{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002053","ANALYSIS_ID":"AN003343","VERSION":"1","CREATED_ON":"January 7, 2022, 8:41 am"},

"PROJECT":{"PROJECT_TITLE":"Resistance to NaFAc of in vitro maturated Toxoplasma gondii bradyzoites in human myotubes","PROJECT_SUMMARY":"The apicomplexan parasite Toxoplasma gondii forms bradyzoite-containing tissue cysts that cause chronic and drug-tolerant infections. Here, we developed a human myotube-based in vitro culture model of functionally mature tissue cysts. In contrast to fast replicating tachyzoite forms of T. gondii these tissue cysts tolerate exposure to the aconitase inhibitor sodium fluoroacetate. These data characterize bradyzoites and tachyzoites treated with NaFAc.","INSTITUTE":"Robert Koch-Institut","LAST_NAME":"Blume","FIRST_NAME":"Martin","ADDRESS":"Seestr. 10, Berlin, Berlin, 13353, Germany","EMAIL":"blumem@rki.de","PHONE":"+49 30 18754 2572"},

"STUDY":{"STUDY_TITLE":"Resistance to NaFAc of in vitro maturated Toxoplasma gondii bradyzoites in human myotubes.","STUDY_SUMMARY":"The apicomplexan parasite Toxoplasma gondii forms bradyzoite-containing tissue cysts that cause chronic and drug-tolerant infections. Here, we developed a human myotube-based in vitro culture model of functionally mature tissue cysts. In contrast to fast replicating tachyzoite forms of T. gondii these tissue cysts tolerate exposure to the aconitase inhibitor sodium fluoroacetate. These data characterize bradyzoites and tachyzoites treated with NaFAc.","INSTITUTE":"Robert Koch-Institut","LAST_NAME":"Blume","FIRST_NAME":"Martin","ADDRESS":"Seestr. 10, Berlin, Berlin, 13353, Germany","EMAIL":"blumem@rki.de","PHONE":"+49 30 18754 2572"},

"SUBJECT":{"SUBJECT_TYPE":"Other organism","SUBJECT_SPECIES":"Toxoplasma gondii","TAXONOMY_ID":"5811"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"PP_invitrocysts_mock_1",
"Factors":{"Factor":"PP_invitrocysts_mock"},
"Additional sample data":{"RAW_FILE_NAME":"PP_invitrocysts_mock_1"}
},
{
"Subject ID":"-",
"Sample ID":"PP_invitrocysts_mock_2",
"Factors":{"Factor":"PP_invitrocysts_mock"},
"Additional sample data":{"RAW_FILE_NAME":"PP_invitrocysts_mock_2"}
},
{
"Subject ID":"-",
"Sample ID":"PP_invitrocysts_mock_3",
"Factors":{"Factor":"PP_invitrocysts_mock"},
"Additional sample data":{"RAW_FILE_NAME":"PP_invitrocysts_mock_3"}
},
{
"Subject ID":"-",
"Sample ID":"PP_invitrocysts_mock_4",
"Factors":{"Factor":"PP_invitrocysts_mock"},
"Additional sample data":{"RAW_FILE_NAME":"PP_invitrocysts_mock_4"}
},
{
"Subject ID":"-",
"Sample ID":"PP_tachys_mock_1",
"Factors":{"Factor":"PP_tachys_mock"},
"Additional sample data":{"RAW_FILE_NAME":"PP_tachys_mock_1"}
},
{
"Subject ID":"-",
"Sample ID":"PP_tachys_mock_2",
"Factors":{"Factor":"PP_tachys_mock"},
"Additional sample data":{"RAW_FILE_NAME":"PP_tachys_mock_2"}
},
{
"Subject ID":"-",
"Sample ID":"PP_tachys_mock_3",
"Factors":{"Factor":"PP_tachys_mock"},
"Additional sample data":{"RAW_FILE_NAME":"PP_tachys_mock_3"}
},
{
"Subject ID":"-",
"Sample ID":"PP_tachys_mock_4",
"Factors":{"Factor":"PP_tachys_mock"},
"Additional sample data":{"RAW_FILE_NAME":"PP_tachys_mock_4"}
},
{
"Subject ID":"-",
"Sample ID":"PP_tachys_mock_5",
"Factors":{"Factor":"PP_tachys_mock_5"},
"Additional sample data":{"RAW_FILE_NAME":"PP_tachys_mock_5"}
},
{
"Subject ID":"-",
"Sample ID":"PP_uninf_mock_1",
"Factors":{"Factor":"PP_uninf_mock"},
"Additional sample data":{"RAW_FILE_NAME":"PP_uninf_mock_1"}
},
{
"Subject ID":"-",
"Sample ID":"PP_uninf_mock_2",
"Factors":{"Factor":"PP_uninf_mock"},
"Additional sample data":{"RAW_FILE_NAME":"PP_uninf_mock_2"}
},
{
"Subject ID":"-",
"Sample ID":"PP_Blank_1",
"Factors":{"Factor":"PP_Blank"},
"Additional sample data":{"RAW_FILE_NAME":"PP_Blank_1"}
},
{
"Subject ID":"-",
"Sample ID":"PP_Blank_2",
"Factors":{"Factor":"PP_Blank"},
"Additional sample data":{"RAW_FILE_NAME":"PP_Blank_2"}
},
{
"Subject ID":"-",
"Sample ID":"PP_Blank_3",
"Factors":{"Factor":"PP_Blank"},
"Additional sample data":{"RAW_FILE_NAME":"PP_Blank_3"}
},
{
"Subject ID":"-",
"Sample ID":"PP_invitrocysts_NaFAc_1",
"Factors":{"Factor":"PP_invitrocysts_NaFAc"},
"Additional sample data":{"RAW_FILE_NAME":"PP_invitrocysts_NaFAc_1"}
},
{
"Subject ID":"-",
"Sample ID":"PP_invitrocysts_NaFAc_2",
"Factors":{"Factor":"PP_invitrocysts_NaFAc"},
"Additional sample data":{"RAW_FILE_NAME":"PP_invitrocysts_NaFAc_2"}
},
{
"Subject ID":"-",
"Sample ID":"PP_invitrocysts_NaFAc_3",
"Factors":{"Factor":"PP_invitrocysts_NaFAc"},
"Additional sample data":{"RAW_FILE_NAME":"PP_invitrocysts_NaFAc_3"}
},
{
"Subject ID":"-",
"Sample ID":"PP_invitrocysts_NaFAc_4",
"Factors":{"Factor":"PP_invitrocysts_NaFAc"},
"Additional sample data":{"RAW_FILE_NAME":"PP_invitrocysts_NaFAc_4"}
},
{
"Subject ID":"-",
"Sample ID":"PP_tachys_NaFAc_1",
"Factors":{"Factor":"PP_tachys_NaFAc"},
"Additional sample data":{"RAW_FILE_NAME":"PP_tachys_NaFAc_1"}
},
{
"Subject ID":"-",
"Sample ID":"PP_tachys_NaFAc_2",
"Factors":{"Factor":"PP_tachys_NaFAc"},
"Additional sample data":{"RAW_FILE_NAME":"PP_tachys_NaFAc_2"}
},
{
"Subject ID":"-",
"Sample ID":"PP_tachys_NaFAc_3",
"Factors":{"Factor":"PP_tachys_NaFAc"},
"Additional sample data":{"RAW_FILE_NAME":"PP_tachys_NaFAc_3"}
},
{
"Subject ID":"-",
"Sample ID":"PP_uninf_NaFAc_1",
"Factors":{"Factor":"PP_uninf_NaFAc"},
"Additional sample data":{"RAW_FILE_NAME":"PP_uninf_NaFAc_1"}
},
{
"Subject ID":"-",
"Sample ID":"PP_uninf_NaFAc_2",
"Factors":{"Factor":"PP_uninf_NaFAc"},
"Additional sample data":{"RAW_FILE_NAME":"PP_uninf_NaFAc_2"}
},
{
"Subject ID":"-",
"Sample ID":"NP_invitrocysts_mock_1",
"Factors":{"Factor":"NP_invitrocysts_mock"},
"Additional sample data":{"RAW_FILE_NAME":"NP_invitrocysts_mock_1"}
},
{
"Subject ID":"-",
"Sample ID":"NP_invitrocysts_mock_2",
"Factors":{"Factor":"NP_invitrocysts_mock"},
"Additional sample data":{"RAW_FILE_NAME":"NP_invitrocysts_mock_2"}
},
{
"Subject ID":"-",
"Sample ID":"NP_invitrocysts_mock_3",
"Factors":{"Factor":"NP_invitrocysts_mock"},
"Additional sample data":{"RAW_FILE_NAME":"NP_invitrocysts_mock_3"}
},
{
"Subject ID":"-",
"Sample ID":"NP_invitrocysts_mock_4",
"Factors":{"Factor":"NP_invitrocysts_mock"},
"Additional sample data":{"RAW_FILE_NAME":"NP_invitrocysts_mock_4"}
},
{
"Subject ID":"-",
"Sample ID":"NP_tachys_mock_1",
"Factors":{"Factor":"NP_tachys_mock"},
"Additional sample data":{"RAW_FILE_NAME":"NP_tachys_mock_1"}
},
{
"Subject ID":"-",
"Sample ID":"NP_tachys_mock_2",
"Factors":{"Factor":"NP_tachys_mock"},
"Additional sample data":{"RAW_FILE_NAME":"NP_tachys_mock_2"}
},
{
"Subject ID":"-",
"Sample ID":"NP_tachys_mock_3",
"Factors":{"Factor":"NP_tachys_mock"},
"Additional sample data":{"RAW_FILE_NAME":"NP_tachys_mock_3"}
},
{
"Subject ID":"-",
"Sample ID":"NP_tachys_mock_4",
"Factors":{"Factor":"NP_tachys_mock"},
"Additional sample data":{"RAW_FILE_NAME":"NP_tachys_mock_4"}
},
{
"Subject ID":"-",
"Sample ID":"NP_tachys_mock_5",
"Factors":{"Factor":"NP_tachys_mock_5"},
"Additional sample data":{"RAW_FILE_NAME":"NP_tachys_mock_5"}
},
{
"Subject ID":"-",
"Sample ID":"NP_uninf_mock_1",
"Factors":{"Factor":"NP_uninf_mock"},
"Additional sample data":{"RAW_FILE_NAME":"NP_uninf_mock_1"}
},
{
"Subject ID":"-",
"Sample ID":"NP_uninf_mock_2",
"Factors":{"Factor":"NP_uninf_mock"},
"Additional sample data":{"RAW_FILE_NAME":"NP_uninf_mock_2"}
},
{
"Subject ID":"-",
"Sample ID":"NP_Blank_1",
"Factors":{"Factor":"NP_Blank"},
"Additional sample data":{"RAW_FILE_NAME":"NP_Blank_1"}
},
{
"Subject ID":"-",
"Sample ID":"NP_Blank_2",
"Factors":{"Factor":"NP_Blank"},
"Additional sample data":{"RAW_FILE_NAME":"NP_Blank_2"}
},
{
"Subject ID":"-",
"Sample ID":"NP_Blank_3",
"Factors":{"Factor":"NP_Blank"},
"Additional sample data":{"RAW_FILE_NAME":"NP_Blank_3"}
},
{
"Subject ID":"-",
"Sample ID":"NP_invitrocysts_NaFAc_1",
"Factors":{"Factor":"NP_invitrocysts_NaFAc"},
"Additional sample data":{"RAW_FILE_NAME":"NP_invitrocysts_NaFAc_1"}
},
{
"Subject ID":"-",
"Sample ID":"NP_invitrocysts_NaFAc_2",
"Factors":{"Factor":"NP_invitrocysts_NaFAc"},
"Additional sample data":{"RAW_FILE_NAME":"NP_invitrocysts_NaFAc_2"}
},
{
"Subject ID":"-",
"Sample ID":"NP_invitrocysts_NaFAc_3",
"Factors":{"Factor":"NP_invitrocysts_NaFAc"},
"Additional sample data":{"RAW_FILE_NAME":"NP_invitrocysts_NaFAc_3"}
},
{
"Subject ID":"-",
"Sample ID":"NP_invitrocysts_NaFAc_4",
"Factors":{"Factor":"NP_invitrocysts_NaFAc"},
"Additional sample data":{"RAW_FILE_NAME":"NP_invitrocysts_NaFAc_4"}
},
{
"Subject ID":"-",
"Sample ID":"NP_tachys_NaFAc_1",
"Factors":{"Factor":"NP_tachys_NaFAc"},
"Additional sample data":{"RAW_FILE_NAME":"NP_tachys_NaFAc_1"}
},
{
"Subject ID":"-",
"Sample ID":"NP_tachys_NaFAc_2",
"Factors":{"Factor":"NP_tachys_NaFAc"},
"Additional sample data":{"RAW_FILE_NAME":"NP_tachys_NaFAc_2"}
},
{
"Subject ID":"-",
"Sample ID":"NP_tachys_NaFAc_3",
"Factors":{"Factor":"NP_tachys_NaFAc"},
"Additional sample data":{"RAW_FILE_NAME":"NP_tachys_NaFAc_3"}
},
{
"Subject ID":"-",
"Sample ID":"NP_uninf_NaFAc_1",
"Factors":{"Factor":"NP_uninf_NaFAc"},
"Additional sample data":{"RAW_FILE_NAME":"NP_uninf_NaFAc_1"}
},
{
"Subject ID":"-",
"Sample ID":"NP_uninf_NaFAc_2",
"Factors":{"Factor":"NP_uninf_NaFAc"},
"Additional sample data":{"RAW_FILE_NAME":"NP_uninf_NaFAc_2"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"For metabolome measurements of tissue cysts and tachyzoites, tachyzoite isolation, cyst maturation and isolation were performed as described above. Metabolites were extracted in 80 % acetonitrile (Carl Roth) and 20 % water (Carl Roth) containing internal standards (phenolphthalein, CAPS, PIPES (Sigma-Aldrich)). Cell pellets were sonicated for 5 min and after centrifugation (21,500 x g, 5 min, 0 °C), the supernatants were transferred to MS vials for immediate LC/MS analysis. 5 µl of each sample were collected to generate a pooled biological quality control (PBQC). 20 µl of the in vitro cysts, bead control and host cell background samples, and 5 µl of the tachyzoite samples were injected. The injection order of the samples was randomized, blanks and PBQCs were injected periodically. The samples were analyzed on a Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific) via 70k MS1 scans, with intermittent 35k data-dependent 35k MS2 scans in positive and negative mode separately. Chromatographic separation was achieved on a Vanquish Flex fitted with an ACQUITY UPLC BEH Amide column (Waters). Running a 25 min linear gradient starting with 90 % eluent A (10 mM ammonium carbonate in acetonitrile) / 10 % eluent B (10 mM ammonium carbonate in water) and ending with 40 % eluent A / 60 % eluent B, followed by washing and equilibration steps. XCalibur 4.2.47 software and Compound Discoverer 3.1 software (Thermo Fisher Scientific) was used for recording and used for peak detection, combination of adducts and compound annotation, respectively. Metabolite identifications were based on either retention time and accurate mass match to an in-house library of 160 authentic standards, or by matching accurate mass and MS2 fragments to m/z cloud database (mirrored offline in m/z vault v 2.1.22.15 in May 2020) (Thermo Fisher Scientific).","SAMPLE_TYPE":"Toxoplasma gondii"},

"TREATMENT":{"TREATMENT_SUMMARY":"Cells were either treated or untreated tachyzoites or treated or untreated bradyzoites (cysts)"},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Uninfected and infected myotubes were prepared. In both cases, two T150 dishes were pooled into one sample. For bradyzoite samples, myotubes were infected with 3.2*106 Pru-tdTomato tachyzoites corresponding to a MOI of 0.3 and cyst formation was induced for indicated time. On the day of harvest, infected samples and uninfected host cell controls were placed on ice, medium was removed and monolayers were washed three times with ice-cold PBS. Cells were then harvested by scraping into 10 ml ice-cold 0.05 % BSA in PBS per T150 dish. Cysts were released from the monolayer via forcing through a 23G needle (Sterican®) 25 times with a syringe and collected via centrifugation (1,200 x g, 10 min, 0 °C). The supernatant was removed, the pellet was resuspended carefully in ice-cold 2 % BSA in PBS containing 200 µl DBA-coupled beads (preparation described below) and samples were incubated for 1 h at 4 °C with gentle shaking. Subsequently, the samples were placed in a magnetic stand on ice, washed five times with 0.1 % BSA in PBS to remove cell debris, followed by two washing steps with PBS to remove residual BSA. Cysts and beads were then collected via centrifugation (1,200 x g, 10 min, 0 °C), shock frozen in liquid nitrogen and stored at -80 °C until extraction. Tachyzoite samples were generated in T150 dishes by infecting myotubes and HFF cells with 3.2*107 tachyzoites corresponding to an MOI of 3 for 48 h. Medium was replaced by ice-cold PBS and monolayers were scraped and passaged through a 27G needle. Tachyzoites were filter-purified through a 3 µm filter (Whatman) and PBS-washed by centrifugation (1,200 x g, 10 min, 0 °C) three times. All samples were extracted simultaneously in 80 % acetonitrile for LC/MS analysis as described below. Bead-only controls were processed equally. Bead-supplemented tachyzoite controls were processed equally to cyst samples, replacing washing steps via magnetic stand by centrifugation (1,200 x g, 10 min, 0 °C). Preparation of beads Coupling of DynabeadsTM MyONETM Streptavdin T1 (Thermo Fisher Scientific) to DBA was done as described in the manufacturer's protocol. Briefly, 200 µl beads/sample were resuspended in 1 ml PBS by vortexing, washed three times with PBS in a magnetic stand and resuspended in 1 ml PBS containing 50 µg DBA / sample. The tube containing the DBA-magnetic bead mixture was incubated on a rotary mixer for 45 min at RT. Uncoupled DBA was removed by washing the coated beads three times with PBS. After washing, the DBA-coated beads were resuspended in 2 ml PBS containing 2 % BSA."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"Chromatographic separation was achieved on a Vanquish Flex fitted with an ACQUITY UPLC BEH Amide column (Waters). Running a 25 min linear gradient starting with 90 % eluent A (10 mM ammonium carbonate in acetonitrile) / 10 % eluent B (10 mM ammonium carbonate in water) and ending with 40 % eluent A / 60 % eluent B, followed by washing and equilibration steps.","CHROMATOGRAPHY_TYPE":"HILIC","INSTRUMENT_NAME":"Thermo Vanquish","COLUMN_NAME":"Waters Acquity BEH Amide (150 x 2.1mm, 1.7um)"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive Plus Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"UNSPECIFIED","MS_COMMENTS":"The injection order of the samples was randomized, blanks and PBQCs were injected periodically. The samples were analyzed on a Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific) via 70k MS1 scans, with intermittent 35k data-dependent 35k MS2 scans in positive and negative mode separately.XCalibur 4.2.47 software and Compound Discoverer 3.1 software (Thermo Fisher Scientific) was used for recording and used for peak detection, combination of adducts and compound annotation, respectively. Metabolite identifications were based on either retention time and accurate mass match to an in-house library of 160 authentic standards, or by matching accurate mass and MS2 fragments to m/z cloud database (mirrored offline in m/z vault v 2.1.22.15 in May 2020) (Thermo Fisher Scientific). Data were exported to Excel for grouping, combination of datasets from positive and negative ionization runs and blank subtraction.."},

"MS_METABOLITE_DATA":{
"Units":"counts",

"Data":[{"Metabolite":"Glycine","PP_invitrocysts_mock_1":"","PP_invitrocysts_mock_2":"","PP_invitrocysts_mock_3":"","PP_invitrocysts_mock_4":"","PP_tachys_mock_1":"","PP_tachys_mock_2":"","PP_tachys_mock_3":"","PP_tachys_mock_4":"","PP_tachys_mock_5":"","PP_uninf_mock_1":"","PP_uninf_mock_2":"","PP_Blank_1":"","PP_Blank_2":"","PP_Blank_3":"","PP_invitrocysts_NaFAc_1":"","PP_invitrocysts_NaFAc_2":"","PP_invitrocysts_NaFAc_3":"","PP_invitrocysts_NaFAc_4":"","PP_tachys_NaFAc_1":"","PP_tachys_NaFAc_2":"","PP_tachys_NaFAc_3":"","PP_uninf_NaFAc_1":"","PP_uninf_NaFAc_2":"","NP_invitrocysts_mock_1":"","NP_invitrocysts_mock_2":"","NP_invitrocysts_mock_3":"","NP_invitrocysts_mock_4":"","NP_tachys_mock_1":"5134818","NP_tachys_mock_2":"4320930","NP_tachys_mock_3":"5465900","NP_tachys_mock_4":"2788015","NP_tachys_mock_5":"3791259","NP_uninf_mock_1":"","NP_uninf_mock_2":"","NP_Blank_1":"","NP_Blank_2":"","NP_Blank_3":"","NP_invitrocysts_NaFAc_1":"","NP_invitrocysts_NaFAc_2":"","NP_invitrocysts_NaFAc_3":"","NP_invitrocysts_NaFAc_4":"","NP_tachys_NaFAc_1":"13792232","NP_tachys_NaFAc_2":"11797835","NP_tachys_NaFAc_3":"12281033","NP_uninf_NaFAc_1":"","NP_uninf_NaFAc_2":""},{"Metabolite":"Crotonic 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5'-monophosphate","retention time (min)":"15.1","MW":"347.0629"},{"Metabolite":"Nervonic acid","retention time (min)":"1.5","MW":"366.3494"},{"Metabolite":"UDPGlucNAc","retention time (min)":"13.7","MW":"607.0816"},{"Metabolite":"Coenzyme Q7","retention time (min)":"2.0","MW":"658.4957"}]
}

}