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

隐孢子虫是一种普遍存在的水生原生动物病原体，可引起世界范围内的腹泻疾病。自.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，正在研发的药物也很少， 确定新的可用药靶点是当务之急。隐孢子虫基因组中编码的是一个单一的 多酮合成酶，CpPKS1。聚酮合成酶，广泛存在于细菌、真菌、原生动物和植物中， 合成具有显著化学结构多样性的聚酮类次级代谢物 生物功能，可能为生产有机体提供了一些生存优势。CpPKS1为 在细胞内感染时上调，但它合成的分子，以及这个分子的功能， 仍然不为人所知。我们研究隐孢子虫聚酮的作用的方法始于 CpPKS1在曲霉中的异源表达，产生两种独特的代谢物。在此R21中 应用我们提出的优化CpPKS1在该系统中的表达，对其结构进行了表征 分子，并探讨其在隐孢子虫宿主-寄生虫相互作用中的作用。因为有许多生物 ，我们广泛地假设CpPK1在这两种寄生虫中都起着关键作用。 发展和/或宿主寄生虫的相互作用。我们将通过完成两个具体的 目标。 目的1：分离曲霉产生的隐孢子虫代谢物并确定其结构。 代谢物。在我们初步表达的cpPKS1中，可能的CpPK1代谢物是 浓度不够高，不能净化。在这里，我们将在nidulans的sms菌株中表达cpPKS1，以减少 内源性代谢物的干扰。代谢物将使用液体进行验证和提纯 色质联用和核磁共振光谱分析。 目的2：阻断CP聚酮代谢物的合成，并考察其缺失对 寄生虫发育与寄主和寄生虫转录本。在这些研究中，我们将抑制合成 使用新描述的条件性基因敲除系统对CpPK1进行基因敲除，并探索由此导致的寄生虫的变化 在支持寄生虫完整生命周期的有机系统中发育(2A)。主机和主机的更改 由于CpPK1缺失而导致的寄生虫转录组将通过RNAseq(2B)进行评估。 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。 探索隐孢子虫特有的一种分子的功能，这可能是寄生虫生物学的基础。 如果CpPK1被证明是寄生虫发育所必需的，未来的研究将检验其治疗的潜力。 合酶的抑制。如果该分子参与宿主和寄生虫的相互作用，RNAseq的研究将 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.