GENE EXPRESSION AND FUNCTION IN ASPERGILLOSIS

描述(申请人提供)：烟曲霉是免疫抑制患者的主要侵袭性霉菌，导致异常高的发病率和死亡率。由于造血干细胞移植或实体器官移植导致的防御能力减弱，导致吸入的孢子在肺部生长和传播。治疗和诊断都是有问题的，这种情况导致了极高的死亡率。我们的目标是在小鼠模型中利用烟曲霉菌在感染过程中的基因表达来确定毒力基因。我们将根据体内表达和宿主反应优先进行基因功能分析，然后通过构建新的突变株和测定其毒力潜力来验证数据的实用性。这些基因产品反过来将成为治疗和诊断开发的高度优先目标。我们将使用纳米串计数器来量化感染组织中特定的烟曲霉菌RNA水平。纳米串对RNA水平的估计比微阵列估计或当前的RNA-Seq能力敏感得多。我们的研究集中在三类基因产物：转录因子、表面和分泌蛋白以及次生代谢物生物合成酶。我们之所以选择转录因子基因，是因为它们的产物可以通过表达谱和染色质免疫沉淀与靶基因和生物学功能相连。我们之所以选择表面和分泌蛋白基因，是因为它们的产品提供了最容易获得的治疗靶点，也因为它们可能释放到液体中使它们成为候选的诊断靶点。我们之所以选择次生代谢物基因，是因为它们的产物可能具有与发病相关的生物活性，如免疫调节。我们将开发一个探索这些基因表达的参考数据集，并通过两个特定的目标通过功能验证来扩展分析。首先，我们将使用一个小鼠模型和两个测序的烟曲霉菌株来确定肺部感染期间烟曲霉的基因表达。其次，我们将通过选择突变菌株的毒力分析来验证来自表达数据的功能推断。我们的发现将提供一套影响感染的调节途径、表面/分泌产物和毒素，从而显著提高对烟曲霉菌感染的理解。这些信息对于确定作为治疗和诊断开发目标的途径和基因产品的优先顺序以及将基础研究与与感染高度相关的基因产品联系起来至关重要。

DESCRIPTION (provided by applicant): Aspergillus fumigatus is the major invasive mold pathogen of immunosuppressed patients, causing exceptionally high morbidity and mortality. Weakened defenses as a consequence of hematopoietic stem cell transplantation or solid organ transplantation allow growth of inhaled spores in the lung and dissemination. Both therapy and diagnosis are problematic, a situation that contributes to extremely high mortality rates. Our objective is to use A. fumigatus gene expression during infection in a murine model to define virulence genes. We will prioritize genes for functional analysis based on in vivo expression and host response, and then validate the utility of the data through construction of new mutant strains and determination of their virulence potential. These gene products in turn will be high priority targets for therapeutic and diagnostic development. We will use a nanoString nCounter to quantify specific A. fumigatus RNA levels in infected tissue. NanoString estimates of RNA levels are much more sensitive than microarray estimates or current RNA-Seq capability. Our proposed studies focus on three classes of gene products: transcription factors, surface and secreted proteins, and secondary metabolite biosynthetic enzymes. We have chosen transcription factor genes because their products can be connected to target genes and biological functions through expression profiling and chromatin immunoprecipitation. We have chosen surface and secreted protein genes because their products present the most accessible therapeutic targets, and because their possible release into fluids makes them candidate diagnostic targets. We have chosen secondary metabolite genes because their products may have biological activity, such as immunomodulation, that is relevant to pathogenesis. We will develop a reference dataset that explores expression of these genes, and extend the analysis with functional validation through two specific aims. First, we will define A. fumigatus gene expression during lung infection, using a murine model and two sequenced A. fumigatus strains. Second, we will validate functional inferences from expression data through virulence assays of select mutant strains. Our findings will significantly improve the understanding of A. fumigatus infection by providing a set of regulatory pathways, surface/secreted products, and toxins that impact infection. The information will be critical for prioritizing pathways and gene products as targets for therapeutic and diagnostic development, and to connect basic research studies to gene products that are highly relevant to infection.