Summary of Study ST002053

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	ST002053
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-Institute
Last Name	Blume
First Name	Martin
Address	Seestr. 10, Berlin, Berlin, 13353, Germany
Email	blumem@rki.de
Phone	+49 30 18754 2572
Submit Date	2022-01-07
Raw Data Available	Yes
Raw Data File Type(s)	raw(Thermo)
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:	SU002135
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
SA193652	NP_Blank_2	NP_Blank
SA193653	NP_Blank_1	NP_Blank
SA193654	NP_Blank_3	NP_Blank
SA193659	NP_invitrocysts_mock_3	NP_invitrocysts_mock
SA193660	NP_invitrocysts_mock_4	NP_invitrocysts_mock
SA193661	NP_invitrocysts_mock_2	NP_invitrocysts_mock
SA193662	NP_invitrocysts_mock_1	NP_invitrocysts_mock
SA193655	NP_invitrocysts_NaFAc_1	NP_invitrocysts_NaFAc
SA193656	NP_invitrocysts_NaFAc_4	NP_invitrocysts_NaFAc
SA193657	NP_invitrocysts_NaFAc_3	NP_invitrocysts_NaFAc
SA193658	NP_invitrocysts_NaFAc_2	NP_invitrocysts_NaFAc
SA193666	NP_tachys_mock_2	NP_tachys_mock
SA193667	NP_tachys_mock_4	NP_tachys_mock
SA193668	NP_tachys_mock_1	NP_tachys_mock
SA193669	NP_tachys_mock_3	NP_tachys_mock
SA193670	NP_tachys_mock_5	NP_tachys_mock_5
SA193663	NP_tachys_NaFAc_1	NP_tachys_NaFAc
SA193664	NP_tachys_NaFAc_2	NP_tachys_NaFAc
SA193665	NP_tachys_NaFAc_3	NP_tachys_NaFAc
SA193673	NP_uninf_mock_2	NP_uninf_mock
SA193674	NP_uninf_mock_1	NP_uninf_mock
SA193671	NP_uninf_NaFAc_1	NP_uninf_NaFAc
SA193672	NP_uninf_NaFAc_2	NP_uninf_NaFAc
SA193675	PP_Blank_3	PP_Blank
SA193676	PP_Blank_2	PP_Blank
SA193677	PP_Blank_1	PP_Blank
SA193682	PP_invitrocysts_mock_1	PP_invitrocysts_mock
SA193683	PP_invitrocysts_mock_4	PP_invitrocysts_mock
SA193684	PP_invitrocysts_mock_3	PP_invitrocysts_mock
SA193685	PP_invitrocysts_mock_2	PP_invitrocysts_mock
SA193678	PP_invitrocysts_NaFAc_2	PP_invitrocysts_NaFAc
SA193679	PP_invitrocysts_NaFAc_1	PP_invitrocysts_NaFAc
SA193680	PP_invitrocysts_NaFAc_3	PP_invitrocysts_NaFAc
SA193681	PP_invitrocysts_NaFAc_4	PP_invitrocysts_NaFAc
SA193689	PP_tachys_mock_4	PP_tachys_mock
SA193690	PP_tachys_mock_3	PP_tachys_mock
SA193691	PP_tachys_mock_2	PP_tachys_mock
SA193692	PP_tachys_mock_1	PP_tachys_mock
SA193693	PP_tachys_mock_5	PP_tachys_mock_5
SA193686	PP_tachys_NaFAc_1	PP_tachys_NaFAc
SA193687	PP_tachys_NaFAc_2	PP_tachys_NaFAc
SA193688	PP_tachys_NaFAc_3	PP_tachys_NaFAc
SA193696	PP_uninf_mock_2	PP_uninf_mock
SA193697	PP_uninf_mock_1	PP_uninf_mock
SA193694	PP_uninf_NaFAc_2	PP_uninf_NaFAc
SA193695	PP_uninf_NaFAc_1	PP_uninf_NaFAc

Showing results 1 to 46 of 46

Showing results 1 to 46 of 46

Collection:

Collection ID:	CO002128
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 ID:	TR002147
Treatment Summary:	Cells were either treated or untreated tachyzoites or treated or untreated bradyzoites (cysts)

Sample Preparation:

Sampleprep ID:	SP002141
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:

SP002141

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	AN003343
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 Q Exactive Plus Orbitrap
Ion Mode	UNSPECIFIED
Units	counts

Chromatography:

Chromatography ID:	CH002475
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:	MS003112
Analysis ID:	AN003343
Instrument Name:	Thermo Q 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