DOI: 10.5061/dryad.69p8cz9b5
title: Part 2: 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-13C6 labeling 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: U-13C6 glucose metabolomic experiment
 Metabolomic analysis and U-13C6 glucose labeling of HFF cells
 infected with <em>T. gondii</em> was performed as previously
 described (Khalifa et al., 2020). HFFs were grown to deep quiescence in 60
 mm dishes in triplicate, then (1) infected with 2 X
 10<sup>6</sup> tachyzoites of <em>T. gondii</em>
 ME49 strain for full infection; (2) equal amount of parasite treated with
 1.5 µM Cytochalasin D for kiss and spit; (3) mock-infected with an equal
 volume of media as negative control and (4) media plus equal concentration
 of cytochalasin D as negative control as well. At 9 hours post-infection
 (HPI), the media was changed to glucose-free RPMI1640 supplemented to 1
 g/L with D-Glucose- U-13C6 (Sigma-Aldrich #389374). After 15 and 30
 minutes of labeling, dishes were washed 3x with ice-cold PBS, then
 quenched with 80:20 HPLC grade Methanol: Water and incubated on dry ice in
 a -80°C for 15 minutes. Plates were scraped, the solution washed twice and
 spun at 2500 x g for 5 minutes at 4°C. Supernatants were combined, dried
 down under N<sub>2</sub> gas manifold, and resuspended in 100
 µL HPLC grade water for analysis on a Thermo-Fisher Vanquish Horizon UHPLC
 joined by electrospray ionization (negative mode) to 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 4̊C, and flow rate was 200 µL/minute.
 Solvent A was 97:3 water/methanol with 9 mM Acetate and 10 mM
 tributylamine (TBA) with a pH of 8.2 (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. The gradient
 was returned to 95% A/5% B over 0.5 minutes and held for 5 minutes to
 re-equilibrate the column. Data analysis was performed using the
 Metabolomics Analysis and Visualization Engine (MAVEN) software. The
 experiment was performed twice in triplicate.
description: # Part 2: Kiss and spit metabolomics highlight the role of host purine
 metabolism during pathogen infection The Data analysis was performed using
 the Metabolomics Analysis and Visualization Engine (MAVEN) software. ###
 U-13C6 labeling of ME49 *T. gondii* kiss and spit and full infection in
 HFF cells
 [https://doi.org/10.5061/dryad.69p8cz9b5](https://doi.org/10.5061/dryad.69p8cz9b5) ## Description of the data and file structure The following samples were run in triplicate: HFF : uninfected cells HFF_CD: uninfected cells plus Cytochalasin D HFF_ME49: infected cells HFF_*CD*_ME49: kiss and spit cells Samples from 1-24 correspond to the first experiment and samples from 25-44 correspond to second experiment. Pre-blank samples and standards are included: * 1_HFF_30.mzXML: Uninfected HFF cell labeled for 30 minutes, Experiment 1, replicate 1 * 2_HFF_30.mzXML: Uninfected HFF cell labeled for 30 minutes, Experiment 1, replicate 2 * 3_HFF_30.mzXML: Uninfected HFF cell labeled for 30 minutes, Experiment 1, replicate 3 * 4_HFF_15.mzXML: Uninfected HFF cell labeled for 15 minutes, Experiment 1,  replicate 1 * 5_HFF_15.mzXML: Uninfected HFF cell labeled for 15 minutes, Experiment 1,  replicate 2 * 6_HFF_15.mzXML: Uninfected HFF cell labeled for 15 minutes, Experiment 1,  replicate 3 * 7_HFF_CD_30.mzXML: uninfected HFF cells plus Cytochalasin D, labeled for 30 minutes, Experiment 1, replicate 1 * 8_HFF_CD_30.mzXML: uninfected HFF cells plus Cytochalasin D, labeled for 30 minutes, Experiment 1, replicate 2 * 9_HFF_CD_30.mzXML :uninfected HFF cells plus Cytochalasin D, labeled for 30 minutes, Experiment 1, replicate 3 * 10_HFF_CD_15.mzXML :uninfected HFF cells plus Cytochalasin D, labeled for 15 minutes, Experiment 1, replicate 1 * 11_HFF_CD_15.mzXML: uninfected HFF cells plus Cytochalasin D, labeled for 15 minutes, Experiment 1, replicate 2 * 12_HFF_CD_15.mzXML: uninfected HFF cells plus Cytochalasin D, labeled for 15 minutes, Experiment 1, replicate 3 * 13_HFF_CD_ME49_30.mzXML: *T. gondii* ME49 kiss and spit- HFF cells, labeled for 30 minutes, Experiment 1, replicate 1 * 14_HFF_CD_ME49_30.mzXML: *T. gondii* ME49 kiss and spit- HFF cells, labeled for 30 minutes, Experiment 1, replicate 2 * 15_HFF_CD_ME49_30.mzXML: *T. gondii* ME49 kiss and spit- HFF cells, labeled for 30 minutes, Experiment 1, replicate 3 * 16_HFF_CD_ME49_15.mzXML: *T. gondii* ME49 kiss and spit- HFF cells, labeled for 15 minutes, Experiment 1, replicate 1 * 17_HFF_CD_ME49_15.mzXML: *T. gondii* ME49 kiss and spit- HFF cells, labeled for 15 minutes, Experiment 1, replicate 2 * 18_HFF_CD_ME49_15.mzXML: *T. gondii* ME49 kiss and spit- HFF cells, labeled for 15 minutes, Experiment 1, replicate 3 * 19_HFF_ME49_30.mzXML: *T. gondii* ME49 infected- HFF cells,labeled for 30 minutes, Experiment 1, replicate 1 * 20_HFF_ME49_30.mzXML: *T. gondii* ME49 infected- HFF cells, labeled for 30 minutes, Experiment 1, replicate 2 * 21_HFF_ME49_30.mzXML: *T. gondii* ME49 infected- HFF cells, labeled for 30 minutes, Experiment 1, replicate 3 * 22_HFF_ME49_15.mzXML: *T. gondii* ME49 infected- HFF cells, labeled for 15 minutes, Experiment 1, replicate 1 * 23_HFF_ME49_15.mzXML: *T. gondii* ME49 infected- HFF cells, labeled for 15 minutes, Experiment 1, replicate 2 * 24_HFF_ME49_15.mzXML: *T. gondii* ME49 infected- HFF cells, labeled for 15 minutes, Experiment 1, replicate 3 * 25_HFF_7_30.mzXML: Uninfected HFF cell labeled for 30 minutes, Experiment 2, replicate 1 * 26_HFF_9_30.mzXML: Uninfected HFF cell labeled for 30 minutes, Experiment 2, replicate 2 * 27_HFF_11_30.mzXML: Uninfected HFF cell labeled for 30 minutes, Experiment 2, replicate 3 * 28_HFF_13_15_f1.mzXML: Uninfected HFF cell labeled for 15 minutes, Experiment 2, replicate 1 * 29_HFF_15_15_f1.mzXML: Uninfected HFF cell labeled for 15 minutes, Experiment 2, replicate 2 * 30_HFF_CD_23_30_f1.mzXML: Uninfected HFF cell labeled for 15 minutes, Experiment 2, replicate 3 * 31_HFF_CD_25_30.mzXML: uninfected HFF cells plus Cytochalasin D, labeled for 30 minutes, Experiment 2, replicate 1 * 32_HFF_CD_27_30.mzXML: uninfected HFF cells plus Cytochalasin D, labeled for 30 minutes, Experiment 2, replicate 2 * 33_HFF_CD_29_15.mzXML: uninfected HFF cells plus Cytochalasin D, labeled for 15 minutes, Experiment 2, replicate 1 * 34_HFF_CD_31_15.mzXML: uninfected HFF cells plus Cytochalasin D, labeled for 15 minutes, Experiment 2, replicate 2 * 35_HFF_CD_ME49_39_30.mzXML: *T. gondii* ME49 kiss and spit- HFF cells, labeled for 30 minutes, Experiment 2, replicate 1 * 36_HFF_CD_ME49_41_30.mzXML *T. gondii* ME49 kiss and spit- HFF cells, labeled for 30 minutes, Experiment 2, replicate 2 * 37_HFF_CD_ME49_43_30.mzXML *T. gondii* ME49 kiss and spit- HFF cells, labeled for 30 minutes, Experiment 2, replicate 3 * 38_HFF_CD_ME49_45_15.mzXML: *T. gondii* ME49 kiss and spit- HFF cells, labeled for 15minutes, Experiment 2, replicate 1 * 39_HFF_CD_ME49_47_15.mzXML: *T. gondii* ME49 kiss and spit- HFF cells, labeled for 15minutes, Experiment 2, replicate 2 * 40_HFF_ME49_55_30.mzXML: *T. gondii* ME49 infected- HFF cells, labeled for 30 minutes, Experiment 2, replicate 1 * 41_HFF_ME49_57_30.mzXML *T. gondii* ME49 infected- HFF cells, labeled for 30 minutes, Experiment 2, replicate 2 * 42_HFF_ME49_59_30.mzXML *T. gondii* ME49 infected- HFF cells, labeled for 30 minutes, Experiment 2, replicate 3 * 43_HFF_ME49_61_15.mzXML *T. gondii* ME49 infected- HFF cells, labeled for 15 minutes, Experiment 2, replicate 1 * 44_HFF_ME49_63_15.mzXML *T. gondii* ME49 infected- HFF cells, labeled for 15 minutes, Experiment 2, replicate 2 * FBP_v415m_f1.mzXML: fructose 1,6, biphosphate standard * S7P_v415m_f1.mzXML : sedoheptulose-7-phosphate standard * NADH_v415m_f1.mzXML: NADH standard * NADPH_v415m_f1.mzXML: NADPH standard * NADplus_v415m_f1.mzXML: NAD standard * NADPplus_v415m_f1.mzXML: NAD standard * PreBlnk1_v415m_f1.mzXML: Blank sample 1 * PreBlnk2_v415m_f1.mzXML: Blank sample 2 * PreBlnk3_v415m_f1.mzXML: Blank sample 3