Thomas P. Wyche

Microbial Strain Prioritization Using Metabolomics Tools for the Discovery of Natural Products

Natural products profoundly impact many research areas, including medicine, organic chemistry, and cell biology. However, discovery of new natural products suffers from a lack of high throughput analytical techniques capable of identifying structural novelty in the face of a high degree of chemical redundancy. Methods to select bacterial strains for drug discovery have historically been based on phenotypic qualities or genetic differences and have not been based on laboratory production of secondary metabolites. Therefore, untargeted LC/MS-based secondary metabolomics was evaluated to rapidly and efficiently analyze marine-derived bacterial natural products using LC/MS-principal component analysis (PCA). A major goal of this work was to demonstrate that LC/MS-PCA was effective for strain prioritization in a drug discovery program. As proof of concept, we evaluated LC/MS-PCA for strain selection to support drug discovery, for the discovery of unique natural products, and for rapid assessment of regulation of natural product production.

Structure and biosynthesis of the antibiotic bottromycin D.

Drug resistant infectious diseases are quickly becoming a global health crisis. While Streptomyces spp. have been a major source of antibiotics over the past 50 years, efficient methods are needed to identify new antibiotics and greatly improve the rate of discovery. LCMS-based metabolomics were applied to analyze extracts of 50 Streptomyes spp. Using this methodology, we discovered bottromycin D and used whole genome sequencing to determine its biosynthesis by a ribosomal pathway.

Forazoline A: Marine-Derived Polyketide with Antifungal In Vivo Efficacy†

Forazoline A, a novel antifungal polyketide with in vivo efficacy against Candida albicans, was discovered using LCMS-based metabolomics to investigate marine-invertebrate-associated bacteria. Forazoline A had a highly unusual and unprecedented skeleton. Acquisition of (13)C-(13)C gCOSY and (13)C-(15)N HMQC NMR data provided the direct carbon-carbon and carbon-nitrogen connectivity, respectively. This approach represents the first example of determining direct (13)C-(15)N connectivity for a natural product. Using yeast chemical genomics, we propose that forazoline A operated through a new mechanism of action with a phenotypic outcome of disrupting membrane integrity.

Peptidolipins B–F, Antibacterial Lipopeptides from an Ascidian-Derived Nocardia sp.

A marine Nocardia sp. isolated from the ascidian Trididemnum orbiculatum was found to produce five new lipopeptides, peptidolipins B-F (1-5), which show distinct similarities to the previously reported L-Val(6) analog of peptidolipin NA. Synthetic modification of peptidolipin E (4) was used to determine the location of an olefin within the lipid chain. The advanced Marfeys method was used to determine the absolute configurations of the amino acids. Peptidolipins B (1) and E (4) demonstrated moderate antibacterial activity against methicillin-resistant Staphylococcus aureus and methicillin-sensitive Staphylococcus aureus.

Stereoselective Preparation of Cyclobutanes with Four Different Substituents: Total Synthesis and Structural Revision of Pipercyclobutanamide A and Piperchabamide G

A general strategy was developed for the diastereo- and enantioselective synthesis of cyclobutanes with four different substituents. It consists of three transition metal-catalyzed reactions — a RhII-catalyzed cyclopropanation, a AgI-catalyzed regioselective and stereospecific ring expansion, and a RhI-catalyzed addition reaction. Structures of pipercyclobutanamide A and piperchabamide G were synthesized and revised.

Species specificity of symbiosis and secondary metabolism in ascidians

Ascidians contain abundant, diverse secondary metabolites, which are thought to serve a defensive role and which have been applied to drug discovery. It is known that bacteria in symbiosis with ascidians produce several of these metabolites, but very little is known about factors governing these ‘chemical symbioses’. To examine this phenomenon across a wide geographical and species scale, we performed bacterial and chemical analyses of 32 different ascidians, mostly from the didemnid family from Florida, Southern California and a broad expanse of the tropical Pacific Ocean. Bacterial diversity analysis showed that ascidian microbiomes are highly diverse, and this diversity does not correlate with geographical location or latitude. Within a subset of species, ascidian microbiomes are also stable over time (R=−0.037, P-value=0.499). Ascidian microbiomes and metabolomes contain species-specific and location-specific components. Location-specific bacteria are found in low abundance in the ascidians and mostly represent strains that are widespread. Location-specific metabolites consist largely of lipids, which may reflect differences in water temperature. By contrast, species-specific bacteria are mostly abundant sequenced components of the microbiomes and include secondary metabolite producers as major components. Species-specific chemicals are dominated by secondary metabolites. Together with previous analyses that focused on single ascidian species or symbiont type, these results reveal fundamental properties of secondary metabolic symbiosis. Different ascidian species have established associations with many different bacterial symbionts, including those known to produce toxic chemicals. This implies a strong selection for this property and the independent origin of secondary metabolite-based associations in different ascidian species. The analysis here streamlines the connection of secondary metabolite to producing bacterium, enabling further biological and biotechnological studies.

Macrotermycins A–D, Glycosylated Macrolactams from a Termite-Associated Amycolatopsis sp. M39

Bioassay-guided metabolomic analyses led to the characterization of four new 20-membered glycosylated polyketide macrolactams, macrotermycins A-D, from a termite-associated actinomycete, Amycolatopsis sp. M39. M39s sequenced genome revealed the macrotermycins putative biosynthetic gene cluster. Macrotermycins A and C had antibacterial activity against human-pathogenic Staphylococcus aureus and, of greater ecological relevance, they also had selective antifungal activity against a fungal parasite of the termite fungal garden.

Solwaric acids A and B, antibacterial aromatic acids from a marine Solwaraspora sp.

Two novel trialkyl-substituted aromatic acids, solwaric acids A and B, were isolated from a marine Solwaraspora sp. cultivated from the ascidian Trididemnum orbiculatum. Solwaric acids A and B were isotopically labeled with U-13C glucose, and analysis of a 13C–13C COSY allowed for unambiguous determination of the location of the phenyl methyl group. The two novel compounds demonstrated antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA).

Selective quantification by 2D HSQC0 spectroscopy of thiocoraline in an extract from a sponge-derived Verrucosispora sp.

We recently developed a 2D 1H-13C HSQC0 approach to quantify individual chemicals in complex mixtures. The HSQC0 approach has been implemented in phase-cycled and gradient-selective versions. As in quantitative 1D NMR, the normalized integrated signal intensities in HSQC0 are proportional to the concentrations of individual chemicals in the mixture. We applied the HSQC0 approach to selectively quantify thiocoraline present at a level of 1% w/w in an extract from a Verrucosispora sp. isolated from the sponge Chondrilla caribensis f. caribensis. We expect that this approach can be used to quantify other natural products of interest in extracts without prior purification.

Host control of symbiont natural product chemistry in cryptic populations of the tunicate Lissoclinum patella.

Natural products (secondary metabolites) found in marine invertebrates are often thought to be produced by resident symbiotic bacteria, and these products appear to play a major role in the symbiotic interaction of bacteria and their hosts. In these animals, there is extensive variation, both in chemistry and in the symbiotic bacteria that produce them. Here, we sought to answer the question of what factors underlie chemical variation in the ocean. As a model, we investigated the colonial tunicate Lissoclinum patella because of its rich and varied chemistry and its broad geographic range. We sequenced mitochondrial cytochrome c oxidase 1 (COXI) genes, and found that animals classified as L. patella fall into three phylogenetic groups that may encompass several cryptic species. The presence of individual natural products followed the phylogenetic relationship of the host animals, even though the compounds are produced by symbiotic bacteria that do not follow host phylogeny. In sum, we show that cryptic populations of animals underlie the observed chemical diversity, suggesting that the host controls selection for particular secondary metabolite pathways. These results imply novel approaches to obtain chemical diversity from the oceans, and also demonstrate that the diversity of marine natural products may be greatly impacted by cryptic local extinctions.