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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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.