DOI: 10.5061/dryad.7d7wm383s
title: Part 4: 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 role of cN-II in <em>T. gondii</em>
 infection, we performed metabolomics on the uninfected and <em>T.
 gondii</em>- infected MDAMB231 parental and cN-II KO cell lines at
 24 and 48 HPI. IMP and GMP, the preferred substrates of the cN-II enzyme,
 tended to accumulate in infected cells in comparison to uninfected cells,
 with the exception of GMP at 24 HPI in cN-II KO cells . The nucleobase
 products of the cN-II reaction, inosine, guanosine, were significantly
 less abundant or not detected in infected cells at 24 HPI. Inosine and
 guanosine were lower in abundance in <em>T.
 gondii</em>-infected cN-II KO cell lines with respect to the
 infected MDAMB231 parental line at 48 HPI. Thus, the genetic deletion of
 cN-II enzyme affected the metabolites IMP, GMP, inosine and guanosine in
 infected cells. MDAMB231 or MDAMB231 KO dishes in
 triplicate were infected with 2 x 10<sup>6</sup> ME49
 tachyzoites. We used as negative controls dishes of uninfected cells. At
 24 and 48 HPI, 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* Infected MDAMB231 cells Metabolomics at 24 and 48 HPI
 [https://doi.org/10.5061/dryad.7d7wm383s](https://doi.org/10.5061/dryad.7d7wm383s) ## Description of the data and file structure The following samples were run in triplicate: MDAMB231 : uninfected MDAM231231 parental cell line MDAMB231_FLORO: uninfected MDAMB231 parental cell line plus Fludarabine MDAMB231_toxo: ME49 *T. gondii* infected MDAM231231 parental cell line MDAMB231_KO: uninfectedcMDAM231231 Cytosolic nucleotidase II (cN-II) Knock-out cell line MDAMB231_KO_toxo: ME49 *T. gondii* infected MDAM231231Cytosolic nucleotidase II (cN-II) Knock-out cell line 24 HPI ( samples from 1-17) or 48 HPI (samples from 18-36) time point are included in the sample name. Pre-blank samples and standards at 10 uM are included.  | 1\_24\_MDAMB231\_FLORO-1.mzML |  Uninfected MDAMB231 cell line treated with Fludarabine for 24 H, replicate 1 | | :----------------------------------- | :----------------------------------------------------------------------------------------- | | 2\_24\_MDAMB231\_FLORO-2.mzML |  Uninfected MDAMB231 cell line treated with Fludarabine for 24 H, replicate 2 | | 3\_24\_MDAMB231\_FLORO-3.mzML |  Uninfected MDAMB231 cell line treated with Fludarabine for 24 H, replicate 3 | | 4\_24\_MDAMB231\_FLORO\_toxo-1.mzML |  ME49 T. gondii-infected MDAMB231 cell line treated with Fludarabine for 24 H, replicate 1 | | 5\_24\_MDAMB231\_FLORO\_toxo-2.mzML |  ME49 T. gondii-infected MDAMB231 cell line treated with Fludarabine for 24 H, replicate 2 | | 6\_24\_MDAMB231\_FLORO\_toxo-3.mzML |  ME49 T. gondii-infected MDAMB231 cell line treated with Fludarabine for 24 H, replicate 3 | | 7\_24\_MDAMB231-1.mzML |  Uninfected MDAMB231 cell line for 24 H, replicate 1 | | 8\_24\_MDAMB231-2.mzML |  Uninfected MDAMB231 cell line for 24 H, replicate 2 | | 9\_24\_MDAMB231-3.mzML |  Uninfected MDAMB231 cell line for 24 H, replicate 3 | | 10\_24\_MDAMB231\_toxo-1.mzML |  ME49 T. gondii-infected MDAMB231 cell line for 24 H, replicate 1 | | 11\_24\_MDAMB231\_toxo-2.mzML |  ME49 T. gondii-infected MDAMB231 cell line for 24 H, replicate 2 | | 12\_24\_MDAMB231\_toxo-3.mzML |  ME49 T. gondii-infected MDAMB231 cell line for 24 H, replicate 3 | | 13\_24\_MDAMB231\_KO-1.mzML |  Uninfected MDAMB231 cN-II Knock-out cell line for 24 H, replicate 1 | | 14\_24\_MDAMB231\_KO-2.mzML |  Uninfected MDAMB231 cN-II Knock-out cell line for 24 H, replicate 2 | | 15\_24\_MDAMB231\_KO-3.mzML |  Uninfected MDAMB231 cN-II Knock-out cell line for 24 H, replicate 3 | | 16\_24\_MDAMB231\_KO\_toxo-1.mzML |  ME49 T. gondii-infected MDAMB231 cN-II Knock-out cell line for 24 H, replicate 1 | | 17\_24\_MDAMB231\_KO\_toxo-2.mzML | ME49 T. gondii-infected MDAMB231 cN-II Knock-out cell line for 24 H, replicate 2 | | 18\_24\_MDAMB231\_KO\_toxo-3.mzML |  ME49 T. gondii-infected MDAMB231 cN-II Knock-out cell line for 24 H, replicate 3 | | 19\_48\_MDAMB231\_FLORO-1.mzML |  Uninfected MDAMB231 cell line treated with Fludarabine for 48 H, replicate 1 | | 20\_48\_MDAMB231\_FLORO-2.mzML |  Uninfected MDAMB231 cell line treated with Fludarabine for 48 H, replicate 2 | | 21\_48\_MDAMB231\_FLORO-3.mzML |  Uninfected MDAMB231 cell line treated with Fludarabine for 48 H, replicate 3 | | 22\_48\_MDAMB231\_FLORO\_toxo-1.mzML |  ME49 T. gondii-infected MDAMB231 cell line treated with Fludarabine for 48 H, replicate 1 | | 23\_48\_MDAMB231\_FLORO\_toxo-2.mzML |  ME49 T. gondii-infected MDAMB231 cell line treated with Fludarabine for 48 H, replicate 2 | | 24\_48\_MDAMB231\_FLORO\_toxo-3.mzML |  ME49 T. gondii-infected MDAMB231 cell line treated with Fludarabine for 48 H, replicate 3 | | 25\_48\_MDAMB231-1.mzML |  Uninfected MDAMB231 cell line for 48 H, replicate 1 | | 26\_48\_MDAMB231-2.mzML |  Uninfected MDAMB231 cell line for 48 H, replicate 2 | | 27\_48\_MDAMB231-2.mzML |  Uninfected MDAMB231 cell line for 48 H, replicate 3 | | 28\_48\_MDAMB231\_toxo-1.mzML |  ME49 T. gondii-infected MDAMB231 cell line for 48 H, replicate 1 | | 29\_48\_MDAMB231\_toxo-2.mzML |  ME49 T. gondii-infected MDAMB231 cell line for 48 H, replicate 2 | | 30\_48\_MDAMB231\_toxo-3.mzML |  ME49 T. gondii-infected MDAMB231 cell line for 48 H, replicate 3 | | 31\_48\_MDAMB231\_KO1-1.mzML |  Uninfected MDAMB231 cN-II Knock-out cell line for 48 H, replicate 1 | | 32\_48\_MDAMB231\_KO1-2.mzML |  Uninfected MDAMB231 cN-II Knock-out cell line for 48 H, replicate 2 | | 33\_48\_MDAMB231\_KO1-3.mzML |  Uninfected MDAMB231 cN-II Knock-out cell line for 48 H, replicate 3 | | 34\_48\_MDAMB231\_KO1\_toxo-1.mzML |  ME49 T. gondii-infected MDAMB231 cN-II Knock-out cell line for 48H, replicate 1 | | 35\_48\_MDAMB231\_KO1\_toxo-2.mzML |  ME49 T. gondii-infected MDAMB231 cN-II Knock-out cell line for 48H, replicate 2 | | 36\_48\_MDAMB231\_KO1\_toxo-3.mzML |  ME49 T. gondii-infected MDAMB231 cN-II Knock-out cell line for 48H, replicate 3 | | AMP\_10uM.mzML |  AMP standard at 10 uM | | dGMP\_10uM.mzML |  dGMP standard at 10 uM | | GDP\_10uM.mzML |  GDP standard at 10 uM | | GMP\_10uM.mzML |  GMP standard at 10 uM | | GTP\_10uM.mzML |  GTP standard at 10 uM | | Guanosine\_10uM.mzML |  Guanosine standard at 10 uM | | IMP\_10uM.mzML |  IMP standard at 10 uM | | Inosine\_10uM.mzML |  Inosine standard at 10 uM | | PreBlnk1.mzML |  Blank sample | | PreBlnk2.mzML |  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