Natural products from a human-asssociated fungal pathogen

说明(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地或水生生物中分离出来的。相比之下，进化成与人类密切相关的微生物尤其有可能合成抑制关键人类靶标的天然产物，并作为新分子的来源仍然未得到充分开发。我们提出了一种系统的方法来鉴定真菌病原体组织胞浆菌的天然产物，组织胞浆菌是一种土壤有机体，大部分时间在哺乳动物的肺中度过。在识别与新的人类靶标结合的新天然产品方面，组织胞浆是一个特别令人信服的选择，原因如下：(1)它已经进化到生活在人类免疫细胞内。虽然其他真菌能够定植免疫受损的宿主，但很少有真菌能够定植免疫活性宿主(这是一项更困难的任务，表明囊状螺旋藻可以操纵正常的先天免疫反应)。(2)组织胞浆的规模化培养可直接用于天然产物的分离。(3)组织胞浆分子遗传学允许直接构建缺少假定的生物合成酶的突变体。(4)尽管对基因组的分析预测，每个组织胞浆体菌株产生10-20种天然产物，但无论是组织胞浆体还是其近亲，都没有通过学术或工业天然产物的发现工作来挖掘新的分子。综上所述，由于组织原体是一种细胞内病原体，通过未知的机制颠覆先天免疫反应以定植宿主免疫细胞，我们假设组织原体天然产物可能富含调节哺乳动物宿主靶标的新化合物。我们将结合SIL实验室在组织胞浆生物学和分子遗传学方面的专业知识与Fischbach实验室在天然产品发现方面的专业知识，全面鉴定和表征组织胞浆分泌的天然产品。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，在概括其自由生活和宿主相关环境的实验室条件下大量培养组织胞浆。利用Fischbach实验室的标准专业知识，我们将通过制备性高效液相色谱法提纯4-6毫克量丰富的天然产物，并使用高分辨率MS和核磁共振来解析它们的结构。(2)利用sIL实验室的专业知识，我们将采取互补的遗传方法，利用RNA干扰来耗尽单个天然产物生物合成基因的组织浆细胞。来自Fischbach实验室的预测算法以及突变菌株上清液中的高效液相色谱/质谱图的结果变化，都将使我们能够将特定的分子与其生物合成基因相关联。(3)我们将进行一系列体外和体内试验，以确定本项目确定的天然产物的生物活性，重点是鉴定与疾病发病机制有关的生物合成基因和天然产物。这些对自然产物生物学的探索性研究在一种无处不在的人类相关微生物中具有很强的潜力，可以发现调节宿主-病原体界面通信的新分子。

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.