Ascaroside Signaling in the Bacterivorous Nematode Caenorhabditis remanei Encodes the Growth Phase of Its Bacterial Food Source Franziska Dolke, †,# Chuanfu Dong, †,#,⊥ Siva Bandi, ‡ Christian Paetz, § Gae ́ tan Glauser, ∥ and Stephan H. von Reuß* ,†,‡,∥ † Department of Bioorganic Chemistry, Max Planck Institute for Chemical Ecology, Hans-Knö ll Straße 8, D-07745 Jena, Germany ‡ Laboratory for Bioanalytical Chemistry, Institute of Chemistry, University of Neuchâ tel, Avenue de Bellevaux 51, CH-2000 Neuchâ tel, Switzerland § Research Group Biosynthesis/NMR, Max Planck Institute for Chemical Ecology, Hans-Knö ll Straße 8, D-07745 Jena, Germany ∥ Neuchâ tel Platform for Analytical Chemistry (NPAC), University of Neuchâ tel, Avenue de Bellevaux 51, CH-2000 Neuchâ tel, Switzerland * S Supporting Information ABSTRACT: A novel class of species-specific modular ascaro- sides that integrate additional fatty acid building blocks was characterized in the nematode Caenorhabditis remanei using a combination of HPLC−ESI−(−)-MS/MS precursor ion scanning, microreactions, HR-MS/MS, MS n , and NMR techniques. The structure of the dominating component carrying a cyclopropyl fatty acid moiety was established by total synthesis. Biogenesis of this female-produced male attractant depends on cyclopropyl fatty acid synthase (cfa), which is expressed in bacteria upon entering their stationary phase. I n their natural habitat of decaying plant material, bacterivorous nematodes such as the model organism Caenorhabditis elegans or its close relative Caenorhabditis remanei depend on their microbiome to convert environmental resources into readily accessible biomass. The majority of their associated bacteria support nematode propagation, but a few are detrimental. 1 Bacterial composition affects nematode popula- tion growth, lifespan, development, metabolism, foraging behavior, and pathogen resistance. 2 Nematodes exhibit preference for beneficial bacteria, but the underlying molecular mechanisms have remained largely unknown. 3 Recent advances in analytical techniques facilitated the characterization of nematode ascarosides, 4 a modular glycolipid library based on the 3,6-dideoxysugar L-ascarylose, which serve as key regulators in nematode chemical ecology. 5 De novo ascaroside biosyn- thesis depends on the co-option of a primary metabolic pathway, the peroxisomal β-oxidation cycle of fatty acid metabolism, to furnish a large diversity of homologous aglycones from very long chain precursors. 4a,6 The resulting short-chain ascarosides are highly conserved in nematodes 7 and further serve as scaffolds for species-specific attachment of additional building blocks from diverse primary metabolic pathways. Some of these building blocks appear to originate from microbial metabolism (Figure 1a) such as the L-tryptophan-derived indole-3-carboxylate unit of indole ascarosides (1, IC-asc-C#; SMID: 8 icas), a group of potent attraction and aggregation signals in the Caenorhabditis 9 or the p-aminobenzoic acid unit of C. elegans’ male-attracting asc-ΔC7-PABA (2, SMID: 8 ascr#8). 10 Ascaroside biosynthesis thus appears to integrate the metabolic state of the producing organism along with environmental factors including bacterial food availability and composition, but this has not yet been experimentally demonstrated. Here, we show that species-specific biogenesis of a potent male-attracting ascaroside signal by females of the gonochoristic C. remanei depends on a bacterial food derived cyclopropyl fatty acid building block (Figure 1), the production of which is initiated upon reaching the stationary growth phase. Comprehensive ascaroside profiling of the C. remanei exometabolome using HPLC−ESI−(−)-MS/MS precursor ion scanning for m/z 73.0 [C 3 H 5 O 2 ] 4a revealed common simple ascarosides along with several yet undescribed modular components (Figure 2a) including a novel class of fatty acid ascarosides (SMID: 8 fasc). ESI-(−)-HR-MS/MS analysis of the dominating component, fasc#1(3), indicated the molecular C 21 H 36 O 8 and suggested the presence of an asc-C4 unit with an uncommon even numbered C4 aglycone along with a hydroxylated C11 fatty acid building block (Figure 2b). Approximately 100 μg of fasc#1(3) was isolated from 1.5 L of the C. remanei culture supernatant using a combination of RP- C18 solid-phase extraction and semipreparative HPLC. Its modular structure including an asc-C4 building block and a 7- Received: June 3, 2019 Letter pubs.acs.org/OrgLett Cite This: Org. Lett. XXXX, XXX, XXX-XXX © XXXX American Chemical Society A DOI: 10.1021/acs.orglett.9b01914 Org. Lett. XXXX, XXX, XXX−XXX Downloaded via NOTTINGHAM TRENT UNIV on July 15, 2019 at 22:15:11 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.