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

真菌病原体对人类健康的影响是毁灭性的。它们感染了数十亿人 全世界每年都有150多万人死亡。最脆弱的是免疫力降低的人 功能，如艾滋病毒携带者或正在接受免疫抑制治疗的癌症或器官患者 移植。最普遍的真菌病原体之一是白色念珠菌，它导致近40%的人死亡 患有血液感染。治疗这些感染是极其困难的，因为真菌与 与人类有关，很少有药物可以在没有宿主毒性的情况下杀死这种真菌。随着……的出现 在抗药性方面，开发新的治疗策略现在至关重要。要解决这一重要问题 临床需求和确定新的抗真菌药物靶点，关键是要揭示使 白念珠菌引起危及生命的人类疾病。 我们是首批获得强大资源的学术实验室之一，这将使我们能够测试功能 几乎所有白念珠菌基因组的基因。此资源包括双条形码的集合 杂合突变体覆盖约90%的基因组，以及菌株集合覆盖约40%的基因组 当基因的剩余野生型等位基因的表达受四环素抗性基因控制时 推动者。这一资源为识别控制关键毒力的基因提供了前所未有的机会。 形态发生等特征。它还能够确定共生关系的决定因素和 毒力，并进一步阐明相关的分子机制。我们已经优化了一个功能 利用下一代测序对真菌毒力性状进行大规模并行分析的基因组学平台 量化每种条形码菌株在混合化验中的相对比例。我们还优化了高 细胞形态和结构的分辨率图像分析，以及识别重要基因的分析 共生性、毒性和与宿主免疫细胞的相互作用。我们的研究将提供第一个全球 白念珠菌形态发生、共生和毒力分析。 我们的研究将：1)开发一个预测白色念珠菌必需基因的计算平台，以及 扩大四环素耐受条件表达菌株的集合，以涵盖大多数非必需的 基因，因为病原菌在体外存活所需的基因几乎不能深入了解寄主的机制 适应或毒力；2)确定关键毒力性状的新调节因子，如形态发生；以及3) 在基因组水平上确定白念珠菌宿主适应和毒力的决定因素。这项工作将提供 扩展的功能基因组学资源，以推动该领域的发展，并将利用这一资源来 阐明控制形态发生和毒力的基因和遗传网络。这将揭示 削弱真菌病原体的新策略，以及改善临床结果的药物靶点。

The impact of fungal pathogens on human health is devastating. They infect billions of people worldwide, and kill more than 1.5 million each year. The most vulnerable are people with reduced immune function, such as those with HIV or those undergoing immune suppressing treatments for cancer or organ transplants. One of the most pervasive fungal pathogens is Candida albicans, which kills 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 resource that will allow us to test the function of almost every gene in the C. albicans genome. This resource includes a collection of double barcoded heterozygous mutants covering ~90% of the genome, and a collection of strains covering ~40% of the genome where the expression of the remaining wild-type allele of a gene is governed by the tetracycline-repressible promoter. This resource provides an unprecedented opportunity to identify genes that control key virulence traits such as morphogenesis. It also enables the identification of determinants of commensalism and virulence, and to further elucidate the molecular mechanisms involved. We have optimized a functional genomics platform for massively parallel analysis of fungal virulence traits using next generation sequencing to quantify the relative proportion of each barcoded strain in pooled assays. We have also optimized high- resolution image analysis of cellular morphology and structures, and assays for identifying genes important for commensalism, virulence, and interaction with host immune cells. Our studies will provide the first global analysis of C. albicans morphogenesis, commensalism, and virulence. Our studies will: 1) develop a computational platform to predict C. albicans essential genes, and expand the collection of tetracycline-repressible conditional expression strains to cover most non-essential genes, since genes required for pathogen viability in vitro provide little insight into mechanisms of 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. This work will provide an expanded functional genomics resource to advance the field, and will leverage this resource to elucidate the genes and genetic networks governing morphogenesis and virulence. This will reveal new strategies to cripple fungal pathogens, and drug targets to improve clinical outcome.