Part 4: 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..., To understand the role of cN-II in T. gondii infection, we performed metabolomics on the uninfected and T. gondii- 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 T. gondii-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 106 ME49 tachyzoites. We used as negative controls dishes of uninfected cells. At 24 and 48 HPI, dishes were washed three times with ice col..., , # 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 |
| :----------------------------------- | :----------------...,