Summary of Study ST002051

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001297. The data can be accessed directly via it's Project DOI: 10.21228/M82H7B This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Study ID	ST002051
Study Title	The apicomplexan parasite Toxoplasma gondii forms bradyzoite-containing tissue cysts that cause chronic and drug-tolerant infections.
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. Metabolomic characterization of purified cysts reveals global changes that comprise increased levels of amino acids and decreased abundance of nucleobase- and tricarboxylic acid cycle-associated metabolites. In contrast to fast replicating tachyzoite forms of T. gondii these tissue cysts tolerate exposure to the aconitase inhibitor sodium fluoroacetate.
Institute	Robert Koch-Institute
Department	NG2
Laboratory	NG2
Last Name	Blume
First Name	Martin
Address	Seestraße 10
Email	blumem@rki.de
Phone	+49 30 18754 2572
Submit Date	2022-01-04
Raw Data Available	Yes
Raw Data File Type(s)	mzML
Analysis Type Detail	LC-MS
Release Date	2022-02-28
Release Version	1

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Project:

Project ID:	PR001297
Project DOI:	doi: 10.21228/M82H7B
Project Title:	In vitro maturation of Toxoplasma gondii bradyzoites in human myotubes and their metabolomic characterization
Project Type:	characterization of in vitro T. gondii stages
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. Metabolomic characterization of purified cysts reveals global changes that comprise increased levels of amino acids and decreased abundance of nucleobase- and tricarboxylic acid cycle-associated metabolites. In contrast to fast replicating tachyzoite forms of T. gondii these tissue cysts tolerate exposure to the aconitase inhibitor sodium fluoroacetate.
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 ID:	SU002133
Subject Type:	Other organism
Subject Species:	Toxoplasma gondii
Taxonomy ID:	5811

Factors:

Subject type: Other organism; Subject species: Toxoplasma gondii (Factor headings shown in green)

mb_sample_id	local_sample_id	Factor
SA193510	0424_Blank_4	Blank
SA193511	0424_Blank_1	Blank
SA193512	0424_Blank_2	Blank
SA193513	0427_Blank_1	Blank
SA193514	0427_Blank_2	Blank
SA193515	0427_Blank_4	Blank
SA193516	0427_Blank_3	Blank
SA193517	0424_Blank_3	Blank
SA193558	0424_NP_T1	NP_bead
SA193559	0427_NP_T1	NP_bead
SA193518	0427_NP_Cysten_T1_2	NP_Cysten_bead
SA193519	0427_NP_Cysten_T1_4	NP_Cysten_bead
SA193520	0424_NP_Cysten_T1_1	NP_Cysten_bead
SA193521	0427_NP_Cysten_T1_3	NP_Cysten_bead
SA193522	0427_NP_Cysten_T1_1	NP_Cysten_bead
SA193523	0424_NP_Cysten_T1_4	NP_Cysten_bead
SA193524	0424_NP_Cysten_T1_3	NP_Cysten_bead
SA193525	0424_NP_Cysten_T1_2	NP_Cysten_bead
SA193526	0427_NP_Tachy_HFF_2	NP_Tachy_HFF
SA193527	0427_NP_Tachy_HFF_4	NP_Tachy_HFF
SA193528	0427_NP_Tachy_HFF_3	NP_Tachy_HFF
SA193529	0424_NP_Tachy_HFF_1	NP_Tachy_HFF
SA193530	0427_NP_Tachy_HFF_1	NP_Tachy_HFF
SA193531	0424_NP_Tachy_HFF_3	NP_Tachy_HFF
SA193532	0424_NP_Tachy_HFF_2	NP_Tachy_HFF
SA193533	0424_NP_Tachy_HFF_4	NP_Tachy_HFF
SA193534	0427_NP_Tachy_HFF_T1_2	NP_Tachy_HFF_bead
SA193535	0427_NP_Tachy_HFF_T1_4	NP_Tachy_HFF_bead
SA193536	0427_NP_Tachy_HFF_T1_3	NP_Tachy_HFF_bead
SA193537	0427_NP_Tachy_HFF_T1_1	NP_Tachy_HFF_bead
SA193538	0424_NP_Tachy_HFF_T1_3	NP_Tachy_HFF_bead
SA193539	0424_NP_Tachy_HFF_T1_1	NP_Tachy_HFF_bead
SA193540	0424_NP_Tachy_HFF_T1_4	NP_Tachy_HFF_bead
SA193541	0424_NP_Tachy_HFF_T1_2	NP_Tachy_HFF_bead
SA193542	0427_NP_Tachy_Tubes_3	NP_Tachy_Tubes
SA193543	0427_NP_Tachy_Tubes_1	NP_Tachy_Tubes
SA193544	0427_NP_Tachy_Tubes_2	NP_Tachy_Tubes
SA193545	0424_NP_Tachy_Tubes_3	NP_Tachy_Tubes
SA193546	0424_NP_Tachy_Tubes_1	NP_Tachy_Tubes
SA193547	0427_NP_Tachy_Tubes_4	NP_Tachy_Tubes
SA193548	0424_NP_Tachy_Tubes_4	NP_Tachy_Tubes
SA193549	0424_NP_Tachy_Tubes_2	NP_Tachy_Tubes
SA193550	0424_NP_Tachy_Tubes_T1_4	NP_Tachy_Tubes_bead
SA193551	0427_NP_Tachy_Tubes_T1_1	NP_Tachy_Tubes_bead
SA193552	0427_NP_Tachy_Tubes_T1_2	NP_Tachy_Tubes_bead
SA193553	0427_NP_Tachy_Tubes_T1_4	NP_Tachy_Tubes_bead
SA193554	0424_NP_Tachy_Tubes_T1_1	NP_Tachy_Tubes_bead
SA193555	0424_NP_Tachy_Tubes_T1_3	NP_Tachy_Tubes_bead
SA193556	0427_NP_Tachy_Tubes_T1_3	NP_Tachy_Tubes_bead
SA193557	0424_NP_Tachy_Tubes_T1_2	NP_Tachy_Tubes_bead
SA193560	0427_NP_uninf_T1_3	NP_uninf_bead
SA193561	0427_NP_uninf_T1_2	NP_uninf_bead
SA193562	0427_NP_uninf_T1_4	NP_uninf_bead
SA193563	0424_NP_uninf_T1_3	NP_uninf_bead
SA193564	0424_NP_uninf_T1_1	NP_uninf_bead
SA193565	0427_NP_uninf_T1_1	NP_uninf_bead
SA193566	0424_NP_uninf_T1_2	NP_uninf_bead
SA193567	0424_NP_uninf_T1_4	NP_uninf_bead
SA193608	0424_PP_T1	PP_bead
SA193609	0427_PP_T1	PP_bead
SA193568	0424_PP_Cysten_T1_2	PP_Cysten_bead
SA193569	0424_PP_Cysten_T1_1	PP_Cysten_bead
SA193570	0427_PP_Cysten_T1_4	PP_Cysten_bead
SA193571	0427_PP_Cysten_T1_3	PP_Cysten_bead
SA193572	0424_PP_Cysten_T1_4	PP_Cysten_bead
SA193573	0424_PP_Cysten_T1_3	PP_Cysten_bead
SA193574	0427_PP_Cysten_T1_1	PP_Cysten_bead
SA193575	0427_PP_Cysten_T1_2	PP_Cysten_bead
SA193576	0427_PP_Tachy_HFF_2	PP_Tachy_HFF
SA193577	0427_PP_Tachy_HFF_4	PP_Tachy_HFF
SA193578	0427_PP_Tachy_HFF_1	PP_Tachy_HFF
SA193579	0427_PP_Tachy_HFF_3	PP_Tachy_HFF
SA193580	0424_PP_Tachy_HFF_2	PP_Tachy_HFF
SA193581	0424_PP_Tachy_HFF_1	PP_Tachy_HFF
SA193582	0424_PP_Tachy_HFF_3	PP_Tachy_HFF
SA193583	0424_PP_Tachy_HFF_4	PP_Tachy_HFF
SA193584	0427_PP_Tachy_HFF_T1_2	PP_Tachy_HFF_bead
SA193585	0427_PP_Tachy_HFF_T1_4	PP_Tachy_HFF_bead
SA193586	0427_PP_Tachy_HFF_T1_1	PP_Tachy_HFF_bead
SA193587	0427_PP_Tachy_HFF_T1_3	PP_Tachy_HFF_bead
SA193588	0424_PP_Tachy_HFF_T1_3	PP_Tachy_HFF_bead
SA193589	0424_PP_Tachy_HFF_T1_1	PP_Tachy_HFF_bead
SA193590	0424_PP_Tachy_HFF_T1_2	PP_Tachy_HFF_bead
SA193591	0424_PP_Tachy_HFF_T1_4	PP_Tachy_HFF_bead
SA193592	0427_PP_Tachy_Tubes_3	PP_Tachy_Tubes
SA193593	0427_PP_Tachy_Tubes_2	PP_Tachy_Tubes
SA193594	0424_PP_Tachy_Tubes_1	PP_Tachy_Tubes
SA193595	0427_PP_Tachy_Tubes_1	PP_Tachy_Tubes
SA193596	0427_PP_Tachy_Tubes_4	PP_Tachy_Tubes
SA193597	0424_PP_Tachy_Tubes_4	PP_Tachy_Tubes
SA193598	0424_PP_Tachy_Tubes_2	PP_Tachy_Tubes
SA193599	0424_PP_Tachy_Tubes_3	PP_Tachy_Tubes
SA193600	0427_PP_Tachy_Tubes_T1_2	PP_Tachy_Tubes_bead
SA193601	0427_PP_Tachy_Tubes_T1_4	PP_Tachy_Tubes_bead
SA193602	0427_PP_Tachy_Tubes_T1_1	PP_Tachy_Tubes_bead
SA193603	0427_PP_Tachy_Tubes_T1_3	PP_Tachy_Tubes_bead
SA193604	0424_PP_Tachy_Tubes_T1_2	PP_Tachy_Tubes_bead
SA193605	0424_PP_Tachy_Tubes_T1_3	PP_Tachy_Tubes_bead
SA193606	0424_PP_Tachy_Tubes_T1_1	PP_Tachy_Tubes_bead
SA193607	0424_PP_Tachy_Tubes_T1_4	PP_Tachy_Tubes_bead

Showing page 1 of 2 Results: 1 2 Next Showing results 1 to 100 of 108

Collection:

Collection ID:	CO002126
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:	Cultured human myotubes with Toxoplasma gondii

Treatment:

Treatment ID:	TR002145
Treatment Summary:	Cells were either uninfected, tachyzoites, tachyzoites with beads, bradyzoites (cysts), beads only

Sample Preparation:

Sampleprep ID:	SP002139
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 3.2106 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 3.2107 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.

Sampleprep ID:

SP002139

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.

Combined analysis:

Analysis ID	AN003338
Analysis type	MS
Chromatography type	HILIC
Chromatography system	Thermo Vanquish
Column	Waters Acquity BEH Amide (150 x 2.1mm,1.7um)
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Exactive Plus Orbitrap
Ion Mode	UNSPECIFIED
Units	counts

Chromatography:

Chromatography ID:	CH002472
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.
Instrument Name:	Thermo Vanquish
Column Name:	Waters Acquity BEH Amide (150 x 2.1mm,1.7um)
Flow Gradient:	Running a 25 min linear gradient starting with 90% A / 10% B and ending with 40% eluent A / 60% eluent B, followed by washing and equilibration steps.
Solvent A:	100% acetonitrile; 10 mM ammonium carbonate
Solvent B:	100% water; 10 mM ammonium carbonate
Chromatography Type:	HILIC

MS:

MS ID:	MS003107
Analysis ID:	AN003338
Instrument Name:	Thermo Exactive Plus Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
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.
Ion Mode:	UNSPECIFIED