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

项目摘要/摘要 隐球菌性脑膜炎是由真菌引起的一种严重的中枢神经系统感染 病原体新生隐球菌，主要存在于免疫系统受损的人群中。CM杀死181,000人 每年都有人，艾滋病患者负担最大。它是第二个最常见的艾滋病杀手 撒哈拉以南非洲的患者，仅次于结核病。CM的治疗和诊断都是 更复杂的是，疾病表现的可变性有很好的记录。患者结局的变化一直是 在一定程度上与新生葡萄球菌的基因有关。我们进行了初步的全基因组关联 对40株临床分离株的全基因组及其相关患者数据进行研究(Gwas)。GWAS揭示了一种 单核苷酸多态(SNPs)和插入/缺失(Indels)与C. 新生杆菌基因与患者预后。我们删除了这些基因的一个子集，并表明它们发挥着 在毒力方面以前未被定性的角色。此外，有证据表明， 新生葡萄球菌基因组的结构变异与患者预后。这项提议将检验这一假设 个体核苷酸变异会导致患者疾病表现的变化。目标1将确定 用扩增序列分析和Will方法检测大批新生葡萄球菌临床分离株中的SNPs/Indels 使用长阅读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 以确定临床上相关的基因组变异。目标2将决定 通过在致病或致病基因中交换基因等位基因在生物学背景下的核苷酸变异 研究背景并分析其发病机制的变化。目标2将定义细胞的生物学功能 通过对初步确定的新基因的鉴定来观察基因组差异 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中，以 定义单一基因变化的影响。这项拟议的研究具有创新性，因为它是第一个 比较新生葡萄球菌的单核苷酸多态和患者预后的差异。建议数 研究将1)确定SNP/indels对新生葡萄球菌表型的作用；2)确定未知的毒力 因素；以及3)建立一种诊断方法来识别感染新生葡萄球菌菌株，从而导致靶向 治疗和提高患者存活率。

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.