Part 2: Kiss and spit metabolomics highlight the role of host purine metabolism during pathogen infection

Research facility: Morgridge Institute for Research

Published Sep 05, 2025 on Dryad. https://doi.org/10.5061/dryad.69p8cz9b5

Abstract

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

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

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

U-13C6 glucose metabolomic experiment

Metabolomic analysis and U-13C6 glucose labeling of HFF cells infected with T. gondii 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 10⁶ tachyzoites of T. gondii 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 N₂ 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.

Part 2: Kiss and spit metabolomics highlight the role of host purine metabolism during pathogen infection

Data files

Abstract

README: Part 2: Kiss and spit metabolomics highlight the role of host purine metabolism during pathogen infection

U-13C6 labeling of ME49 T. gondii kiss and spit and full infection in HFF cells

Description of the data and file structure

Methods