## 2 Results
### Extracellular Metabolic Profiles of A549 Cells after Infection with S. aureus
#### 2.2.1 Glucose and Glutamine Are Consumed by A549 under Control and Infection Conditions  
Similar to other immortalized or cancer cell lines, A549 cells are expected to consume high amounts of glucose and exhibit a high glycolytic rate under aerobic conditions, which is known as the \"[PERSON] effect\" [23; 24]. Furthermore, cancer cell metabolism requires an enhanced glutamine uptake to fuel anabolic pathways via glutaminolysis [25; 26]. Indeed, we observed that 78% of the  
Figure 2: Extracellular metabolic profile during infection. (**A**) Extracellular concentrations changes of metabolites involved in glycolysis and glutaminolysis of control cells (black bars) and infected cells (grey) normalized on the proliferation rate ([c\({}_{\text{trc}}\)-c\({}_{\text{0}}\)])/(cell number\({}_{\text{trc}}\)/cell number\({}_{\text{tr0}}\)). Data are presented as mean with standard deviation (\(n\geq 6\)). (**B**) Extracellular concentrations changes of other amino acids were normalized on the proliferation rate ([c\({}_{\text{trc}}\)-c\({}_{\text{0}}\)])/(cell number\({}_{\text{trc}}\)/cell number\({}_{\text{tr0}}\)). Data are presented as mean (\(n\geq 6\)) in a colour coded chart. Red fields indicate an increase in the extracellular concentration and blue fields indicate a decrease of the metabolite concentration compared to the initial concentration. provided glucose and 87% of the provided glutamine were consumed by A549 cells under non-infection conditions within 24 h (Supplementary Material Table S1). In accordance to the high rates of glycolysis, we detected extracellular lactate in appropriate concentrations up to 18.14 mmol/L. This corresponds to approximately 1.85 molecules of lactate per metabolized glucose molecule. Additionally, extracellular pyruvate and glutamate showed increased concentrations of 0.39 mmol/L and 0.58 mmol/L over time respectively, indicating a secretion of these metabolites. The secreted amount of glutamate corresponds to 33% of the consumed glutamine. Both the pyruvate and the glutamate secretion suggest an intracellular overflow of intermediates of central metabolic carbon pathways. After infection with _S. aureus_, the A549 host cells maintained the glycolytic metabolism but only produced 1.62 molecules lactate per metabolized molecule of glucose. This indicates a slightly different intracellular flux of glucose under infection conditions. We compared the concentration differences between the medium and the sampling time points with regard to the factor with which cells had multiplied (Figure 2A). We found that infected cells use less glucose and produced less lactate within the first 12 h after infection. After that, the consumption of glucose and production of lactate was similar again to non-infected cells even if the growth was strongly reduced. Interestingly, the glutamine uptake was increased in infected cells significantly after 8 h and was elevated even more till 24 h. Moreover, between 2 h and 24 h after infection, pyruvate and glutamate secretion was strongly increased. However, after 48 h and 72 h glutamine uptake as well as glutamate and pyruvate secretion were reduced by approximately 30% and 50%, respectively. This indicates that the elevated secretion of glutamate and pyruvate is infection related and might represent an intracellular excess of these metabolic precursors. 2.2 Amino Acid Uptake Is Reduced in the Infected A549 Cell Culture but the Secretion of Amino Acids Is Enhanced  
Aside from glutamine and glutamate, we were able to quantify 15 more amino acids in the extracellular space. For most of these amino acids, a clear uptake was detected under control conditions in particular for the branched-chain amino acids and methionine (Figure 2B). After infection, however, the amino acid uptake was strongly reduced. Especially for threonine, phenylalanine, lysine and valine no uptake was detected within 24 h. Moreover, we found that concentrations of glycine, aspartate and alanine were increased in the extracellular space.