Causes and Consequences of Hypermutability in Cryptococcus neoformans

新型隐球菌高度突变的原因和后果

• 批准号: 10170252
• 负责人: Shelby Jordan Priest
• 金额: $ 3.48万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2022-05-08
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10170252
• 关键词: Accounting; Acquired Immunodeficiency Syndrome; Affect; Alleles; Animal Model; Antifungal Agents; Biological Assay; Cessation of life; Clinical; Collection; Complement; Complex; Cryptococcal Meningitis; Cryptococcosis; Cryptococcus; Cryptococcus neoformans; DNA Transposable Elements; Defect; Diagnosis; Disadvantaged; Drug Targeting; Drug resistance; Elements; Engineering; Etiology; Evolution; Exhibits; Exons; Face; Fungal Drug Resistance; Genes; Genetic; Genetic Crosses; Genetic Determinism; Genome; Genome Stability; Genomic Instability; Genotype; Geographic Distribution; Goals; Growth; HIV Seropositivity; Health; Human; Immunocompromised Host; In Vitro; Individual; Infection; Inhalation; Laboratories; Liquid substance; Low income; Measures; Microbe; Minimum Inhibitory Concentration measurement; Modeling; Molecular; Mus; Mutation; Nature; Nonsense Mutation; North America; Organism; Pathogenesis; Pathogenicity; Patients; Pharmacotherapy; Phenotype; Phylogenetic Analysis; Population; Prevalence; Production; Proteins; Public Health; Quantitative Trait Loci; RNA Interference; RNA library; Recording of previous events; Resistance; Retrotransposon; Role; Selfish Genes; Sequence Analysis; Small Interfering RNA; Small RNA; Source; Suppressor Mutations; System; Testing; Transcriptional Silencer Elements; Virulence; base; beneficial microorganism; clinically relevant; drug sensitivity; fitness; genome integrity; genome sequencing; immunosuppressed; in vivo; insight; microorganism; mortality; mutant; new therapeutic target; novel; pathogen; pathogenic fungus; pressure; reconstitution; screening; treatment strategy; whole genome

ABSTRACT To successfully survive and compete within their environmental niches, microorganisms must stochastically acquire mutations or face evolutionary stagnation. Although increased mutation rates are often deleterious in multicellular organisms, hypermutation can be beneficial for microbes in the context of a strong selective pressure. To explore how hypermutation arises in nature and elucidate its consequences, we employed a recently assembled collection of 387 sequenced clinical and environmental isolates of Cryptococcus neoformans, a fungal pathogen responsible for approximately 15% of AIDS-related deaths annually. HIV-positive individuals diagnosed with cryptococcal meningitis face unacceptably high mortality rates: up to 70% in low income nations and 30% in North America. This high mortality is attributable to a dearth of antifungal treatment options, so limited because of the conserved homology between many essential fungal and human proteins, and to the high rates of resistance to antifungal drugs. Preliminary screening for the ability of each isolate to acquire resistance to otherwise lethal concentrations of diverse antifungal agents has identified 30 hypermutator strains, including two robust hypermutators. Characterization of the resistant colonies the two isolates produced revealed that insertion of a single transposable element (TE) was largely responsible for de novo drug resistance. Long- read whole genome sequencing (WGS) revealed that both hypermutator genomes encode >600 copies of this TE and harbor a nonsense mutation in the first exon of an RNAi component known to be involved in TE silencing, ZNF3. Quantitative trait loci mapping of F1 segregants from a genetic cross between one of the hypermutators and the laboratory reference strain identified a single significant peak associated with hypermutation that includes the mutant znf3 allele. Therefore, our central hypothesis is that hypermutability due to frequent transposition in these isolates is attributable to the presence of a novel, functional TE in the C. neoformans lineage as well as an RNAi defect. To determine the genetic and molecular basis of this elevated transposition and define its impact on the fitness of these strains and their ability to acquire drug resistance in host-relevant conditions, we propose two specific aims. In aim 1, genetic complementation, deletion, and reconstitution will be used to define the roles of ZNF3 and the identified TE in hypermutation. Analysis of WGS of other isolates in the collection will be conducted to identify suppressor mutations and characterize the evolutionary trajectory of the identified hypermutator alleles. In aim 2, we will determine how these increased mutation rates and transposition contribute to fitness and drug resistance in vitro through competition assays and Etests and in vivo in Galleria mellonella and murine infection models. The combination of this powerful eukaryotic model organism’s extensive history in the lab, pathogenic nature, and well-established sexual cycle along with the availability of this diverse collection of fully sequenced isolates represents a unique opportunity to determine the genetic sources of hypermutation and its phenotypic consequences.

抽象的 为了在其环境生态位中成功生存和竞争，微生物必须 尽管突变率经常增加，但随机获得突变或面临进化停滞。 超突变对多细胞生物体有害，但在强突变的情况下可能对微生物有益。 为了探索自然界中超突变是如何发生并阐明其后果，我们 采用最近收集的 387 个已测序的隐球菌临床和环境分离株 新型隐球菌是一种真菌病原体，每年导致约 15% 的艾滋病毒阳性死亡。 被诊断患有隐球菌性脑膜炎的个体面临着令人难以接受的高死亡率：在低水平地区高达 70% 收入国家和北美的 30% 是由于抗真菌治疗导致的死亡。 由于许多重要的真菌和人类蛋白质之间保守的同源性，选择如此有限，并且 初步筛选每个分离株获得抗真菌药物的高耐药率。 对不同致死浓度的不同抗真菌药物的耐药性已鉴定出 30 种超突变菌株， 包括两个强大的超变子，这两个分离株产生的抗性菌落的特征揭示了。 单个转座元件（TE）的插入在很大程度上导致了新的耐药性。 读取全基因组测序（WGS）显示，两个超变基因组都编码超过 600 个拷贝 TE 并在已知参与 TE 沉默的 RNAi 成分的第一个外显子中存在无义突变， ZNF3。来自其中一个超变体之间遗传杂交的 F1 分离子的数量性状基因座作图。 实验室参考菌株识别出与超突变相关的单个显着峰 包括突变的 znf3 等位基因，因此，我们的中心假设是由于频繁发生的超突变。 这些分离株中的转座归因于新型隐球菌中存在新型功能性 TE 谱系以及 RNAi 缺陷以确定这种升高的转座的遗传和分子基础。 并确定其对这些菌株的适应性及其在宿主相关性中获得耐药性的能力的影响 在目标1中，我们提出了两个具体目标，即遗传互补、删除和重建。 用于定义 ZNF3 和已识别的 TE 在其他分离株的全基因组测序分析中的作用。 将在收集中识别抑制突变并表征进化轨迹 在目标 2 中，我们将确定这些增加的突变率和变化。 通过竞争测定和 Etests 以及体内的转座有助于体外适应性和耐药性 这种强大的真核模型的结合。 该生物体在实验室中的广泛历史、致病性质、以及完善的性周期以及 这种完全测序的分离株的多样性集合的可用性代表了确定 超突变的遗传来源及其表型后果。