## 3 Discussion  
In this work, a metabolomics approach was used to investigate the metabolic consequences of _S. aureus_ exposure in a human airway epithelial cell line. We observed time dependent changes in nutrient uptake, intracellular amino acid content and nucleotide synthesis (Figure 9). By monitoring growth and survival of A549 cells after exposure to _S. aureus_, we observed that the infection induces cell death in a certain proportion of the cells, but also restricts growth in the surviving population. Simultaneously, we observed a reduced uptake of essential nutrients from the cell culture medium. It is noteworthy, that cytochalasin D treated A549 cells also entered growth arrest but without a significant amount of cell death and the reduced uptake of amino acids. This strongly  
Figure 9: Key results of metabolic alterations and deduced pathways involved during infection. The summary graphic shows metabolic differences between control cells and cells after exposure to _S. aureus_ measured at indicated time points. Names of metabolites are written in regular font, metabolic pathways in bold font and cellular processes in italic font. Green coloured labels indicate increased concentrations or higher activity and red labels indicate decreased concentrations or reduced activity. The pentose-phosphate pathway (PP-pathway) is coloured blue to indicate a changed flux of carbon during infection, which was only detectable when azaserine is present. indicates, that growth arrest does not necessarily restrict nutrient uptake. Recently, it was shown that _S. aureus_ infection inhibits proliferation of HeLa cells by a G2/M phase transition delay, most likely induced by the action of phenolsoluble modulin \(\alpha\) (PSM\(\alpha\)) [29; 30]. \(\alpha\)-type PSMs are peptides secreted by community-acquired MRSA such as USA300 that exhibit cytolytic activity and induce neutrophil activation [31]. A549 epithelial cells might also be affected by the action of secreted PSM\(\alpha\). When the intracellular bacterial load was strongly decreased, we observed after 48 h the resumption of proliferation and also the uptake of extracellular nutrients. This indicates that the cause for the growth inhibition is coupled to the amount of intracellular _S. aureus_ cells. A lower amount of intracellular _S. aureus_ would also result in a lower amount of secreted PSM\(\alpha\), which in turn would initiate growth again. Additionally, A549 cells were previously shown to enter growth arrest when infected with measles virus due to the upregulation of the Interferon Regulatory Factor 1 (IRF1) [32]. Moreover, lipoteichoic acids of _S. aureus_ induce TLR2 signalling which leads to IRF1 upregulation [33] and therefore also might contribute to the growth arrest during infection. The early intracellular amino acid profile of infected cells showed an overall decrease in concentration. A similar general decrease in metabolite concentrations was recently observed by our group in airway epithelial cells treated with staphylococcal Hla [13]. Hla is a secreted virulence factor of _S. aureus_, which forms pores in the host cell membrane which has several effects on the host cell ranging from autophagy induction, alterations in cell signalling, cytoskeleton rearrangements and cytokine secretion [34; 35; 36]. It might also be responsible for the intracellular depletion of metabolites in this study, since USA300 is a potent producer of alpha toxin [37; 38]. However, if Hla is responsible, it might also be secreted by _S. aureus_ during the infection time, when bacteria and host cells were cultured together for 2 h. Other pathogens are also known to modulate cellular processes and metabolism from the extracellular space. In the case of pathogenic species from _Chlamydia_, _Xanthomonas_, _Pseudomonas_, _Ralstonia_, _Shigella_, _Salmonella_, _Escherichia_ and _Yersinia_, this is accomplished by translocating effector molecules into the host cell cytosol via the type III secretion systems (reviewed by [39]).