DOI: 10.5061/dryad.b2rbnzsjd
title: Part 1: Kiss and spit metabolomics highlight the role of host purine metabolism during pathogen infection

description: Intracellular bacteria and protists rely on the host cell to supply many
 metabolites, but the mechanisms through which pathogens manipulate host
 metabolism to their benefit are not understood. Here, we demonstrate that
 when the obligate intracellular parasite Toxoplasma gondii secretes its
 rhoptry organelle contents into the host cytoplasm before invasion—a
 process called “kiss and spit”—host cell metabolite abundance is altered
 in nucleotide synthesis, the pentose phosphate pathway, glycolysis, and
 amino acid synthesis. U-13C6clabeling metabolomics confirmed that kiss and
 spit increased the flow of carbon through the pentose phosphate pathway
 and nucleotide synthesis. An increase in 2,3-bisphosphoglycerate abundance
 led us to investigate the activation of host cytosolic nucleosidase II
 (cN-II) to provide purines for the parasite. We found that T.
 gondii manipulates the host cN-II enzyme to dephosphorylate GMP and IMP
 that it needs for replication. Further, we found that the approved
 anti-cancer drug fludarabine, which inhibits cN-II, also
 inhibits Toxoplasma replication. These results
 reveal Toxoplasma host cell manipulation and highlight
 potential therapies for toxoplasmosis. There are several datasets related
 to T. gondii kiss and spit Part 1: Kiss and spit metabolomics
 highlight the role of host purine metabolism during pathogen infection:
 10.5061/dryad.b2rbnzsjd : Time course of T. gondii kiss and spit-HFF cells
 metabolomics Part 2: Kiss and spit metabolomics highlight the
 role of host purine metabolism during pathogen infection:
 10.5061/dryad.69p8cz9b5:  U-13C6 labeling of ME49 T.
 gondii  kiss and spit and full infection in HFF cells Part
 3: Kiss and spit metabolomics highlight the role of host purine
 metabolism during pathogen infection: 10.5061/dryad.9p8cz8wrn:
  Effect of fludarabine on purine metabolism in T.
 gondii infected HFF host cells Part 4: Kiss and spit
 metabolomics highlight the role of host purine metabolism during pathogen
 infection: 10.5061/dryad.7d7wm383s: ME49T. gondii infected
 MDAMB231 cells Metabolomics at 24 and 48 HPI Part 5: Kiss and
 spit metabolomics highlight the role of host purine metabolism during
 pathogen infection: 10.5061/dryad.ghx3ffbxx: Effect of AMP addition on
 purine metabolism in T. gondii infected host cells at 48
 HPI Part 6: Kiss and spit metabolomics highlight the role of host
 purine metabolism during pathogen infection: 10.5061/dryad.zkh1893jn: ME49
 T. gondii Kiss and spit negative controls
description: Kiss and spit time course metabolomics HFFs
 were seeded in 60 mm dishes and allowed to reach confluency. Then, HFF
 dishes in triplicate were treated with 2 x 10<sup>6</sup> ME49
 tachyzoites that had been pre-incubated with 1.5 μM cytochalasin D (Cyt D)
 for 15 minutes (Sigma-Aldrich) or an equal addition of media by volume. An
 additional negative control of media only treated with cytochalasin D was
 added to a separate set of dishes. Cyt D is a reversible actin inhibitor,
 for this reason it was kept in the media all the time during the
 experiments. At time points 1.5, 3, 6, 9, and 12 HPK&S, dishes
 were washed three times with ice cold PBS, then quenched with 80:20 HPLC
 grade Methanol: Water (Sigma-Aldrich). Dishes were incubated on dry ice at
 -80°C for 15 minutes. Plates were scraped, the solution removed, and spun
 at 2500 x g for 5 minutes at 4°C. The supernatant was removed and stored
 on ice, then the pellet was washed again in quenching solution and
 re-spun. Supernatants were combined, dried down under
 N<sub>2</sub>, and stored at -80°C.
  Samples were resuspended in 100 µL HPLC grade water (Fisher
 Optima) for analysis on a Thermo Fisher Vanquish Horizon UHPLC coupled to
 an electrospray ionization source (HESI) part of a hybrid
 quadrupole-Orbitrap high resolution mass spectrometer (Q Exactive
 Orbitrap; Thermo Scientific). Chromatography was performed using a 100 mm
 x 2.1 mm x 1.7 µm BEH C18 column (Acquity) at 30°C. 20 µL of the sample
 was injected via an autosampler at 4°C and flow rate was 200 µL/min.
 Solvent A was 97:3 water/methanol with 10 mM tributylamine (TBA)
 (Sigma-Aldrich) adjusted to a pH of 8.2 using approximately 9 mM Acetate
 (final concentration, Sigma-Aldrich). Solvent B was 100% methanol with no
 TBA (Sigma-Aldrich). Products were eluted in 95% A / 5% B for 2.5 minutes,
 then a gradient of 95% A / 5% B to 5% A / 95% B over 14.5 minutes, then
 held for an additional 2.5 minutes at 5%A / 95%B. Finally, the gradient
 was returned to 95% A / 5% B over 0.5 minutes and held for 5 minutes to
 re-equilibrate the column. MS parameters included: scan in negative mode;
 scan range = 70 - 1000 m/z; Automatic Gain control (AGC) = 1e6, spray
 voltage = 3.0 kV, maximum ion collection time = 40 ms, and capillary
 temperature = 350C. Peaks were matched to known standards for
 identification. Data analysis was performed using the Metabolomics
 Analysis and Visualization Engine (MAVEN) software (Clasquin et al.,
 2012). Data analysis was performed using
 the Metabolomics Analysis and Visualization Engine (MAVEN)
 software.  Clasquin MF, Melamud E, Rabinowitz JD. LC-MS
 data processing with MAVEN: a metabolomic analysis and visualization
 engine. Curr Protoc Bioinformatics [Internet]. 2012 Mar;Chapter
 14(SUPPL.37):Unit14.11. Available from: http://www.ncbi.nlm.nih.gov/pubmed/22389014
description: # *Me49 T. gondii* Kiss and spit in HFF cells metabolomics
 [https://doi.org/10.5061/dryad.b2rbnzsjd](https://doi.org/10.5061/dryad.b2rbnzsjd) We performed a time course mass spectrometry-based analysis of the impact of *T. gondii* on host cell metabolism using a kiss and spit in vitro tissue culture model and a metabolomics protocol that have demonstrated effectiveness specifically for the metabolites of interest in mammalian cell cultures. By pretreating *T. gondii* with the actin polymerization inhibitor cytochalasin D (Cyt D), we allowed *T. gondii* to secrete the contents of the rhoptries into host cells while preventing infection. ## Description of the data and file structure Samples were run in triplicated.  *T. gondii* Kiss and spit HFF cells or Uninfected Cytochalasin D (Cyt D) treated HFF cell samples are indicated in the name of the sample. The HPK&S (hours post kiss and spit) or hours (H) are indicated in the name of the sample.  * 1A.Infected_1.5_HPK_S.mzXML: *T. gondii* -kiss and spit- HFF cells after 1.5 HPK&S  replicate 1 * 1B.Infected_1.5_HPK_S.mzXML: *T. gondii* -kiss and spit- HFF cells after 1.5 HPK&S  replicate 2 * 1C.Infected_1.5_HPK_S.mzXML: *T. gondii* -kiss and spit- HFF cells after 1.5 HPK&S  replicate 3 * 2A.Infected_3_HPK_S.mzXML : *T. gondii* -kiss and spit- HFF cells after 3 HPK&S  replicate 1 * 2B.Infected_3_HPK_S.mzXML: *T. gondii* -kiss and spit- HFF cells after 3 HPK&S  replicate 2 * 2C.Infected_3_HPK_S.mzXML: *T. gondii* -kiss and spit- HFF cells after 3 HPK&S  replicate 3 * 3A.Infected_6_HPK_S.mzXML : *T. gondii* -kiss and spit- HFF cells after 6 HPK&S   replicate 1 * 3B.Infected_6_HPK_S.mzXML mzXML : *T. gondii* -kiss and spit- HFF cells after 6 HPK&S  replicate 2 * 3C.Infected_6_HPK_S.mzXML mzXML : *T. gondii* -kiss and spit- HFF cells after 6 HPK&S  replicate 3 * 4A.Infected_9_HPK_S.mzXML mzXML : *T. gondii* -kiss and spit- HFF cells after 9 HPK&S  replicate 1 * 4B.Infected_9_HPK_S.mzXML mzXML : *T. gondii* -kiss and spit- HFF cells after 6 HPK&S  replicate 2 * 4C.Infected_9_HPK_S.mzXML mzXML : *T. gondii* -kiss and spit- HFF cells after 6 HPK&S  replicate 3 * 5A.Infected_12_HPK_S.mzXML: mzXML : *T. gondii* -kiss and spit- HFF cells after 12 HPK&S  replicate 1 * 5B.Infected_12_HPK_SI.mzXML: *T. gondii* -kiss and spit- HFF cells after 12 HPK&S   replicate 2 * 5C.Infected_12_HPK_S.mzXML: : *T. gondii* -kiss and spit- HFF cells after 12 HPK&S  replicate 3 * 6A.Uninfected_1.5_H.mzXML: Uninfected- Cyt D treated-HFF cells after 1.5 H replicate 1 * 6B.Uninfected_1.5_H.mzXML: Uninfected- Cyt D treated-HFF cells after 1.5 H replicate 2 * 6C.Uninfected_1.5_H.mzXML: Uninfected- Cyt D treated-HFF cells after 1.5 H replicate 3 * 7A.Uninfected_3_H.mzXML: Uninfected- Cyt D treated-HFF cells after 3 H replicate 1 * 7B.Uninfected_3_H.mzXML: Uninfected- Cyt D treated-HFF cells after 3 H replicate 2 * 7C.Uninfected_3_H.mzXML: Uninfected- Cyt D treated-HFF cells after 3 H replicate 3 * 8A.Uninfected_6_H.mzXML: Uninfected- Cyt D treated-HFF cells after 6 H replicate 1 * 8B.Uninfected_6_H.mzXML: Uninfected- Cyt D treated-HFF cells after 6 H replicate 2 * 8C.Uninfected_6_H.mzXML: Uninfected- Cyt D treated-HFF cells after 6 H replicate 3 * 9A.Uninfected_9_H.mzXML: Uninfected- Cyt D treated-HFF cells after 9 H replicate 1 * 9B.Uninfected_9_H.mzXML: Uninfected- Cyt D treated-HFF cells after 9 H replicate 2 * 9C.Uninfected_9_H.mzXML: Uninfected- Cyt D treated-HFF cells after 9 H replicate 3 * 10A.Uninfected_12_H.mzXML: Uninfected- Cyt D treated-HFF cells after 12 H replicate 1 * 10B.Uninfected_12_H.mzXML: Uninfected- Cyt D treated-HFF cells after 12 H replicate 2 * 10C.Uninfected_12_H.mzXML: Uninfected- Cyt D treated-HFF cells after 12 H replicate 3 ### **Code/Software** Data analysis was performed using the Metabolomics Analysis and Visualization Engine (MAVEN) software