DOI: 10.5061/dryad.ghx3ffbxx
title: Part 5: 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: <em>T. gondii </em>preferentially uses AMP salvage
 pathway to obtain purines. So, it is reasonable to think that the
 parasites treated with fludarabine or with genetic deletion of cN-II, use
 AMP to compensate for inosine and guanosine reduction. Thus, we performed
 target metabolomics in uninfected and infected MDAMB231 parental and cN-II
 KO cells, compensated with different concentrations of AMP (0, 1, 2, 4 µM)
 during 48 HPI. MDAMB231 cells dishes in triplicate were
 treated with 2 x 10<sup>6</sup> ME49 tachyzoites and
 supplemented with AMP.  At 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: # Effect of AMP addition on purine metabolism in *T. gondii* infected host
 cells at 48 HPI
 [https://doi.org/10.5061/dryad.ghx3ffbxx](https://doi.org/10.5061/dryad.ghx3ffbxx) ## Description of the data and file structure The following samples were run in triplicate: MDAMB231 : uninfected MDAM231231 parental cell line Infected MDAMB231: ME49 *T. gondii* infected MDAMB231 parental cell line Infected MDAMB231 4mM AMP: ME49 *T. gondii* infected MDAM231231 parental cell line plus 4 uM of AMP Infected MDAMB231 2mM AMP: ME49 *T. gondii* infected MDAM231231 parental cell line plus 2 uM of AMP Infected MDAMB231 1mM AMP: ME49 *T. gondii* infected MDAM231231 parental cell line plus 1 uM of AMP MDAMB231 KO: uninfected MDAM231231 cytosolic nucleotidase II (cN-II) Knock-out cell line Infected MDAMB231KO: ME49 *T. gondii* infected MDAMB231 cytosolic nucleotidase II (cN-II) Knock-out cell line Infected MDAMB231 KO 4mM AMP: ME49 *T. gondii* infected MDAM231231 cytosolic nucleotidase II (cN-II) Knock-out cell line plus 4 uM of AMP Infected MDAMB231 KO 2mM AMP: ME49 *T. gondii* infected MDAM231231 cytosolic nucleotidase II (cN-II) Knock-out cell line plus 2 uM of AMP Infected MDAMB231 KO 1mM AMP: ME49 *T. gondii* infected MDAM231231 cytosolic nucleotidase II (cN-II) Knock-out cell line plus 1 uM of AMP Standard an pre-Blank are included. | 02.mzML | 02.MDAMB231.mzXML: uninfected MDAM231231 parental cell line, replicate 1 | | :------------------- | :------------------------------------------------------------------------------------------------------------------------------------------- | | 04.mzML | 04.MDAMB231.mzXML: uninfected MDAM231231 parental cell line, replicate 2 | | 06.mzML | 06.MDAMB231.mzXML: uninfected MDAM231231 parental cell line, replicate 3 | | 08.mzML | 08.Infected\_MDAMB231.mzXML: ME49 T. gondii infected MDAM231231 parental cell line, replicate 1 | | 10.mzML | 10.Infected\_MDAMB231.mzXML: ME49 T. gondii infected MDAM231231 parental cell line, replicate 2 | | 12.mzML | 12.Infected\_MDAMB231.mzXML: ME49 T. gondii infected MDAM231231 parental cell line, replicate 3 | | 14.mzML | 14.Infected\_MDAMB231\_4mM\_AMP.mzXML: ME49 T. gondii infected MDAM231231 parental cell line, supplemented with 4 uM of AMP,  replicate 1 | | 16.mzML | 16.Infected\_MDAMB231\_4mM\_AMP.mzXML: ME49 T. gondii infected MDAM231231 parental cell line, supplemented with 4 uM of AMP,  replicate 2 | | 18.mzML | 18.Infected\_MDAMB231\_4mM\_AMP.mzXML: ME49 T. gondii infected MDAM231231 parental cell line, supplemented with 4 uM of AMP,  replicate 3 | | 20.mzML | 20.Infected\_MDAMB231\_2mM\_AMP.mzXML: ME49 T. gondii infected MDAM231231 parental cell line, supplemented with 2 uM of AMP,  replicate 1 | | 22.mzML | 22.Infected\_MDAMB231\_2mM\_AMP.mzXML: ME49 T. gondii infected MDAM231231 parental cell line, supplemented with 2 uM of AMP,  replicate 2 | | 24.mzML | 24.Infected\_MDAMB231\_2mM\_AMP.mzXML: ME49 T. gondii infected MDAM231231 parental cell line, supplemented with 2 uM of AMP,  replicate 3 | | 26.mzML | 26.Infected\_MDAMB231\_1mM\_AMP.mzXML: ME49 T. gondii infected MDAM231231 parental cell line, supplemented with 1 uM of AMP,  replicate 1 | | 28.mzML | 28.Infected\_MDAMB231\_1mM\_AMP.mzXML: ME49 T. gondii infected MDAM231231 parental cell line, supplemented with 1 uM of AMP,  replicate 2 | | 30.mzML | 30.Infected\_MDAMB231\_1mM\_AMP.mzXML: ME49 T. gondii infected MDAM231231 parental cell line, supplemented with 1 uM of AMP,  replicate 3 | | 32.mzML | 32.MDAMB231\_KO.mzXML: uninfected MDAM231231 cN-II KO cell line, replicate 1 | | 34.mzML | 34.MDAMB231\_KO.mzXML: uninfected MDAM231231 cN-II KO cell line, replicate 2 | | 36.mzML | 36.MDAMB231\_KO.mzXML: uninfected MDAM231231 cN-II KO cell line, replicate 3 | | 38.mzML | 38.Infected\_MDAMB231\_KO.mzXML: ME49 T. gondii infected MDAM231231 cN-II KO cell line, replicate 1 | | 40.mzML | 40.Infected\_MDAMB231\_KO.mzXML: ME49 T. gondii infected MDAM231231 cN-II KO cell line, replicate 2 | | 42.mzML | 42.Infected\_MDAMB231\_KO.mzXML: ME49 T. gondii infected MDAM231231 cN-II KO cell line, replicate 3 | | 44.mzML | 44.Infected\_MDAMB231\_KO\_4mM\_AMP.mzXML: ME49 T. gondii infected MDAM231231 cN-II KO cell line, supplemented with 4 uM of AMP, replicate 1 | | 46.mzML | 46.Infected\_MDAMB231\_KO\_4mM\_AMP.mzXML: ME49 T. gondii infected MDAM231231 cN-II KO cell line, supplemented with 4 uM of AMP, replicate 2 | | 48.mzML | 48.Infected\_MDAMB231\_KO\_4mM\_AMP.mzXML: ME49 T. gondii infected MDAM231231 cN-II KO cell line, supplemented with 4 uM of AMP, replicate 3 | | 50.mzML | 50.Infected\_MDAMB231\_KO\_2mM\_AMP.mzXML: ME49 T. gondii infected MDAM231231 cN-II KO cell line, supplemented with 2 uM of AMP, replicate 1 | | 52.mzML | 52.Infected\_MDAMB231\_KO\_2mM\_AMP.mzXML: ME49 T. gondii infected MDAM231231 cN-II KO cell line, supplemented with 2 uM of AMP, replicate 2 | | 54.mzML | 54.Infected\_MDAMB231\_KO\_2mM\_AMP.mzXML: ME49 T. gondii infected MDAM231231 cN-II KO cell line, supplemented with 2 uM of AMP, replicate 3 | | 56.mzML | 56.Infected\_MDAMB231\_KO\_1mM\_AMP.mzXML: ME49 T. gondii infected MDAM231231 cN-II KO cell line, supplemented with 1 uM of AMP, replicate 1 | | 58.mzML | 58.Infected\_MDAMB231\_KO\_1mM\_AMP.mzXML: ME49 T. gondii infected MDAM231231 cN-II KO cell line, supplemented with 1 uM of AMP, replicate 2 | | 60.mzML | 60.Infected\_MDAMB231\_KO\_1mM\_AMP.mzXML: ME49 T. gondii infected MDAM231231 cN-II KO cell line, supplemented with 1 uM of AMP, replicate 3 | | AMP\_10uM.mzML | AMP\_10uM.raw\_v296\_f1.mzXML: AMP standard at 10 uM | | GDP\_10uM.mzML | GDP\_10uM.raw\_v296\_f1.mzXML: GDP standard at 10 uM | | GMP\_10uM.mzML | GMP\_10uM.raw\_v296\_f1.mzXML: GMP standard at 10 uM | | GTP\_10uM.mzML | GTP\_10uM.raw\_v296\_f1.mzXML: GTP standard at 10 uM | | Guanosine\_10uM.mzML | Guanosine\_10uM.raw\_v296\_f1.mzXML: Guanosine standard at 10 uM | | IMP\_10uM.mzML | IMP\_10uM.raw\_v296\_f1.mzXML: IMP standard at 10 uM | | Inosine\_10uM.mzML | Inosine\_10uM.raw\_v296\_f1.mzXML: Inosine standard at 10 uM | | PreBlnk1.mzML | PreBlnk1.raw\_v296\_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