Systematic Analysis of Morphogenesis, Commensalism, and Virulence in a Leading Human Fungal Pathogen

摘要/摘要 真菌病原体对人类健康构成毁灭性威胁，感染全球数十亿人， 每年造成150多万人死亡。白色念珠菌是最普遍的真菌病原体之一， 导致近40%的血液感染患者死亡。治疗这些感染是极其困难的， 因为真菌与人类关系密切，很少有药物可以在没有宿主毒性的情况下杀死真菌。使用 耐药性的出现，开发新的治疗策略现在至关重要。致信地址 这一重要的临床需求和确定新的抗真菌药物靶点，对于揭示其作用机制至关重要 使白色念珠菌能够引起危及生命的人类疾病。 我们是首批获得强大功能基因组资源的学术实验室之一 独一无二的定位，可以扩展，使我们能够测试白念珠菌基因组中几乎每一个基因的功能。 该资源包括一组条件表达菌株，覆盖约40%的基因组 目标基因的等位基因在二倍体病原体中缺失，剩余的野生型等位基因的表达是 由四环素可抑制的启动子控制。在之前的资助期内，我们开发了一条管道，以 将资源扩展到基因组规模，优化了大规模并行分析的功能基因组学平台 使用下一代测序和混合分析来量化真菌毒力性状的相对比例 每个菌株，都用分子条形码唯一标记；优化的高分辨率图像分析 细胞形态和结构；并开发出鉴定对共生关系重要的基因的方法， 毒力以及与宿主免疫细胞的相互作用。我们高效的高吞吐量分析确定了 系统的基因分析，以发现新的生物学见解，而这些见解是无法基于 当前的范例，并实现了对管理宿主的关键机制的聚焦、假设驱动的剖析- 病原体相互作用。我们的研究将首次对白色念珠菌的形态发生进行全球分析， 共生性和毒性，并将揭示基本的生物机制 在没有系统遗传方法的情况下预测的。 我们的研究将：1)完成四环素耐药条件表达菌株的收集，以 涵盖非必需基因，因为病原体在体外存活所需的基因对宿主的了解很少 适应或毒力；2)确定关键毒力性状的新调节因素，如形态发生；以及3)确定 白念珠菌的决定因素在基因组范围内具有适应性和毒力。我们的综合品系 将向社区提供表型资料的资源和简编，推动这一领域的发展 具有可公开访问的数据库和解释性机器学习模型，以最大限度地提高洞察力。这项工作将 为任何真菌病原体提供最全面的功能基因组学资源，并将揭示 控制宿主适应的基因，揭示了削弱真菌病原体的新策略。

SUMMARY/ABSTRACT Fungal pathogens pose a devastating threat to human health, infecting billions of people worldwide and causing more than 1.5 million deaths each year. Candida albicans is one of the most pervasive fungal pathogens, killing almost 40% of people suffering from bloodstream infections. Treating these infections is extremely difficult, as fungi are closely related to humans and there are very few drugs that kill the fungus without host toxicity. With the emergence of drug resistance, the development of new therapeutic strategies is now crucial. To address this important clinical need and identify new antifungal drug targets, it is critical to uncover mechanisms that enable C. albicans to cause life-threatening human disease. We are one of the first academic labs to obtain a powerful functional genomics resource that we are uniquely positioned to expand to allow us to test the function of almost every gene in the C. albicans genome. This resource includes a collection of conditional expression strains that covers ~40% of the genome where one allele of a target gene is deleted in the diploid pathogen, and expression of the remaining wild-type allele is governed by the tetracycline-repressible promoter. During the prior funding period we: developed a pipeline to expand the resource to genome scale, optimized a functional genomics platform for massively parallel analysis of fungal virulence traits using next generation sequencing with pooled assays to quantify the relative proportion of each strain, which are uniquely marked with molecular barcodes; optimized high-resolution image analysis of cellular morphology and structures; and developed assays for identifying genes important for commensalism, virulence, and interaction with host immune cells. Our efficient high-throughput analyses established the power of systematic genetic analysis to uncover new biological insights that could not have been predicted based on current paradigms and enabled focused, hypothesis-driven dissection of key mechanisms governing host- pathogen interactions. Our studies will provide the first global analysis of C. albicans morphogenesis, commensalism, and virulence, and will reveal fundamental biological mechanisms that could not be predicted without a systematic genetic approach. Our studies will: 1) complete the collection of tetracycline-repressible conditional expression strains to cover non-essential genes, since genes required for pathogen viability in vitro provide little insight into host adaptation or virulence; 2) identify novel regulators of key virulence traits such as morphogenesis; and 3) identify determinants of C. albicans host adaptation and virulence on a genome scale. Our comprehensive strain resources and compendium of phenotypic profiles will be made available to the community, advancing the field with a publicly accessible database and interpretive machine learning model to maximize insight. This work will provide the most comprehensive functional genomics resource for any fungal pathogen and will reveal genes governing host adaptation, revealing new strategies to cripple fungal pathogens.