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

隐孢子虫的聚酮化合物次生代谢物：外源产生、化合物表征和细胞内发育中的功能。

• 批准号: 10657338
• 负责人: NANCY P KELLER
• 金额: $ 23.54万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10657338
• 关键词: Ablation; Air; Aspergillus; Aspergillus fumigatus; Aspergillus nidulans; Bacteria; Biological; Biological Process; Biology; Cell Wall; Cells; Chemical Structure; Codon Nucleotides; Cryptosporidium; Data; Development; Dinophyceae; Disease; Exhibits; Funding Mechanisms; Future; Genes; Genome; Genus Mycobacterium; Infection; Investigation; Joints; Life Cycle Stages; Liquid substance; Mass Spectrum Analysis; Mus; NMR Spectroscopy; Natural Products Chemistry; Organism; Organoids; Parasites; Parasitology; Pharmaceutical Preparations; Phylogenetic Analysis; Plants; Play; Production; Protozoa; Role; Structure; Support System; System; Techniques; Testing; Therapeutic; Toxin; Vaccines; Virulence Factors; antimicrobial; conditional knockout; diarrheal disease; fungus; high risk; immunoregulation; innovation; liquid chromatography mass spectrometry; new therapeutic target; pathogen; pharmacologic; polyketide synthase; polyketides; response; secondary metabolite; small molecule; therapeutically effective; transcriptome; transcriptome sequencing; waterborne

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.

隐孢子虫是一种普遍存在的水生原虫病原体，可在世界范围内引起腹泻病。自从 既没有疫苗也没有有效的疗法来治疗这种疾病，正在研发的药物也很少， 确定新的药物靶点势在必行。隐孢子虫基因组中编码的是单个 聚酮合酶，CpPKS1。聚酮合酶广泛存在于细菌、真菌、原生动物和植物中， 合成具有显着化学结构多样性的聚酮化合物次级代谢产物 生物功能，大概为生产生物体提供了一些生存优势。 CpPKS1 是 在细胞内感染期间上调，但它合成的分子以及该分子的功能， 仍然未知。我们研究隐孢子虫聚酮化合物的作用的方法始于 cpPKS1 在曲霉中的异源表达，产生两种独特的代谢物。在这个R21 我们建议在该系统中优化 CpPKS1 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 聚酮化合物具有的活性，我们广泛假设 CpPK1 在这两种寄生虫中都发挥着关键作用 发育和/或宿主寄生虫相互作用。我们将通过完成两个特定的任务来检验这个假设 目标。 目标 1：分离曲霉中产生的隐孢子虫代谢物并阐明其结构 代谢物。在我们对构巢曲霉中 cpPKS1 的初步表达中，推定的 CpPK1 代谢物是 浓度不够高，无法纯化。在这里，我们将在构巢曲霉的 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纯化 色谱-质谱法和核磁共振波谱法。 目标 2：消除 Cp 聚酮化合物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，我们将抑制合成 使用新描述的条件敲除系统对 CpPK1 进行修饰，并探索由此产生的寄生虫变化 支持完整寄生虫生命周期的类器官系统的开发（2A）。主机的变化和 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分子的功能，这可能是寄生虫生物学的基础。 如果 CpPK1 被证明对寄生虫发育至关重要，未来的研究将探讨其治疗潜力 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。