Causes and Consequences of Hypermutability in Cryptococcus neoformans

摘要 为了成功地在环境中生存和竞争，微生物必须 随机获得突变或面临进化停滞。虽然突变率的增加往往 超突变在多细胞生物体中是有害的，但在强超突变的背景下，超突变对微生物是有益的。 选择性压力为了探索自然界中超突变是如何产生的，并阐明其后果，我们 使用了最近收集的387个已测序的隐球菌临床和环境分离株 新生儿是一种真菌病原体，每年造成约15%的艾滋病相关死亡。hiv阳性 被诊断患有隐球菌性脑膜炎的人面临着不可接受的高死亡率：低死亡率高达70%。 收入国家和北美的30%。这种高死亡率是由于缺乏抗真菌治疗 选择，由于许多必需的真菌和人类蛋白质之间的保守同源性而受到限制， 抗真菌药物的高耐药性。初步筛选每个分离株获得 对其它致死浓度的不同抗真菌剂的抗性已经鉴定出30种超变菌株， 包括两个强大的超变子两个分离株产生的抗性菌落的特征显示， 单个转座因子（TE）的插入是导致新生耐药性的主要原因。长- 读取全基因组测序（WGS）显示，两个超变基因组编码>600个拷贝的这种 TE，并且在已知参与TE沉默的RNAi组分的第一外显子中具有无义突变， ZNF 3。一个超突变体与F1分离体的数量性状基因座定位 实验室参考菌株鉴定出与超突变相关的单个显著峰， 包括突变的ZnF 3等位基因。因此，我们的中心假设是，由于频繁的 这些分离株中的转座归因于C.新生 以及RNAi缺陷。为了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疾病中获得耐药性的能力的影响。 在这种情况下，我们提出了两个具体目标。在目标1中，遗传互补、缺失和重建将 用于确定ZNF 3和已鉴定的TE在超突变中的作用。其他分离株的WGS分析 将进行收集，以确定抑制突变和特征的进化轨迹 超变等位基因在目标2中，我们将确定这些增加的突变率， 转座通过竞争测定和Etest在体外和体内有助于适应性和耐药性 在Galleria mellonella和鼠感染模型中。这个强大的真核生物模型 微生物在实验室中的广泛历史，致病性，以及沿着的良好的性周期， 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。

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.