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病原体，引起极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，使得吸入孢子在肺部生长和传播。治疗和诊断都有问题，这种情况导致极高的死亡率。我们的目标是在小鼠模型感染期间使用烟曲霉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考虑基因的功能分析，然后通过构建新的突变株和确定其毒力潜力来验证数据的实用性。这些基因产物反过来将成为治疗和诊断发展的优先目标。我们将使用nanoString nCounter来量化感染组织中特定的烟曲霉RNA水平。纳米链对RNA水平的估计比微阵列估计或当前RNA- seq能力要敏感得多。我们提出的研究重点是三类基因产物：转录因子，表面和分泌蛋白，次生代谢产物生物合成酶。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联系起来。我们之所以选择表面和分泌蛋白基因，是因为它们的产物是最容易获得的治疗靶点，而且它们可能释放到液体中，使它们成为候选的诊断靶点。我们选择次级代谢物基因是因为它们的产物可能具有生物活性，如免疫调节，这与发病机制有关。我们将开发一个参考数据集来探索这些基因的表达，并通过两个特定的目标扩展功能验证分析。首先，我们将使用小鼠模型和两个测序的烟曲霉菌株来确定烟曲霉基因在肺部感染中的表达。其次，我们将通过选择突变株的毒力测定来验证从表达数据得出的功能推断。我们的发现将通过提供一套影响感染的调节途径、表面/分泌产物和毒素，显著提高对烟曲霉感染的理解。这些信息对于优先考虑途径和基因产物作为治疗和诊断开发的目标，以及将基础研究与与感染高度相关的基因产物联系起来至关重要。

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.