Functional Analysis of Natural Variation in the Pathogen Candida albicans

真菌白色念珠菌引起多种感染，发病率和死亡率很高。侵入性感染- 在美国，这些疾病每年造成10，000人死亡，在全世界，估计每年有400，000人死亡。的 微生物仍然是一个威胁，原因有很多，包括抗真菌医疗设备的范围有限， 耐药性的发生，生物体作为生物膜生长的能力，以及毒力的多样性 这些决定因素使其能够感染几乎任何组织。 我们对C.白色念珠菌感染机制主要来自一种临床分离株，菌株SC 5314， 及其衍生物。一个长期的知识差距是从SC 5314分析得出的结论在多大程度上 可以推广到其他临床分离株。我们在初步结果中讨论了这个问题， 在另外四个临床分离株中，对控制生物膜和菌丝产生的基因进行了功能测定， 怨恨四个主要的系统进化分支。我们的研究结果表明，简单删除的影响，以及- 所研究的基因在C.白色念珠菌菌株。我们寻求将我们的工作扩展到更大的小组， 菌株，扩大我们的重点，包括主机相互作用表型，并利用我们的研究结果， 利用自然变异的基因发现策略。在我们的第一个目标，我们将解决两个关键 问题：第一，监管网络架构在C.白色念珠菌的分支之间是什么样的？ 第二，是一个调节网络的物种范围内的共同元素丰富的功能相关的目标， 基因？我们将扩大我们的初步研究，包括15个额外的临床分离株，代表主要的 进化枝我们将使用生物学表型来评估突变影响的一致性，基因表达， 表达测定以评估基因调控网络变异。物种范围分析的效用 将用EFG 1基因进行详细测试;我们将定义菌株非依赖性靶基因，并评估靶基因。 基因功能通过生物膜和菌丝生产的测定。在我们的第二个目标中，我们将研究生物膜- 菌丝调节因子从毒力的角度，以确定是否和如何不同的影响穆塔- 延伸到致病性。我们将分析参数的主机相互作用和感染轨迹通过ex 体内和体内分析。在我们的第三个目标，我们将实施一个新的补充克隆战略，以确定- 从选定的临床分离株中确定独特变异表型的因果突变。我们将使用基于克隆库的 C.白色本身 这些研究的总体结果将为C. 白色念珠菌跨越自然菌株多样性的范围。它将有助于优先考虑途径和基因产物， 潜在的治疗靶点，因为它们对C.白色念珠菌菌株。该工作还将 提供了两种广泛适用的策略，应变无关网络分析和C。白色的COM- 完成克隆，为解决C.白色 毒性

The fungus Candida albicans causes diverse infections with substantial morbidity and mortality. Invasive infec- tions cause 10,000 deaths per year in the US, and an estimated 400,000 deaths per year worldwide. The organism remains a threat for many reasons, including the limited scope of the antifungal armamentarium, the occurrence of drug resistance, the ability of the organism to grow as biofilm, and the diverse array of virulence determinants that enable it to infect almost any tissue. Our understanding of C. albicans infection mechanisms comes mainly from one clinical isolate, strain SC5314, and its derivatives. One chronic knowledge gap is the extent to which conclusions from analysis of SC5314 can be generalized to other clinical isolates. We have addressed this question in preliminary results through functional assays of genes that govern production of biofilm and hyphae in four additional clinical isolates, rep- resenting four major phylogenetic clades. Our findings indicate that the impact of simple deletions of well- studied genes is highly variable among C. albicans strains. We seek to extend our work to a larger panel of strains, to extend our focus to include host interaction phenotypes, and to use our findings to develop broadly applicable gene discovery strategies that exploit natural variation. In our first aim, we will address two key questions: First, is regulatory network architecture as diverse within C. albicans clades as it is between clades? Second, are species-wide common elements of a regulatory network enriched for functionally relevant target genes? We will extend our preliminary studies to include 15 additional clinical isolates that represent the major clades. We will use biological phenotypes to assess the uniformity of impact of the mutations, and gene ex- pression assays to make an appraisal of gene regulatory network variation. The utility of species-wide analysis will be tested in detail with the EFG1 gene; we will define strain-independent target genes and assess target gene function through assays of biofilm and hypha production. In our second aim, we will look at the biofilm- hyphal regulators from the perspective of virulence to determine whether and how the diverse impact of muta- tions extends to pathogenicity. We will analyze parameters of host interaction and infection trajectory via ex vivo and in vivo analyses. In our third aim, we will implement a new complementation-cloning strategy to iden- tify causal mutations for unique variant phenotypes from select clinical isolates. We will use clone library-based complementation in C. albicans itself. The overall results of these studies will provide a new view of key virulence-associated gene functions in C. albicans that spans the range of natural strain diversity. It will help to prioritize pathways and gene products as potential therapeutic targets due to their uniformly strong impact across C. albicans strains. The work will also provide two broadly applicable strategies, strain-independent network analysis and C. albicans-based com- plementation cloning, that yield new avenues to address multiple questions in the study of C. albicans virulence.