Single nucleotide variations account for strain specific virulence changes in Cryptococcus neoformans

项目总结/摘要 隐球菌性脑膜炎（CM）是一种严重的中枢神经系统（CNS）感染，由真菌引起。 病原体新型隐球菌，主要发生在免疫系统受损的人群中。CM杀死181，000人 艾滋病患者的负担最重。它是艾滋病的第二大杀手 在撒哈拉以南非洲，仅次于肺结核。CM的治疗和诊断都是 疾病表现的变异性是复杂的。患者结局的变化 部分与C的基因型相关。新人类我们进行了初步的全基因组关联 研究（GWAS）对40个临床分离株的全基因组与相关的患者数据。GWAS揭示了一个 单核苷酸多态性（SNPs）和插入/缺失（INDEL）之间的关联。 新生儿基因和患者结局。我们删除了这些基因的一个子集，并表明它们在基因组中起着重要的作用。 以前未描述的毒性作用。此外，有一个记录的关联， C.结构变异新生儿基因组和患者结局。这项提案将检验这一假设 单个核苷酸变异会导致患者疾病表现的变化。目标1将确定 SNPs/INDEL在大群体C.通过使用扩增子测序， 使用长读段测序来表征临床分离株中的结构变异。我们将比较这些数据 以确定临床相关的基因组变异。目标2将决定 在生物学背景下通过交换毒性或无毒遗传学基因中的基因等位基因的核苷酸变异 背景，然后分析发病机制的变化。目标2将定义 通过对初步GWAS中鉴定的新基因进行表征， 分析.最后，我们将使用CRISPR将单核苷酸变异引入参考基因等位基因， 定义单个基因变化的影响。这项研究的创新之处在于，它是第一个 比较C.新生儿和患者结局的差异。拟议 研究将1）确定SNP/INDEL在C.新形虫表型; 2）鉴定未知毒力 3）建立一种诊断方法，用于鉴定感染性C.新型菌株，导致针对 治疗和提高患者生存率。

PROJECT SUMMARY/ABSTRACT Cryptococcal meningitis (CM) is a severe central nervous system (CNS) infection caused by the fungal pathogen Cryptococcus neoformans, primarily in people with compromised immune systems. CM kills 181,000 people annually, with the largest burden on AIDS patients. It is the second most common killer of AIDS patients in sub-Saharan Africa, behind only tuberculosis. Both the treatment and diagnosis of CM is complicated by a well-documented variability in disease presentation. Changes in patient outcome have been associated, in part, with the genotype of C. neoformans. We performed a preliminary genome wide association study (GWAS) on 40 whole genomes of clinical isolates with associated patient data. The GWAS revealed an association between single nucleotide polymorphisms (SNPs) and insertions/deletions (INDELs) in C. neoformans genes and patient outcome. We deleted a sub-set of these genes and showed they play a previously uncharacterized role in virulence. Furthermore, there is a documented association between structural variation in the C. neoformans genome and patient outcome. This proposal will test the hypothesis that individual nucleotide variations cause changes in patient disease presentation. Aim 1 will identify SNPs/INDELs in a large population of C. neoformans clinical isolates by using amplicon sequencing and will use long-read sequencing to characterize structural variants in the clinical isolates. We will compare these data to patient outcome to identify clinically relevant genomic variants. Aim 2 will determine the role of the nucleotide variations in a biological context by exchanging gene alleles in virulent or avirulent genetic backgrounds and then analyzing changes in pathogenesis. Aim 2 will define the biological function of the observed genomic differences through characterization of novel genes identified in the preliminary GWAS analysis. Finally, we will introduce single nucleotide variations into the reference gene alleles using CRISPR to define the impact of single genetic changes. The proposed research is innovative in that it is the first to compare single nucleotide polymorphisms in C. neoformans and differences in patient outcome. The proposed research will 1) determine the role of SNP/INDELs on C. neoformans phenotype; 2) identify unknown virulence factors; and 3) build a diagnostic method for identifying the infecting C. neoformans strain, leading to targeted therapy and improved patient survival.