#METABOLOMICS WORKBENCH ThomasS_20210809_195740_mwtab.txt DATATRACK_ID:2792 STUDY_ID:ST001900 ANALYSIS_ID:AN003088 PROJECT_ID:000000 VERSION 1 CREATED_ON August 10, 2021, 3:58 pm #PROJECT PR:PROJECT_TITLE Systemic host inflammation induces stage-specific transcriptomic modification PR:PROJECT_TITLE and slower maturation in malaria parasites PR:PROJECT_TYPE MS untargeted metabolomics analysis PR:PROJECT_SUMMARY Previous reports suggest that the maturation rate of malaria parasites within PR:PROJECT_SUMMARY red blood cells (RBC) is not constant for a given species in vivo. For instance, PR:PROJECT_SUMMARY maturation can be influenced by host nutrient status or circadian rhythm. Here PR:PROJECT_SUMMARY we observed in mice that systemic host inflammation, induced by PR:PROJECT_SUMMARY lipopolysaccharide (LPS) conditioning or ongoing acute malaria infection, slowed PR:PROJECT_SUMMARY the progression of a single cohort of parasites from one generation of RBC to PR:PROJECT_SUMMARY the next. LPS-conditioning and acute infection both triggered substantial PR:PROJECT_SUMMARY changes to the metabolomic composition of plasma in which parasites circulated. PR:PROJECT_SUMMARY This altered plasma directly slowed parasite maturation in a manner that could PR:PROJECT_SUMMARY not be rescued by supplementation, consistent with the presence of inhibitory PR:PROJECT_SUMMARY factors. Single-cell transcriptomic assessment of mixed parasite populations, PR:PROJECT_SUMMARY exposed to a short period of systemic host inflammation in vivo, revealed PR:PROJECT_SUMMARY specific impairment in the transcriptional activity and translational capacity PR:PROJECT_SUMMARY of trophozoites compared to rings or schizonts. Thus, we provide in vivo PR:PROJECT_SUMMARY evidence of transcriptomic and phenotypic plasticity of asexual blood-stage PR:PROJECT_SUMMARY Plasmodium parasites when exposed to systemic host inflammation PR:INSTITUTE QIMR Berghofer Medical Research Institute PR:DEPARTMENT Cell & Molecular Biology Department PR:LABORATORY Precision & Systems Biomedicine PR:LAST_NAME Stoll PR:FIRST_NAME Thomas PR:ADDRESS 300 Herston Road, Herston QLD 4006, Australia PR:EMAIL thomas.stoll@qimrberghofer.edu.au PR:PHONE +61 7 3845 3992 #STUDY ST:STUDY_TITLE Systemic host inflammation induces stage-specific transcriptomic modification ST:STUDY_TITLE and slower maturation in malaria parasites (part II) ST:STUDY_SUMMARY Previous reports suggest that the maturation rate of malaria parasites within ST:STUDY_SUMMARY red blood cells (RBC) is not constant for a given species in vivo. For instance, ST:STUDY_SUMMARY maturation can be influenced by host nutrient status or circadian rhythm. Here ST:STUDY_SUMMARY we observed in mice that systemic host inflammation, induced by ST:STUDY_SUMMARY lipopolysaccharide (LPS) conditioning or ongoing acute malaria infection, slowed ST:STUDY_SUMMARY the progression of a single cohort of parasites from one generation of RBC to ST:STUDY_SUMMARY the next. LPS-conditioning and acute infection both triggered substantial ST:STUDY_SUMMARY changes to the metabolomic composition of plasma in which parasites circulated. ST:STUDY_SUMMARY This altered plasma directly slowed parasite maturation in a manner that could ST:STUDY_SUMMARY not be rescued by supplementation, consistent with the presence of inhibitory ST:STUDY_SUMMARY factors. Single-cell transcriptomic assessment of mixed parasite populations, ST:STUDY_SUMMARY exposed to a short period of systemic host inflammation in vivo, revealed ST:STUDY_SUMMARY specific impairment in the transcriptional activity and translational capacity ST:STUDY_SUMMARY of trophozoites compared to rings or schizonts. Thus, we provide in vivo ST:STUDY_SUMMARY evidence of transcriptomic and phenotypic plasticity of asexual blood-stage ST:STUDY_SUMMARY Plasmodium parasites when exposed to systemic host inflammation ST:INSTITUTE QIMR Berghofer Medical Research Institute ST:DEPARTMENT Cell & Molecular Biology Department ST:LABORATORY Precision & Systems Biomedicine ST:LAST_NAME Stoll ST:FIRST_NAME Thomas ST:ADDRESS 300 Herston Road ST:EMAIL thomas.stoll@qimrberghofer.edu.au ST:NUM_GROUPS 5 ST:TOTAL_SUBJECTS 30 ST:STUDY_TYPE Study part 2 of 2 (independent experiment 2; replication experiment of Study ST:STUDY_TYPE part 1) ST:PHONE +61 7 3845 3992 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN C57BL/6J, C57BL/6J.rag1−/− SU:AGE_OR_AGE_RANGE 6-8 weeks SU:GENDER Female #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - MP01 Genotype:WT Acute | Treatment:Infected Replicate=1; RAW_FILE_NAME=191015LL_MetHILICpos_MP01; RAW_FILE_NAME=191016LL_MetHILICneg_MP01 SUBJECT_SAMPLE_FACTORS - MP02 Genotype:WT Acute | Treatment:Infected Replicate=2; RAW_FILE_NAME=191015LL_MetHILICpos_MP02; RAW_FILE_NAME=191016LL_MetHILICneg_MP02 SUBJECT_SAMPLE_FACTORS - MP03 Genotype:WT Naïve | Treatment:Control Replicate=6; RAW_FILE_NAME=191015LL_MetHILICpos_MP03; RAW_FILE_NAME=191016LL_MetHILICneg_MP03 SUBJECT_SAMPLE_FACTORS - MP05 Genotype:WT LPS | Treatment:LPS treatment Replicate=2; RAW_FILE_NAME=191015LL_MetHILICpos_MP05; RAW_FILE_NAME=191016LL_MetHILICneg_MP05 SUBJECT_SAMPLE_FACTORS - MP06 Genotype:KO Acute | Treatment:Immune deficient infected control Replicate=3; RAW_FILE_NAME=191015LL_MetHILICpos_MP06; RAW_FILE_NAME=191016LL_MetHILICneg_MP06 SUBJECT_SAMPLE_FACTORS - MP07 Genotype:WT Naïve | Treatment:Control Replicate=1; RAW_FILE_NAME=191015LL_MetHILICpos_MP07; RAW_FILE_NAME=191016LL_MetHILICneg_MP07 SUBJECT_SAMPLE_FACTORS - MP08 Genotype:KO Acute | Treatment:Immune deficient infected control Replicate=5; RAW_FILE_NAME=191015LL_MetHILICpos_MP08; RAW_FILE_NAME=191016LL_MetHILICneg_MP08 SUBJECT_SAMPLE_FACTORS - MP09 Genotype:KO Naïve | Treatment:Immune deficient control Replicate=4; RAW_FILE_NAME=191015LL_MetHILICpos_MP09; RAW_FILE_NAME=191016LL_MetHILICneg_MP09 SUBJECT_SAMPLE_FACTORS - MP10 Genotype:WT LPS | Treatment:LPS treatment Replicate=1; RAW_FILE_NAME=191015LL_MetHILICpos_MP10; RAW_FILE_NAME=191016LL_MetHILICneg_MP10 SUBJECT_SAMPLE_FACTORS - MP11 Genotype:KO Acute | Treatment:Immune deficient infected control Replicate=1; RAW_FILE_NAME=191015LL_MetHILICpos_MP11; RAW_FILE_NAME=191016LL_MetHILICneg_MP11 SUBJECT_SAMPLE_FACTORS - MP12 Genotype:WT LPS | Treatment:LPS treatment Replicate=3; RAW_FILE_NAME=191015LL_MetHILICpos_MP12; RAW_FILE_NAME=191016LL_MetHILICneg_MP12 SUBJECT_SAMPLE_FACTORS - MP13 Genotype:WT Naïve | Treatment:Control Replicate=5; RAW_FILE_NAME=191015LL_MetHILICpos_MP13; RAW_FILE_NAME=191016LL_MetHILICneg_MP13 SUBJECT_SAMPLE_FACTORS - MP14 Genotype:WT Naïve | Treatment:Control Replicate=4; RAW_FILE_NAME=191015LL_MetHILICpos_MP14; RAW_FILE_NAME=191016LL_MetHILICneg_MP14 SUBJECT_SAMPLE_FACTORS - MP15 Genotype:KO Naïve | Treatment:Immune deficient control Replicate=1; RAW_FILE_NAME=191015LL_MetHILICpos_MP15; RAW_FILE_NAME=191016LL_MetHILICneg_MP15 SUBJECT_SAMPLE_FACTORS - MP16 Genotype:WT Acute | Treatment:Infected Replicate=5; RAW_FILE_NAME=191015LL_MetHILICpos_MP16; RAW_FILE_NAME=191016LL_MetHILICneg_MP16 SUBJECT_SAMPLE_FACTORS - MP18 Genotype:KO Acute | Treatment:Immune deficient infected control Replicate=2; RAW_FILE_NAME=191015LL_MetHILICpos_MP18; RAW_FILE_NAME=191016LL_MetHILICneg_MP18 SUBJECT_SAMPLE_FACTORS - MP19 Genotype:WT LPS | Treatment:LPS treatment Replicate=4; RAW_FILE_NAME=191015LL_MetHILICpos_MP19; RAW_FILE_NAME=191016LL_MetHILICneg_MP19 SUBJECT_SAMPLE_FACTORS - MP21 Genotype:WT Acute | Treatment:Infected Replicate=3; RAW_FILE_NAME=191015LL_MetHILICpos_MP21; RAW_FILE_NAME=191016LL_MetHILICneg_MP21 SUBJECT_SAMPLE_FACTORS - MP22 Genotype:WT Acute | Treatment:Infected Replicate=4; RAW_FILE_NAME=191015LL_MetHILICpos_MP22; RAW_FILE_NAME=191016LL_MetHILICneg_MP22 SUBJECT_SAMPLE_FACTORS - MP24 Genotype:KO Acute | Treatment:Immune deficient infected control Replicate=4; RAW_FILE_NAME=191015LL_MetHILICpos_MP24; RAW_FILE_NAME=191016LL_MetHILICneg_MP24 SUBJECT_SAMPLE_FACTORS - MP26 Genotype:WT LPS | Treatment:LPS treatment Replicate=6; RAW_FILE_NAME=191015LL_MetHILICpos_MP26; RAW_FILE_NAME=191016LL_MetHILICneg_MP26 SUBJECT_SAMPLE_FACTORS - MP27 Genotype:KO Acute | Treatment:Immune deficient infected control Replicate=6; RAW_FILE_NAME=191015LL_MetHILICpos_MP27; RAW_FILE_NAME=191016LL_MetHILICneg_MP27 SUBJECT_SAMPLE_FACTORS - MP28 Genotype:KO Naïve | Treatment:Immune deficient control Replicate=6; RAW_FILE_NAME=191015LL_MetHILICpos_MP28; RAW_FILE_NAME=191016LL_MetHILICneg_MP28 SUBJECT_SAMPLE_FACTORS - MP29 Genotype:WT LPS | Treatment:LPS treatment Replicate=5; RAW_FILE_NAME=191015LL_MetHILICpos_MP29; RAW_FILE_NAME=191016LL_MetHILICneg_MP29 SUBJECT_SAMPLE_FACTORS - MP30 Genotype:WT Acute | Treatment:Infected Replicate=6; RAW_FILE_NAME=191015LL_MetHILICpos_MP30; RAW_FILE_NAME=191016LL_MetHILICneg_MP30 SUBJECT_SAMPLE_FACTORS - MP31 Genotype:KO Naïve | Treatment:Immune deficient control Replicate=3; RAW_FILE_NAME=191015LL_MetHILICpos_MP31; RAW_FILE_NAME=191016LL_MetHILICneg_MP31 SUBJECT_SAMPLE_FACTORS - MP33 Genotype:KO Naïve | Treatment:Immune deficient control Replicate=2; RAW_FILE_NAME=191015LL_MetHILICpos_MP33; RAW_FILE_NAME=191016LL_MetHILICneg_MP33 SUBJECT_SAMPLE_FACTORS - MP34 Genotype:WT Naïve | Treatment:Control Replicate=3; RAW_FILE_NAME=191015LL_MetHILICpos_MP34; RAW_FILE_NAME=191016LL_MetHILICneg_MP34 SUBJECT_SAMPLE_FACTORS - MP36 Genotype:KO Naïve | Treatment:Immune deficient control Replicate=5; RAW_FILE_NAME=191015LL_MetHILICpos_MP36; RAW_FILE_NAME=191016LL_MetHILICneg_MP36 SUBJECT_SAMPLE_FACTORS - MP37 Genotype:WT Naïve | Treatment:Control Replicate=2; RAW_FILE_NAME=191015LL_MetHILICpos_MP37; RAW_FILE_NAME=191016LL_MetHILICneg_MP37 SUBJECT_SAMPLE_FACTORS - QC_MP_01 Genotype:QC | Treatment:QC Replicate=1; RAW_FILE_NAME=191015LL_MetHILICpos_QC_MP_01; RAW_FILE_NAME=191016LL_MetHILICneg_QC_MP_01 SUBJECT_SAMPLE_FACTORS - QC_MP_02 Genotype:QC | Treatment:QC Replicate=2; RAW_FILE_NAME=191015LL_MetHILICpos_QC_MP_02; RAW_FILE_NAME=191016LL_MetHILICneg_QC_MP_02 SUBJECT_SAMPLE_FACTORS - QC_MP_03 Genotype:QC | Treatment:QC Replicate=3; RAW_FILE_NAME=191015LL_MetHILICpos_QC_MP_03; RAW_FILE_NAME=191016LL_MetHILICneg_QC_MP_03 SUBJECT_SAMPLE_FACTORS - QC_MP_04 Genotype:QC | Treatment:QC Replicate=4; RAW_FILE_NAME=191015LL_MetHILICpos_QC_MP_04; RAW_FILE_NAME=191016LL_MetHILICneg_QC_MP_04 SUBJECT_SAMPLE_FACTORS - QC_MP_05 Genotype:QC | Treatment:QC Replicate=5; RAW_FILE_NAME=191015LL_MetHILICpos_QC_MP_05; RAW_FILE_NAME=191016LL_MetHILICneg_QC_MP_05 SUBJECT_SAMPLE_FACTORS - QC_MP_06 Genotype:QC | Treatment:QC Replicate=6; RAW_FILE_NAME=191015LL_MetHILICpos_QC_MP_06; RAW_FILE_NAME=191016LL_MetHILICneg_QC_MP_06 SUBJECT_SAMPLE_FACTORS - MP_Blank_01 Genotype:Blank | Treatment:Blank Replicate=1; RAW_FILE_NAME=191015LL_MetHILICpos_MP_Blank_01; RAW_FILE_NAME=191016LL_MetHILICneg_MP_Blank_01 SUBJECT_SAMPLE_FACTORS - MP_Blank_02 Genotype:Blank | Treatment:Blank Replicate=2; RAW_FILE_NAME=191015LL_MetHILICpos_MP_Blank_02; RAW_FILE_NAME=191016LL_MetHILICneg_MP_Blank_02 SUBJECT_SAMPLE_FACTORS - MP_Blank_03 Genotype:Blank | Treatment:Blank Replicate=3; RAW_FILE_NAME=191015LL_MetHILICpos_MP_Blank_03; RAW_FILE_NAME=191016LL_MetHILICneg_MP_Blank_03 #COLLECTION CO:COLLECTION_SUMMARY Two independent experiments were conducted, each with 6 mice per treatment group CO:COLLECTION_SUMMARY (30 individuals in total). Mice were euthanized by CO2 asphyxiation and their CO:COLLECTION_SUMMARY blood was taken by cardiac puncture into lithium-heparin coated tubes. Samples CO:COLLECTION_SUMMARY were spun for 5 min at 5000 rpm (approx. 7,043 × g) and plasma was immediately CO:COLLECTION_SUMMARY aliquoted into 1.5 mL tubes. In addition, a global sample pool containing equal CO:COLLECTION_SUMMARY volumes of each sample was prepared as quality control (QC) and four aliquots CO:COLLECTION_SUMMARY were transferred into 1.5 mL tubes. Finally, collection tube blank extractions CO:COLLECTION_SUMMARY were performed in triplicate by adding 1x PBS (same volume as blood collection) CO:COLLECTION_SUMMARY to lithium-heparin tubes and then transferring an aliquot into a 1.5 mL tube CO:SAMPLE_TYPE Blood (plasma) #TREATMENT TR:TREATMENT_SUMMARY Genotype: Treatment C57BL/6J Naïve: Control, C57BL/6J mice were TR:TREATMENT_SUMMARY intraperitoneally injected (200 uL) with saline (0.9%) 9 hours prior to plasma TR:TREATMENT_SUMMARY acquisition. C57BL/6J Acute: Infected, C57BL/6J mice were infected with 10^5 TR:TREATMENT_SUMMARY Plasmodium berghei ANKA parasitised red blood cells 5 days prior to plasma TR:TREATMENT_SUMMARY acquisition and intraperitoneally injected (200 uL) with saline (0.9%) 9 hours TR:TREATMENT_SUMMARY prior to plasma acquisition for analysis. C57BL/6J LPS: LPS treatment, C57BL/6J TR:TREATMENT_SUMMARY mice were intraperitoneally injected (200 uL) with lipopolysaccharides (LPS) TR:TREATMENT_SUMMARY (0.75 mg/mL), from E.coli O127:B8, 9 hours prior to plasma acquisition. rag1-/- TR:TREATMENT_SUMMARY Naïve: Immune deficient control, C57BL/6J.rag1-/- mice were intraperitoneally TR:TREATMENT_SUMMARY injected (200 uL) with saline (0.9%) 9 hours prior to plasma acquisition. TR:TREATMENT_SUMMARY rag1-/- Acute: Immune deficient infected control, C57BL/6J.rag1-/- mice were TR:TREATMENT_SUMMARY infected with 10^5 Plasmodium berghei ANKA parasitised red blood cells 5 days TR:TREATMENT_SUMMARY prior to plasma acquisition and intraperitoneally injected (200 uL) with saline TR:TREATMENT_SUMMARY 9 hours prior to plasma acquisition for analysis. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Ten-times the sample volume of ice-cold butanol/methanol (1:1) containing 50 SP:SAMPLEPREP_SUMMARY µg/mL antioxidant 2,6-di-tert-butyl-4-methylphenol (BHT) was added to each SP:SAMPLEPREP_SUMMARY sample and vortexed for 10 s. Samples were snap frozen and transported on dry SP:SAMPLEPREP_SUMMARY ice. Subsequently, samples were thawed on ice and labelled in a randomized SP:SAMPLEPREP_SUMMARY order. Samples were sonicated for 15 min in an ice-cold water bath sonicator, SP:SAMPLEPREP_SUMMARY stored for 2 hrs at -30oC and then centrifuged for 15 min at 16,000 × g (4oC). SP:SAMPLEPREP_SUMMARY Lastly, samples were aliquoted, dried down using a vacuum concentrator and SP:SAMPLEPREP_SUMMARY stored at -80oC until LC/MS analysis #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Metabolite separation was performed on a Zorbax HILIC Plus RRHD (95Å, 1.8 µm, CH:CHROMATOGRAPHY_SUMMARY 2.1x100mm) analytical column connected to a 3 x 5 mm Zorbax HILIC Plus UHPLC CH:CHROMATOGRAPHY_SUMMARY guard column. The autosampler and column temperature were set to 4°C and 40°C, CH:CHROMATOGRAPHY_SUMMARY respectively. In positive and negative mode, eluent A was 10 mM ammonium acetate CH:CHROMATOGRAPHY_SUMMARY (pH neutral) in acetonitrile/milliQ water (95:5, v/v) and eluent B was 10 mM CH:CHROMATOGRAPHY_SUMMARY ammonium acetate (pH neutral) in acetonitrile/milliQ water (50:50, v/v). Total CH:CHROMATOGRAPHY_SUMMARY method runtime was 12 min with the following gradient for both modes: 0 min (1% CH:CHROMATOGRAPHY_SUMMARY eluent B) - 3.5 min (50% B) - 5.5 min (99%B) - 6.5 min (99% B) - 6.7 min (1% B) CH:CHROMATOGRAPHY_SUMMARY - 12 min (1% B). Flow rate was set to 0.5 mL/min. CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Agilent 1290 Infinity II CH:COLUMN_NAME Agilent Zorbax HILIC Plus RRHD (95Å, 1.8 µm, 2.1x100mm) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6545 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS MS acquisition: The LC/MS platform consisted of a 1290 Infinity II UHPLC coupled MS:MS_COMMENTS to a 6545 QTOF mass spectrometer via Dual AJS ESI source (Agilent, Santa Clara, MS:MS_COMMENTS USA) and was controlled using MassHunter data acquisition software (v.10.1). MS:MS_COMMENTS Assessment of MS instrument performance and usage of reference ions were also MS:MS_COMMENTS performed as described previously. Full scan MS data (m/z 50-1700) was acquired MS:MS_COMMENTS at a scan rate of 2.5 spectra/sec (equals 3224 transients/spectrum) with the MS:MS_COMMENTS following source conditions: Gas temperature 250°C, gas flow 13 L/min, sheath MS:MS_COMMENTS gas temperature and flow at 400°C and 12 L/min, respectively, nebulizer 30 psi, MS:MS_COMMENTS fragmentor 135, capillary voltage at +4500 V and -4000 V, nozzle voltage was MS:MS_COMMENTS zero. Data processing: Positive and negative mode data was analysed separately. MS:MS_COMMENTS Data files (30 sample files, 6 QC files and 3 tube blank extraction files) were MS:MS_COMMENTS loaded into MassHunter Profinder (v 10 SP1, Agilent) and assigned to sample MS:MS_COMMENTS groups. Spectral feature extraction was performed using the recursive feature MS:MS_COMMENTS extraction method employing default settings with minor adjustments: Peak MS:MS_COMMENTS extraction was restricted to retention time (Rt) range 0-6.5 min, compound MS:MS_COMMENTS binning and alignment tolerances were set to 1% + 0.3 min for Rt and 20 ppm + 2 MS:MS_COMMENTS mDa for mass, integrator Agile 2 was used for peak integration, peak filters MS:MS_COMMENTS were set to at least 2500 counts and features must have satisfied filter MS:MS_COMMENTS conditions in at least 75 % of files in at least one sample group. Feature peak MS:MS_COMMENTS area was exported and data cleaning was performed using an in-house R script MS:MS_COMMENTS compiled of the following steps. Features were deleted if they: had a mean MS:MS_COMMENTS QC/tube blank area ratio of < 10; were absent across all QC samples; and had MS:MS_COMMENTS duplicates present. In addition, samples with a TIC scaling factor more than 50% MS:MS_COMMENTS above or below the median TIC were removed. MS:MS_RESULTS_FILE ST001900_AN003088_Results.txt UNITS:peak area Has m/z:Neutral masses Has RT:Yes RT units:Minutes #END