DOI: 10.5061/dryad.zkh1893jn
title: Part 6: 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: Kiss and Spit Heat-Killed Parasite Control: Low pass ME49
 parasites were lysed from host cells, counted, and heat-killed by
 incubation at 85°C for 30 minutes. As a negative control, empty media was
 also heated for the same amount of time. 2 x 10<sup>6</sup>
 heat-killed parasites, or an equivalent volume of empty media was then
 added to confluent and quiescent dishes of HFFs in triplicate. Dishes were
 incubated for 12 hours at 37°C before having their metabolites extracted
 and analyzed using the below metabolomic methodology.
 Kiss and Spit Conditioned Media Control: Conditioned media was
 taken from heavily infected (MOI 0.75) cells prior to host cell lysis and
 the release of parasites into the media. Media from uninfected paired
 dishes of host cells served as the negative control. Confluent and
 quiescent dishes of HFFs were incubated in each media condition for 12
 hours at 37°C before having their metabolites extracted and analyzed using
 the the below metabolomic methodology. Kiss and
 Spit-Cytochalasin D Negative Control: To ensure that the changes we
 observed in host metabolism were not attributable to the <em>T.
 gondii</em> remaining in the dish we performed a control to measure
 the metabolic contribution of the parasites. 2 X
 10<sup>6</sup> ME49 <em>T. gondii</em> parasites
 were incubated in media with 1.5 μM cytochalasin D at 37°C  for 12 hours
 before pelleting the parasites, washing them to remove the media, and then
 extracting metabolites using the below metabolomic methodology. A blank
 control (media with cytochalasin D but no parasites) was treated
 identically. Metabolites were quantified using HPLC-MS and metabolites
 were identified with known standards. 
 <strong>Metabolomic:</strong> At
 the specific time points, 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
description: # Me49 *T. gondii* Kiss and spit negative controls
 [https://doi.org/10.5061/dryad.zkh1893jn](https://doi.org/10.5061/dryad.zkh1893jn) ## Description of the data and file structure The following samples were run in triplicate in each experiment: Heat Killed : Kiss and spit -Me49 *T. gondii* Heat- Killed parasite control Heat Media: Kiss and spit -Heat- media control Conditioned: Kiss and spit -Me49 *T. gondii* conditioned-media control Media conditioned: Kiss and spit -conditioned-media control Parasite Cyt D: Kiss and spit -Me49 *T. gondii* Cytochalasin D treated- parasite control Media Cyt D:  Kiss and spit - Cytochalasin D treated- media control Samples from A to U correspond to the first experiment. Samples from Ab to Ub correspond to the second experiment. Pre-Blank samples are included: * A.Heat_killed.mzXML: Kiss and spit -Me49 *T. gondii* Heat- Killed parasite control, experiment 1, replicate 1 * B.Heat_killed.mzXML: Kiss and spit -Me49 *T. gondii* Heat- Killed parasite control, experiment 1, replicate 2 * C.Heat_killed.mzXML: Kiss and spit -Me49 *T. gondii* Heat- Killed parasite control, experiment 1, replicate 3 * Ab.Heat_killed.mzXML: Kiss and spit -Me49 *T. gondii* Heat- Killed parasite control, experiment 2, replicate 1 * Bb.Heat_killed.mzXML:  Kiss and spit -Me49 *T. gondii* Heat- Killed parasite control, experiment 2, replicate 2 * Cb.Heat_killed.mzXML:  Kiss and spit -Me49 *T. gondii* Heat- Killed parasite control, experiment 2, replicate 3 * D.Heat_media.mzXML:  Kiss and spit -Heat- media control, experiment 1, replicate 1 * E.Heat_media.mzXML:  Kiss and spit -Heat- media control, experiment 1, replicate 2 * F.Heat_media.mzXML:  Kiss and spit -Heat- media control, experiment 1, replicate 3 - Db.Heat_media.mzXML:  Kiss and spit -Heat- media control, experiment 2, replicate 1 - Eb.Heat_media.mzXML:  Kiss and spit -Heat- media control, experiment 2, replicate 2 - Fb.Heat_media.mzXML:  Kiss and spit -Heat- media control, experiment 2, replicate 3 - G.Conditioned.mzXML: Kiss and spit -Me49 *T. gondii* conditioned-media control, experiment 1, replicate 1 - H.Conditioned.mzXML: Kiss and spit -Me49 *T. gondii* conditioned-media control, experiment 1, replicate 2 - I.Conditioned.mzXML: Kiss and spit -Me49 *T. gondii* conditioned-media control, experiment 1, replicate 3 - Hb.Conditioned_.mzXML: Kiss and spit -Me49 *T. gondii* conditioned-media control, experiment 2, replicate 1 - Gb.Conditioned.mzXML: Kiss and spit -Me49 *T. gondii* conditioned-media control, experiment 2, replicate 2 - Ib.Conditioned.mzXML: Kiss and spit -Me49 *T. gondii* conditioned-media control, experiment 2, replicate 3 - J.Media_conditioned.mzXML: Kiss and spit -conditioned-media control, experiment 1, replicate 1 - K.Media_conditioned.mzXML: Kiss and spit -conditioned-media control, experiment 1, replicate 2 - L.Media_conditioned.mzXML: Kiss and spit -conditioned-media control, experiment 1, replicate 3 - Kb.Media_conditioned.mzXML: Kiss and spit -conditioned-media control, experiment 2, replicate 1 - Jb.Media_conditioned.mzXML: Kiss and spit -conditioned-media control, experiment 2, replicate 2 - Lb.Media_conditioned.mzXML: Kiss and spit -conditioned-media control, experiment 2, replicate 3 * M.Parasite_Cyt_D.mzXML: Kiss and spit -Me49 *T. gondii* Cytochalasin D treated- parasite control, experiment 1, replicate 1 * N.Parasite_Cyt_D.mzXML: Kiss and spit -Me49 *T. gondii* Cytochalasin D treated- parasite control, experiment 1, replicate 2 * O.Parasite_Cyt_D.mzXML: Kiss and spit -Me49 *T. gondii* Cytochalasin D treated- parasite control, experiment 1, replicate 3 * Mb.Parasite_Cyt_D.mzXML: Kiss and spit -Me49 *T. gondii* Cytochalasin D treated- parasite control, experiment 2, replicate 1 * Nb.Parasite_Cyt_D.mzXML: Kiss and spit -Me49 *T. gondii* Cytochalasin D treated- parasite control, experiment 2, replicate 2 * Ob.Parasite_Cyt_D.mzXML: Kiss and spit -Me49 *T. gondii* Cytochalasin D treated- parasite control, experiment 2, replicate 3 * P.Media_Cyt_D.mzXML: Kiss and spit - Cytochalasin D treated- media control, experiment 1, replicate 1 * Q.Media_Cyt_D.mzXML: Kiss and spit - Cytochalasin D treated- media control, experiment 1, replicate 2 * R.Media_Cyt_D.mzXML: Kiss and spit - Cytochalasin D treated- media control, experiment 1, replicate 3 * Pb.Media_Cyt_D.mzXML: Kiss and spit - Cytochalasin D treated- media control, experiment 2, replicate 1 * Qb.Media_Cyt_D.mzXML: Kiss and spit - Cytochalasin D treated- media control, experiment 2, replicate 2 * Rb.Media_Cyt_D.mzXML: Kiss and spit - Cytochalasin D treated- media control, experiment 2, replicate 3 * S.PreBlnk1_v415m_f1.mzXML: blank sample 1, experiment 1 * T.PreBlnk2_v415m_f1.mzXML: blank sample 2, experiment 1 * U.PreBlnk3_v415m_f1.mzXML: blank sample 3, experiment 1 * Sb.PreBlnk1_v415m_f1.mzXML: blank sample 1, experiment 2 * Tb.PreBlnk2_v415m_f1.mzXML: blank sample 2, experiment 2 * Ub.PreBlnk3_v415m_f1.mzXML: blank sample 2, experiment 2 ## Code/Software Peaks were matched to known standards for identification. Data analysis was performed using the Metabolomics Analysis and Visualization Engine (MAVEN) software