Project 3 Population studies of major human fungal pathogens: genomic and transcriptomic analyses of interactions with the host and microbiome and the rise of antifungal resistance

真菌病原体对人类健康有重大影响；缺乏有效的抗真菌治疗，种类繁多 在感染人类的物种中，新血统的出现、物种和抗药性是主要的 对治疗的挑战。这项提案将研究两种导致最大数量的机会主义的物种 侵袭性霉菌病和第三种代表地方性二型性霉菌病的主要原因。我们会 比较每种病原体的临床分离株，以确定高毒力或抗真菌的遗传基础 耐药菌株，并区分引起侵袭性感染的菌株与低毒力菌株的区别 或者是健康真菌菌群的一部分。在第一个目标中，我们将研究阿尔茨海默病发病的遗传学基础。 通过结合大规模表型、全基因组测序和自然分离株的转录分析，新生隐球菌是免疫受损个体感染的主要原因。我们会 描述自然分离株在肺部感染期间体内生长和传播到 并将利用这些措施进行全基因组关联筛查 病原体；同时，我们将测量超过4,000个隐球菌基因缺失集合的增长 菌株。突出分离株之间基因调控的变化，并精确定位高表达基因 在不同的感染阶段，我们将进行体内阶段和参与真菌的关键细胞的RNA-Seq 互动。我们还将研究这种病原体在宿主感染期间的微观进化，并将其与 在动物模型中观察到的变化。在第二个目标中，我们将解决一个主要的问题，即 血液念珠菌感染，这是免疫功能低下患者死亡的主要原因。我们会 检测肠道和皮肤共生部位的念珠菌和分离株的宿主内多样性 从念珠菌血症患者身上追踪这些血液感染的来源。我们将适应新的浓缩 提高后基因组样本中念珠菌读数比例的方法。最后，我们将测试分离物 它们更多地与血液来源有关，以确认它们在动物中的相对毒性 模特。在目标3中，我们将检测从患者身上收集的马尔尼菲青霉菌分离株。 最近的临床试验比较了两种抗真菌疗法。这是最大的临床分离株集合。 为临床试验收集的关于地方性真菌病的患者元数据。我们将使用全基因组和RNA-Seq分析来确定与高死亡率的耐药性和侵袭性感染相关的特性。 虽然这三个目标代表了独立的项目来检查真菌的发病机制，但每个目标都利用了大规模的 ‘组学方法，以扩展和开发研究和理解真菌毒力的新范式 自然种群变异的背景。这项工作将提供史无前例的全基因组视角 与临床样本相关的病原体，以及对其发现的综合，将提出新的治疗方法 对个别患者的诊断和治疗及危险因素进行前瞻性监测。

Fungal pathogens have a major impact on human health; the lack of effective antifungal therapies, the diversity of species infecting humans, and the emergence of new lineages, species and drug resistance represent major challenges to treatment. This proposal will examine two species causing the largest number of opportunistic invasive mycoses and a third species representing a major cause of endemic dimorphic mycoses. We will compare clinical isolates from each pathogen to determine the genetic basis of highly virulent or antifungal resistant isolates, and to discriminate how isolates that cause invasive infections differ from those of low virulence or that are part of a healthy mycoflora. In the first aim, we will study the genetic basis of pathogenesis of Cryptococcus neoformans, a major cause of infection in immunocompromised individuals, by combining large-scale phenotyping, whole genome sequencing, and transcriptional analysis of natural isolates. We will characterize how natural isolates differ in in vivo growth during pulmonary infection and dissemination to the central nervous system and will use these measures to carry out genome-wide association screens of the pathogen; in parallel, we will measure the growth of the Cryptococcus gene deletion collection of over 4,000 strains. To highlight changes in gene regulation between isolates and pinpoint genes that are highly expressed at different infection stages, we will carry out RNA-Seq of in vivo stages and key cells involved in fungal interactions. We will also examine the microevolution of this pathogen during host infection and compare that to changes observed in an animal model. In the second aim, we will address a major question about the origin of bloodstream Candida infections, which are a major cause of mortality in immunocompromised patients. We will examine the intra-host diversity of Candida isolates between commensal sites in the gut and the skin and isolates from patients with candidemia to trace the origin of these bloodstream infections. We will adapt new enrichment approaches to increase the proportion of Candida reads in metagenomic samples. Lastly, we will test isolates that are more frequently associated with the bloodstream origin to confirm their relative virulence in an animal model. In aim 3, we will examine isolates of Talaromyces (Penicillium) marneffei collected from patients in a recent clinical trial comparing two antifungal treatments. This represents the largest collection of clinical isolates collected for a clinical trial with patient metadata for an endemic mycosis. We will use whole genome and RNA-Seq analyses to define properties associated with drug resistance and aggressive infections with high mortality. While these three aims represent independent projects to examine fungal pathogenesis, each utilizes large-scale ‘omics approaches to extend and develop new paradigms for studying and understanding fungal virulence in the context of natural population variation. This work will provide an unprecedented whole-genome view into pathogens associated with clinical samples, and a synthesis of its findings will suggest new approaches for diagnosis and treatment for individual patients and risk factors to monitor prospectively.