Integrated metabolomic and microbial community analysis of estuarine sediments and their potential as sources of bioactive natural products

ABSTRACT Estuaries are dynamic environments in which the variability of abiotic and biotic factors support highly diverse microbial communities in sediments, including seagrass rhizospheres. A high likelihood of chemically mediated interactions in these complex, competitive communities makes estuaries an exciting potential source of new biologically active compounds. New pharmaceuticals are needed to combat drug resistance in the treatment of infectious and chronic diseases. Compounds targeting the protein secretory pathway at the endoplasmic reticulum (ER) display potent anticancer, antiviral, and antibacterial activities. Within this Sec61 translocon - mediated pathway, protein chaperone GRP78 is emerging as a potential druggable target with inhibitors showing efficacy against drug resistant cancers and viral and bacterial infection. Environmental sampling of sediments provides access to metabolites from complex microbial communities, for investigating both their role in the environment and potential biomedical relevance. Solid phase adsorbent resin samplers have been used extensively to monitor toxins from harmful algal blooms and have recently begun to be applied to the detection of natural products in marine sediments. The central hypothesis of this project is that microbial communities in estuarine sediments are valuable sources of novel bioactive NPs, with potential for the treatment and management of human disease, that may be accessed by in situ environmental sampling using adsorption resins. Sediments in eelgrass beds and nearby unvegetated areas across an estuarine gradient in Oregon will be sampled both by deploying adsorbent resin samplers and collecting small sediment scoops (Aim 1). The adsorbent resin samplers facilitate direct sampling of sediment metabolites from the environment for chemical and biological characterization. The sediment scoops enable parallel 16S rRNA microbial community analysis and comparison of metabolites obtained by solvent extraction of sediment versus adsorbent resin. High resolution LC-MS/MS and computational tools will be used to identify trends in metabolite production at sampling sites with and without eelgrass and support biological assays (Aim 2) in prioritization of samples for further investigation of new bioactive natural products. In Aim 2, a high-throughput magnetic microbead affinity selection screening assay (MagMASS) will be used to identify ligands of GRP78 from the complex mixtures in sediment extracts. When possible, hits identified in this assay will be verified using a cellular thermal shift assay to test GRP78 binding in cells. Extracts will additionally be screened against a panel of bacterial and fungal pathogens and bioactive components of complex mixtures will be predicted using computational methods (NPAnalyst). In Aim 3, laboratory culturing of stabilized microbial communities from estuarine sediments and monocultures of putative natural product producers will assist in linking natural products to their microbial producers and facilitate resupply of prioritized natural products for comprehensive chemical characterization and advanced biological testing.