## 3 Discussion
We identified their potential target sites due to the amounts of educts and products of glutamine utilizing reactions. Our data suggest that especially reactions in purine and pyrimidine synthesis were inhibited by DON and azaserine. Similar metabolic patterns were previously described in mouse leukaemia cells for the CTP-synthease, PPAT and PFAS [27]. For other target sites we observed contrary results. Azaserine and DON are frequently described as potent inhibitors of the hexosamine biosynthesis pathway by inhibiting GFAT [54; 55; 27; 56]. Our study confirmed the inhibitory action in this pathway for DON, whereas treatment with azaserine showed only minor effects. We additionally identified the GMP-synthease as a potential target site of DON by detecting the educt XMP in elevated concentrations when the inhibitor was present. The inhibitory action of DON under control conditions results in depletion of CTP, GlcNAc and their derivatives, which is likely to lead to growth inhibition. The cause of azaserine induced growth inhibition however is not as clear. Although the FGAR-synthease was found to be inhibited, purine and pyrimidine amounts were not different compared to control cells. However, we detected a so far unknown metabolite (Supplementary Material Table S3) that was strongly increased due to azaserine treatment, but further studies are needed to identify this metabolite and resolve its role in cell proliferation. Post infection, however, the inhibitory effect was only marginally visible. Moreover, the CTP amount was still elevated in inhibited infected cells compared to control cells. By using a labelling approach we were able to demonstrate that in A549 cells: (i) the carbon flux into the nucleotide synthesizing pathways is strongly reduced; and (ii) glutamine utilizing enzymes in purine and pyrimidine pathways including the CTP-synthease show significantly lower activity. This may also explain the secretion of glycine and aspartate by infected cells. Glycine production contributes to the THF-C\({}_{1}\)-pool and is especially needed for synthesis of purines. Moreover, aspartate is an important precursor for both purine and pyrimidine synthesis. If utilization of these amino acids is strongly reduced, the excess amounts might rather be secreted. Our data therefore strongly indicate that the growth arrest induced by _S. aureus_ is not due to an intracellular nutrient limitation, and that nucleotide de novo synthesis and utilization of nucleotides is strongly reduced in cells after being exposed to _S. aureus_. Moreover, we could demonstrate that a reduced growth rate of A549 cells is not sufficient to explain the drastically lower flux of carbon into de novo nucleotide synthesis and other infection related regulations are most likely responsible. However, this does not apply to the hexosamine biosynthesis pathway. It remained clearly inhibited in infected cells treated with DON and moreover was found to be active in our labelling approach. UDP-GlcNAcAc is the end product of the HBP and the substrate for the \(O\)-GlcNAcNAc transferase, which transfers the GlcNAc moiety to serines or threonine of target proteins, thereby facilitating the post-translational modification of \(O\)-GlcNAcAcylation [57]. The HBP relies on glucose and glutamine to build up UDP-GlcNAcAc and is therefore a proposed sensor of the nutrient situation [58]. In cancer cells, such as those used in this study, uptake of glucose and glutamine is strongly elevated leading to an increased flux into the HBP and driving O-GlcNAcAcylation [58; 59]. Exposure of A549 cancer cells to _S. aureus_ did not change the nutrient availability and also glycolysis and glutamine uptake were still active. The increased glutamine uptake and the constant uptake of glucose and secretion of alanine, pyruvate and lactate support this idea. We therefore suggest that the HBP is not affected by the _S. aureus_ infection or the resulting host response, but rather by the presence of its precursors.