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

总结/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， 每年造成超过150万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。治疗这些感染非常困难， 因为真菌与人类密切相关，并且很少有药物在没有宿主毒性的情况下杀死真菌。与 随着耐药性的出现，开发新的治疗策略现在至关重要。解决 这一重要的临床需求和确定新的抗真菌药物靶点，揭示机制至关重要 使C.白色念珠菌导致危及生命的人类疾病。 我们是最早获得强大功能基因组学资源的学术实验室之一， 独特的定位，以扩大，使我们能够测试几乎每一个基因的功能，在C。白色念珠菌基因组。 该资源包括条件表达菌株的集合，其覆盖约40%的基因组，其中一个 靶基因的等位基因在二倍体病原体中缺失，并且剩余的野生型等位基因的表达被 由四环素阻遏型启动子控制。在上一个融资期间，我们：开发了一个管道， 将资源扩展到基因组规模，优化功能基因组学平台以进行大规模并行分析 使用下一代测序和合并测定来定量相对比例， 每种菌株，其中唯一标记的分子条形码;优化的高分辨率图像分析， 细胞形态和结构;并开发了用于鉴定对细胞分裂症重要的基因的测定法， 毒力和与宿主免疫细胞的相互作用。我们高效的高通量分析确立了 系统的遗传分析，以揭示新的生物学见解，无法预测的基础上， 当前的范例和使重点，假设驱动的解剖的关键机制，管理主机， 病原体相互作用我们的研究将提供第一个全球性的C。白色念珠菌形态发生， 以及毒性，并将揭示基本的生物学机制， 没有系统的遗传学方法预测。 我们的研究将：1）完成四环素抑制性条件表达菌株的收集， 涵盖非必需基因，因为病原体体外存活所需的基因几乎不能深入了解宿主 适应性或毒力; 2）鉴定关键毒力性状如形态发生的新调节因子;和3）鉴定 C的决定因素白念珠菌宿主适应性和毒力的基因组规模。我们的综合菌株 将向社区提供资源和表型概况纲要， 利用可公开访问的数据库和解释性机器学习模型来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.