Causes and Consequences of Hypermutability in Cryptococcus neoformans

摘要 为了在它们的环境利基中成功地生存和竞争，微生物必须 随机获得突变，否则将面临进化停滞。尽管突变率的增加通常是 在多细胞生物中有害的，超突变可以在强突变的背景下对微生物有利 选择性压力。为了探索超突变是如何在自然界中产生的并阐明其后果，我们 使用了最近组装的387个已测序的隐球菌临床和环境分离株的集合 新生杆菌是一种真菌病原体，每年约占艾滋病相关死亡人数的15%。HIV阳性 被诊断为隐球菌性脑膜炎的人面临着令人无法接受的高死亡率：低至70% 收入国家和北美分别为30%和30%。如此高的死亡率是由于缺乏抗真菌治疗。 选择如此有限，因为许多基本真菌和人类蛋白质之间的保守同源性，以及 对抗真菌药物的高耐药率。各菌株获得致病能力的初步筛选 对其他致死浓度的多种抗真菌药物的抗药性已经鉴定出30株超突变菌株， 包括两个健壮的超级变种人。对这两个分离株产生的抗性菌落进行了鉴定 单一转座元件(TE)的插入在很大程度上导致了从头耐药。长- Read全基因组测序(WGS)显示，两个变异体基因组都编码了600个拷贝的这个 并在已知参与TE沉默的RNAi组件的第一个外显子中存在无义突变， ZNF3.超变异体间遗传杂交F1分离株的数量性状基因座定位 实验室的参考菌株发现了一个与超突变相关的显著峰 包括突变的znf3等位基因。因此，我们的中心假设是由于频繁的 这些分离株中的转座可归因于新生葡萄球菌中存在一种新的、具有功能的TE 血统以及RNAi缺陷。为了确定这种高位转位的遗传和分子基础 并确定其对这些菌株的适合性和它们在宿主相关的环境中获得抗药性的能力的影响 条件下，我们提出了两个具体目标。在目标1中，遗传互补、缺失和重组将 用来确定ZNF3和已鉴定的TE在高突变中的作用。其他分离株的WGS分析 在收集中将进行鉴定抑制子突变并表征进化轨迹 已鉴定的超变种等位基因。在目标2中，我们将确定这些增加的突变率和 通过竞争分析、Etest和体内试验，转座在体外有助于适合性和耐药性 在梅隆氏菌和小鼠感染模型中。这种强大的真核生物模型的结合 生物体在实验室的广泛历史，致病性质，以及成熟的性周期以及 这种完全测序的不同分离株集合的可用性代表着一个独特的机会来确定 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.