#METABOLOMICS WORKBENCH DrewRJones_20190923_124206 DATATRACK_ID:1820 STUDY_ID:ST001258 ANALYSIS_ID:AN002087 PROJECT_ID:PR000844 VERSION 1 CREATED_ON September 30, 2019, 8:33 pm #PROJECT PR:PROJECT_TITLE Modeling the metabolic interplay between a parasitic worm and its bacterial PR:PROJECT_TITLE endosymbiont allows the identification of novel drug targets PR:PROJECT_SUMMARY The filarial nematode Brugia malayi represents a leading cause of disability in PR:PROJECT_SUMMARY the developing world, causing lymphatic filariasis in nearly 40 million people. PR:PROJECT_SUMMARY Currently available drugs are not well-suited to mass drug administration PR:PROJECT_SUMMARY efforts, so new treatments are urgently required. One potential vulnerability is PR:PROJECT_SUMMARY the endosymbiotic bacteria Wolbachia—present in many filariae—which is vital PR:PROJECT_SUMMARY to the worm. Genome scale metabolic networks have been used to study prokaryotes PR:PROJECT_SUMMARY and protists and have proven valuable in identifying therapeutic targets, but PR:PROJECT_SUMMARY only recently have been applied to eukaryotic organisms. Here, we present PR:PROJECT_SUMMARY iDC625, the first compartmentalized metabolic model of a parasitic worm. We used PR:PROJECT_SUMMARY this model to show how metabolic pathway usage allows the worm to adapt to PR:PROJECT_SUMMARY different environments, and predict a set of 99 reactions essential to the PR:PROJECT_SUMMARY survival of B. malayi. We validated three of those reactions with drug tests and PR:PROJECT_SUMMARY demonstrated novel antifilarial properties for all three compounds. PR:INSTITUTE NYU Langone Health PR:LAST_NAME Jones PR:FIRST_NAME Drew PR:ADDRESS 430 E29th Street, WT635A PR:EMAIL drew.jones@nyulangone.org PR:PHONE 6465012054 #STUDY ST:STUDY_TITLE Modeling the metabolic interplay between a parasitic worm and its bacterial ST:STUDY_TITLE endosymbiont allows the identification of novel drug targets ST:STUDY_SUMMARY The filarial nematode Brugia malayi represents a leading cause of disability in ST:STUDY_SUMMARY the developing world, causing lymphatic filariasis in nearly 40 million people. ST:STUDY_SUMMARY Currently available drugs are not well-suited to mass drug administration ST:STUDY_SUMMARY efforts, so new treatments are urgently required. One potential vulnerability is ST:STUDY_SUMMARY the endosymbiotic bacteria Wolbachia—present in many filariae—which is vital ST:STUDY_SUMMARY to the worm. Genome scale metabolic networks have been used to study prokaryotes ST:STUDY_SUMMARY and protists and have proven valuable in identifying therapeutic targets, but ST:STUDY_SUMMARY only recently have been applied to eukaryotic organisms. Here, we present ST:STUDY_SUMMARY iDC625, the first compartmentalized metabolic model of a parasitic worm. We used ST:STUDY_SUMMARY this model to show how metabolic pathway usage allows the worm to adapt to ST:STUDY_SUMMARY different environments, and predict a set of 99 reactions essential to the ST:STUDY_SUMMARY survival of B. malayi. We validated three of those reactions with drug tests and ST:STUDY_SUMMARY demonstrated novel antifilarial properties for all three compounds. ST:INSTITUTE Hospital for Sick Children, University of Toronto, NYU Langone Health ST:LAST_NAME Jones ST:FIRST_NAME Drew ST:ADDRESS 430 E29th Street, WT635A ST:EMAIL drew.jones@nyulangone.org ST:PHONE 6465012054 #SUBJECT SU:SUBJECT_TYPE Invertebrate SU:SUBJECT_SPECIES Brugia malayi SU:TAXONOMY_ID 6279 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS S00869 1 20AF Group:AF SUBJECT_SAMPLE_FACTORS S00870 2 20AF Group:AF SUBJECT_SAMPLE_FACTORS S00871 3 20AF Group:AF SUBJECT_SAMPLE_FACTORS S00872 4 40AM Group:AM SUBJECT_SAMPLE_FACTORS S00873 5 40AM Group:AM SUBJECT_SAMPLE_FACTORS S00874 6 40AM Group:AM SUBJECT_SAMPLE_FACTORS S00875 7 2e6 Mf Group:Mf SUBJECT_SAMPLE_FACTORS S00876 8 2e6 Mf Group:Mf SUBJECT_SAMPLE_FACTORS S00877 9 2e6 Mf Group:Mf SUBJECT_SAMPLE_FACTORS S00878 10 200 L3 Group:L3 SUBJECT_SAMPLE_FACTORS S00879 11 200 L3 Group:L3 SUBJECT_SAMPLE_FACTORS S00880 12 200 L3 Group:L3 SUBJECT_SAMPLE_FACTORS SQ93_B_1 Blank_1 Group:Blank SUBJECT_SAMPLE_FACTORS SQ93_B_2 Blank_2 Group:Blank SUBJECT_SAMPLE_FACTORS SQ93_B_3 Blank_3 Group:Blank SUBJECT_SAMPLE_FACTORS SQ93_B_4 Blank_4 Group:Blank SUBJECT_SAMPLE_FACTORS SQ93_B_5 Blank_5 Group:Blank #COLLECTION CO:COLLECTION_SUMMARY All parasites were obtained from FR3 (Filariasis Research Reagent Resource CO:COLLECTION_SUMMARY Center; BEI Resources, Manassas, VA, USA) where they were isolated and separated CO:COLLECTION_SUMMARY by sex from infected gerbils (Meriones unguiculatus) or mosquitoes (Aedes CO:COLLECTION_SUMMARY aegypti). Worms were flash-frozen and shipped to the New York Blood Center for CO:COLLECTION_SUMMARY processing. Stages used for metabolomics analysis included L3 larvae from CO:COLLECTION_SUMMARY mosquitoes, adult male and female worms at 120dpi, and microfilaria. The number CO:COLLECTION_SUMMARY of worms per sample were 20 adult female worms, 40 adult males, 2X106 CO:COLLECTION_SUMMARY microfilariae, and 200 L3 larvae per biological replicate. Samples were washed CO:COLLECTION_SUMMARY in 1x PBS and run in triplicate. Adult male and female worms were picked CO:COLLECTION_SUMMARY individually from PBS and each biological was weighed. The microfilaria and L3 CO:COLLECTION_SUMMARY samples were spun down, the PBS pipetted off, and weighed directly into a CO:COLLECTION_SUMMARY metabolomics 2mL screw cap vial with total amounts ranging from 1.3 mg (adult CO:COLLECTION_SUMMARY males) to 15.8 mg (microfilaria). Metabolites were extracted and the data CO:COLLECTION_SUMMARY analyzed as described in the Supplementary Information. CO:SAMPLE_TYPE Worms #TREATMENT TR:TREATMENT_SUMMARY All parasites were obtained from FR3 (Filariasis Research Reagent Resource TR:TREATMENT_SUMMARY Center; BEI Resources, Manassas, VA, USA) where they were isolated and separated TR:TREATMENT_SUMMARY by sex from infected gerbils (Meriones unguiculatus) or mosquitoes (Aedes TR:TREATMENT_SUMMARY aegypti). Worms were flash-frozen and shipped to the New York Blood Center for TR:TREATMENT_SUMMARY processing. Stages used for metabolomics analysis included L3 larvae from TR:TREATMENT_SUMMARY mosquitoes, adult male and female worms at 120dpi, and microfilaria. The number TR:TREATMENT_SUMMARY of worms per sample were 20 adult female worms, 40 adult males, 2X106 TR:TREATMENT_SUMMARY microfilariae, and 200 L3 larvae per biological replicate. Samples were washed TR:TREATMENT_SUMMARY in 1x PBS and run in triplicate. Adult male and female worms were picked TR:TREATMENT_SUMMARY individually from PBS and each biological was weighed. The microfilaria and L3 TR:TREATMENT_SUMMARY samples were spun down, the PBS pipetted off, and weighed directly into a TR:TREATMENT_SUMMARY metabolomics 2mL screw cap vial with total amounts ranging from 1.3 mg (adult TR:TREATMENT_SUMMARY males) to 15.8 mg (microfilaria). Metabolites were extracted and the data TR:TREATMENT_SUMMARY analyzed as described in the Supplementary Information. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Metabolite extraction – The mass of the weighed worm samples was used to scale SP:SAMPLEPREP_SUMMARY the metabolite extraction to a ratio of 16.5 mg / 1 mL extraction solvent. SP:SAMPLEPREP_SUMMARY Freezing 80% acetonitrile was added directly to each vial containing the SP:SAMPLEPREP_SUMMARY samples, along with zirconium disruption beads (0.5 mm, RPI) and homogenized for SP:SAMPLEPREP_SUMMARY 3 min at 4°C in a BeadBlasterTM with a 30 sec on, 30 sec off pattern. The SP:SAMPLEPREP_SUMMARY resulting lysate was centrifuged at 21,000 x g for 3 min, and 90% of the SP:SAMPLEPREP_SUMMARY supernatant volume was transferred to a 1.5 mL microfuge tube for speed vacuum SP:SAMPLEPREP_SUMMARY concentration, no heating. The dry extracts were resolublized in a volume of SP:SAMPLEPREP_SUMMARY LCMS grade water 1/10th of that used for the homogenization step, sonicated in a SP:SAMPLEPREP_SUMMARY water bath for 3 min, and transferred to a glass insert for analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 RS CH:COLUMN_NAME SeQuant ZIC- pHILIC (150 x 2.1mm, 5um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive HF hybrid Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE UNSPECIFIED MS:MS_COMMENTS Positive/Negative Polarity Switching #END