Functional Analysis of Natural Variation in the Pathogen Candida albicans

真菌白色念珠菌引起多种感染，发病率和死亡率都很高。侵入性感染- 流感每年在美国造成10,000人死亡，全球估计每年有400,000人死亡。这个 由于许多原因，生物体仍然是一种威胁，包括抗真菌设施的有限范围， 耐药性的发生，生物体作为生物膜生长的能力，以及不同的毒力阵列 使其能够感染几乎任何组织的决定因素。 我们对白色念珠菌感染机制的认识主要来源于一株临床分离株SC5314， 以及它的衍生品。一个长期的知识差距是从SC5314分析得出的结论的程度 可推广到其他临床分离株。我们已经通过以下方式在初步结果中解决了这个问题 对另外四株临床分离株中控制生物膜和菌丝产生的基因进行了功能分析。 憎恨四个主要的系统发育支系。我们的发现表明，简单删除井-的影响- 所研究的基因在不同的白色念珠菌菌株中差异很大。我们寻求将我们的工作扩展到一个更大的小组 菌株，将我们的重点扩展到包括宿主相互作用表型，并利用我们的发现进行广泛的开发 利用自然变异的适用的基因发现策略。在我们的第一个目标中，我们将解决两个关键问题 问题：首先，白色念珠菌分支内的监管网络架构是否像分支之间那样多样化？ 第二，监管网络的物种范围的共同要素是否丰富了与功能相关的目标 基因?我们将扩大我们的初步研究，以包括另外15个代表主要 克拉兹。我们将使用生物学表型来评估突变的影响的一致性，以及基因ex-2。 PRESS分析对基因调控网络的变异进行评价。物种范围分析的实用性 将与EFG1基因一起进行详细测试；我们将定义与菌株无关的目标基因并评估目标 通过生物被膜和菌丝产生的分析来实现基因功能。在我们的第二个目标中，我们将观察生物膜- 从菌丝调节器的毒力角度来确定突变是否以及如何产生不同的影响。 TIONS延伸到致病性。我们将通过EX分析宿主相互作用的参数和感染轨迹 活体和活体分析。在我们的第三个目标中，我们将实施新的互补-克隆战略，以识别- 确定精选临床分离株独特变异表型的因果突变。我们将使用基于克隆库的 白念珠菌本身的互补性。 这些研究的总体结果将为研究与致病相关的关键基因功能提供新的视角。 白念珠菌跨越自然菌株多样性的范围。这将有助于优先考虑途径和基因产物 潜在的治疗靶点，因为它们对白色念珠菌菌株的影响一致很强。这项工作还将 提供了两种广泛适用的策略，菌株独立网络分析和基于白色念珠菌的联合 实现克隆，为解决白色念珠菌研究中的多个问题提供了新的途径 致命性。

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.