## 2 Results
### De novo Purine and Pyrimidine Synthesis during Infection Is Strongly Reduced  
By applying metabolic inhibitors, we were not able to reduce the intracellular nucleotide amounts under infection conditions. We therefore presume that either the inhibitor is not able to reach its target or the nucleotide synthesis is not active under infection conditions. To test if the purine and pyrimidine synthesis are active under infection conditions, we performed a labelling approach in which l-glutamine-amide-\({}^{15}\)N or d-glucose-\({}^{13}\)C\({}_{6}\) was supplied to non-infected and infected cells. Figure 6: The hexosamine-pathway can be inhibited in infected cells. Control cells and infected cells were treated with either DON or azaserine (AZA) for 12 h. Successor metabolites of the glucosamine-fructose-6-phosphate aminotransferase reaction (GFAT) (EC 2.6.1.16) are displayed in relative concentrations as mean with standard deviation normalized on \(10^{7}\) cells (\(n\geq 7\) out of at least four independent experiments). The glutamine-amide group is transferred during the de novo synthesis of uridine once, adenosine and cytidine twice and three times in the case of guanosine. In addition, \(N\)-acetylation of glucose to GlcNAc does also require glutamine as a nitrogen donor. We analysed the metabolites with regard to the total labelling of these compounds and detected labelled nucleotides (ATP, ADP, UTP, UDP, GTP, GDP, CTP, CDP), GlcNAc-P and UDP-sugars (exact masses of analysed isotopes are provided in Supplementary Material Table S4) (Figure 8A). Between 60% and 95% of each nucleotide were found to be labelled after 12 h of cultivation. UDP-GlcNAcNAc was detected in its complete labelled form and additionally with a labelled GlcNAc moiety only. However, in infected cells we could detect only minor amounts of the labelled nucleotides (1%-7%) at the same time of cultivation. Nevertheless, labelled isotopes of GlcNAc-P and UDP-GlcNAcNAc with a labelled GlcNAc moiety were still found in similar or higher amounts compared to the control. This indicates that glutamine is still used for the formation of GlcN, but not for the nucleotide de novo synthesis. Figure 7: TCA-cycle intermediates are affected by DON and PPP intermediates are affected by azasering during infection. Control cells and infected cells were treated with either DON or azasering (AZA) for 12 h. Metabolites involved in: (**A**) glutaminolysis; and (**B**) carbon conversion pathways are displayed. Data are displayed in relative concentration as mean with standard deviation normalized on 10\({}^{\prime\prime}\) cells (\(n\geq 7\) out of at least four independent experiments). Since glucose delivers the carbon units for the ribose moiety of nucleotides via the PP-pathway, the production of nucleotides should also result in an absolute mass M + 5 in the labelling approach with d-glucose-\({}^{13}\)C\({}_{6}\) (Supplementary Material Table S4). Indeed, the nucleotides ATP, UTP, GTP and CTP were dominantly present as the M + 5 labelled isotope (Figure 8B). Furthermore, glucose derived metabolites like Frc-1,6-bP, PRPP and GlcNAc-P were found in fully labelled state in high amounts up to 99%. In accordance with this, for metabolites with a glucose moiety such as UDP-Glc, GDP-Glc, and derivatives like UDP-GlcNAc and UDP-GlcNAc we identified the isotope M+6 (indicating a labelled sugar moiety) as well as the isotope M + 11 (indicating a labelled sugar moiety and ribose moiety). Twelve hours after infection, the nucleotide sugars were mainly found as M + 6 isotopes, whereas nucleotides were mainly detected in their unlabelled form. These data support the idea that nucleotide synthesis in infected A549 cells is switched off, while the formation of nucleotide sugars and pathways like glycolysis and the PP-pathway are still active. Interestingly, growth inhibited A549 cells due to cytochalasin D treatment showed purine and pyrimidine labelling pattern in which CTP and UTP labelling decreased by 40% and 23%, respectively, but ATP and GTP only by 11% and 12% (Figure S1B).