Natural products from a human-asssociated fungal pathogen

描述（由申请人提供）：天然产品通常是从与人类生物学无关的自由生活的陆生或水生生物中分离出来的。相比之下，已经进化到与人类密切相关的微生物特别有可能合成抑制关键人类靶点的天然产物，并且作为新分子的来源仍然未得到充分探索。我们提出了一个系统的方法来确定天然产物的真菌病原体组织胞浆菌，这是一种土壤生物，花了很多时间在哺乳动物的肺。在鉴定结合新的人类靶点的新的天然产物方面，组织胞浆菌是一个特别引人注目的选择，原因如下：（1）它已经进化到生活在人类免疫细胞内。虽然其他真菌能够在免疫功能低下的宿主中定植，但很少有真菌能够在免疫功能正常的宿主中定植（这是一项更困难的任务，表明H。荚膜可以操纵正常的先天免疫应答）。(2)直接培养组织胞浆菌的生产规模培养物用于天然产物分离。(3)组织胞浆菌分子遗传学允许直接构建缺乏推定的生物合成酶的突变体。(4)虽然基因组分析预测每株组织胞浆菌产生10-20种天然产物，但学术界或工业界的天然产物发现工作从未挖掘过组织胞浆菌或其任何近亲的新分子。总之，由于组织胞浆菌是一种细胞内病原体，其通过未知的机制破坏先天免疫应答以定殖宿主免疫细胞，因此我们假设组织胞浆菌天然产物可能富含调节哺乳动物宿主中靶点的新型化合物。我们将联合收割机结合Sil实验室在组织胞浆菌生物学和分子遗传学方面的专业知识和Fischbach实验室在天然产物发现方面的专业知识，全面鉴定和表征组织胞浆菌分泌的天然产物。我们提出了一个三管齐下的方法：（1）基于我们的初步数据，我们将采取微生物方法，在实验室条件下培养大量的组织胞浆菌，重现其自由生活和宿主相关的环境。利用Fischbach实验室的标准专业知识，我们将通过制备型HPLC纯化毫克量的4-6种丰富的天然产物，并使用高分辨率MS和NMR解析其结构。(2)利用Sil实验室的专业知识，我们将采取互补的遗传方法，通过使用RNA干扰来消除单个天然产物生物合成基因的组织浆细胞。来自Fischbach实验室的预测算法和突变菌株上清液提取物中HPLC/MS图谱的变化将使我们能够将特定分子与其生物合成基因相关联。(3)我们将执行一系列体外和体内试验，以确定本项目鉴定的天然产物的生物活性，重点是鉴定有助于疾病发病机制的生物合成基因和天然产物。这些探索性研究的天然产物生物学在一个无处不在的，人类相关的微生物有很大的潜力，发现新的分子，调节通信的主机-病原体接口。

DESCRIPTION (provided by applicant): Natural products are usually isolated from free-living terrestrial or aquatic organisms that have no connection with human biology. In contrast, microbes that have evolved to live in close association with humans are especially likely to synthesize natural products that inhibit key human targets, and remain underexplored as a source of novel molecules. We propose a systematic approach to identify natural products from the fungal pathogen Histoplasma capsulatum, which is a soil organism that spends much of its time in the mammalian lung. In terms of identifying new natural products that bind novel human targets, Histoplasma is a particularly compelling choice for the following reasons: (1) It has evolved to live within human immune cells. Although other fungi are capable of colonizing immunocompromised hosts, very few can colonize immunocompetent hosts (a more difficult task that indicates that H. capsulatum can manipulate the normal innate immune response). (2) It is straightforward to grow production-scale cultures of Histoplasma for natural product isolation. (3) Histoplasma molecular genetics allows the straightforward construction of mutants that lack putative biosynthetic enzymes. (4) Although analysis of the genome predicts that each strain of Histoplasma produces 10-20 natural products, neither Histoplasma nor any of its close relatives have ever been mined for novel molecules by an academic or industrial natural product discovery effort. In sum, since Histoplasma is an intracellular pathogen that subverts the innate immune response to colonize host immune cells by unknown mechanisms, we hypothesize that Histoplasma natural products may be enriched for novel compounds that modulate targets in mammalian hosts. We will combine the expertise of the Sil lab in Histoplasma biology and molecular genetics with the expertise of the Fischbach lab in natural product discovery to comprehensively identify and characterize natural products secreted by Histoplasma. We propose a three-pronged approach: (1) Based on our preliminary data, we will take a microbial approach, growing large quantities of Histoplasma under laboratory conditions that recapitulate its free-living and host-associated environments. Utilizing standard expertise in the Fischbach lab, we will purify milligram quantities of 4-6 abundant natural products by preparative HPLC, and use high-resolution MS and NMR to solve their structures. (2) Taking advantage of expertise in the Sil lab, we will take a complementary genetic approach by using RNA interference to deplete Histoplasma cells of individual natural product biosynthetic genes. Both predictive algorithms from the Fischbach lab and the resultant changes in the HPLC/MS profile in supernatant extracts from the mutant strains will allow us to correlate particular molecules with their biosynthetic genes. (3) We will execute a series of in vitro and in vivo assays to determine the bioactivity of natural products identified by this project, with an emphasis on the identification of biosynthetic genes and natural products that contribute to disease pathogenesis. These exploratory studies of natural product biology in a ubiquitous, human-associated microbe have strong potential for uncovering new molecules that modulate communication at the host-pathogen interface.