Frank C. Schroeder - US grants

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Small molecules form the basis of communication, defense, and behavior in many organisms. We have elucidated several of the chemical cues in the nematode Caenorhabditis elegans that control behavior, including dauer formation and mating. These signaling molecules add to the wealth of biological data already established for C. elegans, providing the key components to learning more about nematode ecology and behavior. Nematodes are the most abundant animals on earth, occupy virtually every ecological niche, and thus provide an outstanding opportunity for comparative studies of animal behavior. This information is important to human health, because billions of people and large numbers of crops in the world are infected with parasitic nematodes.Our current studies focus on two groups of small molecules, the ascarosides and a family of steroidal bile acids, which serve important functions in behavior and development of nematodes. Central to the proposed research is the use of new NMR-spectroscopic methodology (DANS) that permits the analysis of complex small molecule mixtures and greatly accelerates both the structure elucidation process and the functional characterization of the detected compounds.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Small molecules form the basis of communication, defense, and behavior in many organisms. We have elucidated several of the chemical cues in the nematode Caenorhabditis elegans that control behavior, including dauer formation and mating. These signaling molecules add to the wealth of biological data already established for C. elegans, providing the key components to learning more about nematode ecology and behavior. Nematodes are the most abundant animals on earth, occupy virtually every ecological niche, and thus provide an outstanding opportunity for comparative studies of animal behavior. This information is important to human health, because billions of people and large numbers of crops in the world are infected with parasitic nematodes.Our current studies focus on two groups of small molecules, the ascarosides and a family of steroidal bile acids, which serve important functions in behavior and development of nematodes. Central to the proposed research is the use of new NMR-spectroscopic methodology (DANS) that permits the analysis of complex small molecule mixtures and greatly accelerates both the structure elucidation process and the functional characterization of the detected compounds.

DESCRIPTION (provided by applicant): The widespread dependence on antibiotics in medicine and agriculture have resulted in the continued emergence of antibiotic resistance, raising the specter of the end of the antibiotic era. This crisis has underscored the need for the identification of new antibiotics and anti-infective strategies. Our research proposal focuses on the exploration of an untapped potential reservoir of antimicrobials-compounds that we hypothesize nematodes employ as chemical defense against bacteria and fungi, in habitats characterized by highly complex microbiomes, including compost soil, rotting fruit, and decomposing insect carcasses. Our proposal brings together the highly complementary expertise from the field of Caenorhabditis elegans innate immunity and host-microbe interactions (D.K.), and from the field of C. elegans metabolomics and small- molecule signaling (F.S.). We will focus on metabolomic analysis of the nematode species C. elegans and Pristionchus pacificus, each of which feed on bacteria and are exposed to a wide variety of non-pathogenic and pathogenic bacteria and fungi in their natural environments, but which also exhibit differences in susceptibility to bacteria. Our recent studies of the metabolomes of these nematodes have identified several novel classes of small molecules of yet undetermined function that have chemical structures suggestive of interactions with bacteria. We propose to develop nematode metabolite libraries that are enriched for compounds produced by these nematode hosts in response to pathogenic bacteria and fungi, including the human pathogens Pseudomonas aeruginosa and Staphylococcus aureus. We will then screen these small molecule metabolite libraries for antimicrobial activity, taking advantage of established pathogenesis assays that follow microbial proliferation and survival of the nematode host, with subsequent definitive identification of active compounds via comparative metabolomics, and chemical synthesis. We anticipate finding compounds that directly affect bacterial and fungal viability as well as metabolites that function through the modulation of microbial colonization and virulence mechanisms. Our project has the potential to identify new classes of antimicrobial compounds and potentially novel anti-infective mechanisms that may help stem the tide of antibiotic resistant organisms.

SUMMARY Individuals can respond to diverse nutrients and dietary restrictions in markedly different ways. Some people easily gain weight, but others remain thin no matter what they eat. Additionally, metabolic diseases can differ dramatically among individuals in a population, for both rare single-gene Mendelian diseases and common multifactorial metabolic diseases such as obesity and type 2 diabetes. In large part, this variability suggests that individual genetic differences greatly affect the likelihood to get sick as well as the severity of the illness for both rare and common metabolic diseases across a population. It would be extremely valuable if one could identify both rare and common variants that contribute to individual responses to diet and to the acquisition of different types of metabolic diseases. Rare variants are usually identified by linkage mapping and whole- genome sequencing using families with affected individuals. By contrast, common variants are usually identified by genome-wide association studies using large populations of people with and without a disease. We will develop personalized metabolic network models for a large set of genetic individuals of the nematode C. elegans, both representing healthy metabolic state and mimicking an inborn error of human metabolism. With our experimental system and approach we will be able to derive predictions of both rare and common variation in a variety of metabolic traits influenced by nutrition. We will extensively validate such predictions using CRISPR/Cas9-mediated genome editing.

Overall: Our project combines the significant advantages of a genetic model organism, sophisticated pathway mapping tools, high-throughput and accurate quantum chemistry (QM), and state-of-the-art experimental measurements. The result will be an efficient and cost-effective approach for unknown compound identification in metabolomics, which is one of the major limitations facing this growing field of medical science. Caenorhabditis elegans has several advantages for this study, including over 10,000 available genetic mutants, well-developed CRISPR/Cas9 technology, and a panel of over 500 wild C. elegans isolates with complete genomes. Half of C. elegans genes have homologs to human disease genes, making this model organism an outstanding choice to improve our understanding of metabolic pathways in human disease. We will develop an automated pipeline for sample preparation to reproducibly measure tens of thousands of unknown features by UHPLC-MS/MS. We will use the wild isolates to conduct metabolome-wide genetic association studies (m-GWAS), and SEM-path to locate unknowns in pathways using partial correlations. The relevance of the unknown metabolites to specific pathways will be tested by measuring UHPLC-MS/MS data from genetic mutants of those pathways. Molecular formula and pathway information will be the inputs for automated quantum mechanical calculations of all possible structures, which will be used to accurately calculate NMR chemical shifts that will be matched to experimental data. The correct structures will be validated by comparing them with 2D NMR data of the same compound. The validated computed structures will then be used to improve QM-based MS/MS fragment prediction, using the experimental UHPLC-MS/MS data. The Experimental Core (EC) will be responsible for the preparation and spectral data collection for several different types of C. elegans metabolome samples. This includes (i) a large-scale reference sample of the common laboratory strain ?N2?, (ii) a set of over 100 wild C. elegans isolates, representing a set of genetically diverse but homozygous ?individuals?, which will be used for mapping conserved biochemical pathways using a genome-wide association (m-GWAS) approach, and (iii) a set of deletion mutants that will be used to validate gene function predictions and characterize unknown features in known genetic pathways. These samples will be characterized by taking advantage of the complementary strengths of LC-MS/MS (speed and broad metabolite coverage), high-resolution FTMS (direct determination of experimental molecular formulas), and NMR (atomic-level structural data). When analyzed with approaches described in the Computational Core, the generated spectral data will be used to develop an automatic pipeline of unknown compound identification that will be generally applicable to a wide range of diverse model systems, including higher animals and human samples.