透明细胞状态之间相互转换的必要性， 最近构建的TLO-无效菌株与med 3 β/β和野生型菌株相比。此外，我们将评估 TLO完全丧失对信息素反应和不透明细胞交配效率的表型影响， 因为异位诱导成不透明状态的MED 3突变体也遭受降低的交配效率。到 确定特定的TLO旁系同源物是否负责细胞状态转换和交配的改变，Aim 2将 产生一组含有单个表位标记的TLO质粒及其天然启动子的菌株 重新引入空背景。将测试这些菌株的野生型表型恢复， 目标1中确定的特征。这些TLO再引入菌株也将用于鉴定旁系同源物特异性结合的TLO。 通过染色质免疫沉淀测序（ChIP-Seq）检测DNA靶点，并通过RNA检测受影响的调节子 测序（RNA-Seq）。这项工作将确定TLO基因家族和个体旁系同源物在肿瘤发生中的作用。 拟性周期白色念珠菌此外，它将开始确定职能多样化的程度 在一个大的旁系同源基因家族的成员中，除了基因重复之外，这在很大程度上是未经测试的， 提供了更好的理解基因如何被保留在紧密排列的进化空间中。