DOI: 10.5061/dryad.9p8cz8wrn
title: Part 3: 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: To understand the mechanism of fludarabine growth inhibition, we
 performed metabolomics on the treated infected HFF cells. We performed
 metabolomics of infected HFF cells comparing infections with the parental
 Pru WT and PruΔHXGPRT <em>T. gondii</em> strains in the
 presence and absence of fludarabine. HFF were seeded in
 60 mm dishes in triplicate and allowed to reach confluency. 36h before the
 procedure, the DMEM media was changed to metabolomic media. Then, each
 dish was infected with 2 x 10<sup>6</sup>  Pru or PruΔHXGPRT
 tachyzoites and treated with 50 μM fludarabine or solvent only
 control.  At 24 hours post infection, dishes were
 washed three times with PBS, spun, and then metabolites were extracted and
 analyzed using the previously published methodology 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. 10 μM of AMP, GMP, IMP, adenosine, adenine, Guanine,
 Guanosine, and Inosine -HPLC standard were analyzed simultaneously. Data
 analysis was performed using the Metabolomics Analysis and Visualization
 Engine (MAVEN) software Fludarabine affected the
 abundance of purines in <em>T. gondii</em> infected-HFF cells
 at 24 HPI. IMP and GMP, the preferred substrates of the cN-II enzyme,
 accumulated when treated with fludarabine, as we expected. The nucleobase
 products of the cN-II reaction, inosine, guanosine, were significantly
 less abundant with fludarabine treatment. 
description: # Effect of fludarabine on purine metabolism in *T. gondii* infected HFF
 host cells
 [https://doi.org/10.5061/dryad.9p8cz8wrn](https://doi.org/10.5061/dryad.9p8cz8wrn) ## Description of the data and file structure The following samples were run in triplicate: HFF : uninfected cells HFF_Fludarabine: uninfected cells plus fludarabine HFF_*Pru*_wt: Pru WT *T. gondii* Infected cells HFF_*Pru_wt*_Fludarabine: Pru WT *T. gondii* Infected cells plus fludarabine HFF_*Pru_*Delta: Pru delta HXGPRT *T. gondii* Infected cells HFF_*Pru_Delta*_Fludarabine: Pru delta HXGPRT *T. gondii* Infected cells plus fludarabine Samples without any additional letters correspond to the first experiment and samples from Aa to Xa correspond to second experiment. Pre-blank samples and standards at 10 uM are included. * HFF1_v412m_f1.mzXML: Uninfected HFF cells, Experiment 1, replicate 1 * HFF2_v412m_f1.mzXML: Uninfected HFF cells, Experiment 1, replicate 2 * HFF3_v412m_f1.mzXML: Uninfected HFF cells, Experiment 1, replicate 3 * HFF_Fludarabine1_v412m_f1.mzXML: Uninfected HFF cells treated with fludarabine, Experiment 1, replicate 1 * HFF_Fludarabine2_v412m_f1.mzXML: Uninfected HFF cells treated with fludarabine, Experiment 1, replicate 2 * HFF_Fludarabine3_v412m_f1.mzXML: Uninfected HFF cells treated with fludarabine, Experiment 1, replicate 3 * HFF_Pru_wt1_v412m_f1.mzXML:Pru wt *T. gondii* infected HFF cells, Experiment 1, replicate 1 * HFF_Pru_wt2_v412m_f1.mzXML:Pru wt *T. gondii* infected HFF cells, Experiment 1, replicate 2 * HFF_Pru_wt3_v412m_f1.mzXML:Pru wt *T. gondii* infected HFF cells, Experiment 1, replicate 3 * HFF_Pru_wt_Fludarabine1_v412m_f1.mzXML: Pru wt *T. gondii* infected HFF cells treated with fludarabine, Experiment 1, replicate 1 * HFF_Pru_wt_Fludarabine2_v412m_f1.mzXML :Pru wt *T. gondii* infected HFF cells treated with fludarabine, Experiment 1, replicate 2 * HFF_Pru_wt_Fludarabine3_v412m_f1.mzXML :Pru wt *T. gondii* infected HFF cells treated with fludarabine, Experiment 1, replicate 3 * HFF_Pru_Delta1_v412m_f1.mzXML: Pru delta HXGPRT *T. gondii* infected HFF cells, Experiment 1, replicate 1 * HFF_Pru_Delta2_v412m_f1.mzXML: Pru delta HXGPRT *T. gondii* infected HFF cells, Experiment 1, replicate 2 * HFF_Pru_Delta3_v412m_f1.mzXML: Pru delta HXGPRT *T. gondii* infected HFF cells, Experiment 1, replicate 3 * HFF_Pru_Delta_Fludarabine1_v412m_f1.mzXML: Pru delta HXGPRT *T. gondii* infected HFF cells treated with fludarabine, Experiment 1, replicate 1 * HFF_Pru_Delta_Fludarabine2_v412m_f1.mzXML: Pru delta HXGPRT *T. gondii* infected HFF cells treated with fludarabine, Experiment 1, replicate 2 * HFF_Pru_Delta_Fludarabine3_v412m_f1.mzXML: Pru delta HXGPRT *T. gondii* infected HFF cells treated with fludarabine, Experiment 1, replicate 3   * Aa_HFF1_v412m_f1.mzXML: Uninfected HFF cells, Experiment 2, replicate 1 * Ba_HFF2_v412m_f1.mzXML: Uninfected HFF cells, Experiment 2, replicate 2 * Ca_HFF3_v412m_f1.mzXML: Uninfected HFF cells, Experiment 2, replicate 3 * Da_HFF_Fludarabine1_v412m_f1.mzXML:Uninfected HFF cells treated with fludarabine, Experiment 2, replicate 1 * Ea_HFF_Fludarabine2_v412m_f1.mzXML:Uninfected HFF cells treated with fludarabine, Experiment 2, replicate 2 * Fa_HFF_Fludarabine3_v412m_f1.mzXML:Uninfected HFF cells treated with fludarabine, Experiment 2, replicate 3 * Ma_HFF_Pru_wt1_v412m_f1.mzXML:Pru wt *T. gondii* infected HFF cells, Experiment 2, replicate 1 * Na_HFF_Pru_wt2_v412m_f1.mzXML:Pru wt *T. gondii* infected HFF cells, Experiment 2, replicate 2 * Oa_HFF_Pru_wt3_v412m_f1.mzXML:Pru wt *T. gondii* infected HFF cells, Experiment 2, replicate 3 * Pa_HFF_Pru_wt_Fludarabine1_v412m_f1.mzXML: Pru wt *T. gondii* infected HFF cells treated with fludarabine, Experiment 2, replicate 1 * Qa_HFF_Pru_wt_Fludarabine2_v412m_f1.mzXML: Pru wt *T. gondii* infected HFF cells treated with fludarabine, Experiment 2, replicate 2 * Ra_HFF_Pru_wt_Fludarabine3_v412m_f1.mzXML: Pru wt *T. gondii* infected HFF cells treated with fludarabine, Experiment 2, replicate 3 * Sa_HFF_Pru_Delta1_v412m_f1.mzXML: Pru delta HXGPRT *T. gondii* infected HFF cells, Experiment 2, replicate 1 * Ta_HFF_Pru_Delta2_v412m_f1.mzXML: Pru delta HXGPRT *T. gondii* infected HFF cells, Experiment 2, replicate 2 * Ua_HFF_Pru_Delta3_v412m_f1.mzXML: Pru delta HXGPRT *T. gondii* infected HFF cells, Experiment 2, replicate 3 * Va_HFF_Pru_Delta_Fludarabine1_v412m_f1.mzXML: Pru delta HXGPRT *T. gondii* infected HFF cells treated with fludarabine, Experiment 1, replicate 1 * Wa_HFF_Pru_Delta_Fludarabine2_v412m_f1.mzXML: Pru delta HXGPRT *T. gondii* infected HFF cells treated with fludarabine, Experiment 1, replicate 2 * Xa_HFF_Pru_Delta_Fludarabine3_v412m_f1.mzXML: Pru delta HXGPRT *T. gondii* infected HFF cells treated with fludarabine, Experiment 1, replicate 3 * Adenosine_10uM_v412m_f1.mzXML: Adenosine standard at 10 uM * AMP_10uM_v412m_f1.mzXML: AMP standard at 10 uM * GDP_10uM_v412m_f1.mzXML: GDP standard at 10 uM * GMP_10uM_v412m_f1.mzXML: GMP standard at 10 uM * GTP_10uM_v412m_f1.mzXML: GTP standard at 10 uM * Guanine_10uM_v412m_f1.mzXML: Guanine standard at 10 uM * Guanosine_10uM_v412m_f1.mzXML: Guanosine standard at 10 uM * dGMP_10uM_v412m_f1.mzXML: dGMP standard at 10 uM * IMP_10uM_v412m_f1.mzXML: IMP standard at 10 uM * Inosine_10uM_v412m_f1.mzXML: Inosine standard at 10 uM * Blank1_v412m_f1.mzXML: blank sample * Blank2_v412m_f1.mzXML: blank sample ## Code/Software Peaks were matched to known standards for identification. Data analysis was performed using the Metabolomics Analysis and Visualization Engine (MAVEN) software