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

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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.