Part 3: Kiss and spit metabolomics highlight the role of host purine metabolism during pathogen infection

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

Methods
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 T. gondii 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 106  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 N2, 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 T. gondii 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.