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方法， 感染人类的物种的增加，以及新谱系、物种和耐药性的出现， 治疗的挑战。这项建议将审查两个物种造成最大数量的机会主义 侵袭性真菌病和第三种代表地方性二型真菌病的主要原因。我们将 比较每种病原体的临床分离株，以确定高毒力或抗真菌药物的遗传基础。 耐药菌株，并区分如何分离，造成侵入性感染的不同，从那些低毒力 或者是健康菌群的一部分。在第一个目标，我们将研究发病机制的遗传基础， 新型隐球菌，免疫功能低下的个人感染的主要原因，通过结合大规模的表型，全基因组测序，和自然分离物的转录分析。我们将 描述自然分离株在肺部感染和传播过程中体内生长的差异 中枢神经系统，并将使用这些措施进行全基因组关联屏幕的 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 菌株为了突出分离株之间基因调控的变化，并查明高表达的基因 在不同的感染阶段，我们将进行真菌感染的体内阶段和关键细胞的RNA-Seq， 交互.我们还将研究这种病原体在宿主感染过程中的微进化，并将其与 在动物模型中观察到的变化。在第二个目标中，我们将讨论一个关于 血液念珠菌感染，这是免疫功能低下患者死亡的主要原因。我们将 检查肠道和皮肤中念珠菌分离株的宿主内多样性， 来追踪这些血流感染的来源。我们将适应新的丰富 增加宏基因组样品中念珠菌读数比例的方法。最后，我们将测试分离物 更经常与血流来源相关，以确认它们在动物中的相对毒力 模型在目标3中，我们将检查从患者中收集的马尔尼菲篮状菌（青霉菌）分离株， 最近的临床试验比较两种抗真菌治疗。这代表了临床分离株的最大集合 收集用于地方性真菌病的患者元数据的临床试验。我们将使用全基因组和RNA-Seq分析来定义与耐药性和高死亡率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，以检查真菌的发病机制，每个利用大规模的 组学方法，以扩展和开发新的范式，用于研究和理解真菌的毒力， 人口自然变化的背景。这项工作将提供一个前所未有的全基因组视图， 与临床样本相关的病原体，其研究结果的综合将提出新的方法， 对个体患者的诊断和治疗以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.