Cryptosporidium's polyketide secondary metabolite: exogenous production, compound characterization and function in intracellular development.

隐孢子虫是一种广泛存在于水中的原生动物病原体，可引起世界范围内的疟疾。以来 目前既没有疫苗也没有有效的治疗方法来治疗这种疾病，而且很少有药物在研制中， 必须确定新的药物靶点。在隐孢子虫基因组中编码的是一个 聚酮合酶，CpPKS 1。聚酮酶广泛存在于细菌、真菌、原生动物和植物中， 合成聚酮化合物次级代谢物，其显示出显著多样性化学结构， 生物学功能，可能为生产生物体提供一些生存优势。CpPKS 1是 在细胞内感染过程中上调，但它合成的分子，以及这个分子的功能， 仍然未知。我们研究隐孢子虫聚酮化合物作用的方法始于 cpPKS 1在曲霉中的异源表达产生两种独特的代谢产物。在R21中 应用，我们建议优化CpPKS 1在该系统中的表达，表征该系统的结构， 分子，并探讨其在隐孢子虫宿主-寄生虫相互作用中的功能。由于许多生物 活性的聚酮化合物，我们广泛假设，CpPK 1起着关键作用，无论是寄生虫 发展和/或宿主寄生虫相互作用。我们将通过完成两个具体的测试来验证这一假设。 目标。 目的1：分离曲霉隐孢子虫代谢产物，并对其结构进行鉴定 代谢物。我们在A.假定的CpPK 1代谢产物是 浓度不足以提纯在这里，我们将在A. nidulans减少 内源性代谢物的干扰。将使用液体验证和纯化代谢物 色谱-质谱法和NMR光谱法。 目的2：消除Cp聚酮代谢物的合成，并检查其缺失对 寄生虫发育以及宿主和寄生虫转录组。在这些研究中，我们将抑制 使用一种新描述的条件性敲除系统的CpPK 1，并探讨由此产生的变化，寄生虫 在支持完整寄生虫生命周期的类器官系统中发育（2A）。主机和 将通过RNAseq评估由于CpPK 1缺失而导致的寄生虫转录组（2B）。 这些研究采用了寄生虫学和天然产物化学领域的高度创新技术 探索隐孢子虫独特分子的功能，这可能是寄生虫生物学的基础。 如果CpPK 1被证明是寄生虫发育所必需的，未来的研究将检查治疗的潜力。 合成酶的抑制。如果该分子参与宿主和寄生虫的相互作用，RNAseq研究将 为有针对性的调查提供初步数据。

Cryptosporidium is a ubiquitous water-born protozoal pathogen that causes diarrheal disease world-wide. Since there are neither vaccines nor effective therapeutics to treat this disease, and very few drugs in the pipeline, identification of new druggable targets is imperative. Encoded within the Cryptosporidium genome is a single polyketide synthase, CpPKS1. Polyketide synthases, found widely in bacteria, fungi, protozoa, and plants, synthesize polyketide secondary metabolites that exhibit a remarkable diversity of chemical structures and biologic functions, presumably providing the producing organism with some survival advantage. CpPKS1 is upregulated during intracellular infection but the molecule it synthesizes, and the function of this molecule, remain unknown. Our approach to investigating the role of the Cryptosporidium polyketide began with heterologous expression of cpPKS1 in Aspergillus which produced two unique metabolites. In this R21 application we propose to optimize expression of CpPKS1 in this system, characterize the structure of this molecule and explore its function in Cryptosporidium host-parasite interactions. Because of the many biological activities possessed by polyketides, we broadly hypothesize that CpPK1 plays a critical role in either parasite development and/or host parasite interactions. We will test this hypothesis through the completion of two specific aims. Aim 1: Isolate the Cryptosporidium metabolite produced in Aspergillus and elucidate the structure of the metabolite. In our preliminary expression of cpPKS1 in A. nidulans the putative CpPK1 metabolites were not in high enough concentration to purify. Here we will express cpPKS1 in SMs- strains of A. nidulans to reduce interference from endogenous metabolites. Metabolites will be validated and purified using liquid chromatography-mass spectrometry and NMR spectroscopy. Aim 2: To ablate synthesis of the Cp polyketide metabolite and examine the effects of its absence on parasite development and host and parasite transcriptomes. In these studies, we will inhibit the synthesis of CpPK1 using a newly described conditional knockout system and explore the resulting changes to parasite development in an organoid system that supports the complete parasite life cycle (2A). Changes in host and parasite transcriptome due to the absence of CpPK1 will be evaluated by RNAseq (2B). These studies employ highly innovative techniques from the fields of parasitology and natural product chemistry to explore the function of a molecule unique to Cryptosporidium that could be fundamental to parasite biology. Should CpPK1 prove essential for parasite development, future studies will examine the potential for therapeutic inhibition of the synthase. If the molecule is involved in host and parasite interactions, the RNAseq studies will provide preliminary data for targeted investigations.