Part 5: 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 approv..., T. gondii 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 106 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 N2, and stored at -80°C.
Samples w..., , # 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-I...,