Abstract:
The opportunistic human pathogen Staphylococcus aureus can cause a wide range of infections and is a growing threat to the healthcare systems due to the development of antibiotic resistance. To survive and multiply under unfavourable conditions, S. aureus has evolved strategies to sense and respond to various environmental stresses, such as nutrient limitation or antibiotic-induced cell wall stress. The stringent response is a bacterial stress response characterised by the synthesis of the alarmones (p)ppGpp, which enables bacterial survival under nutrient starvation, antibiotic exposure or other external stresses by transcriptional reprogramming and growth arrest. The functional consequences of the stringent response in S. aureus have been studied in previous work under various conditions, including transcriptionally or chemically induced amino acid limitation, and during infection in human macrophages and animal models. In this thesis, the role of the stringent response in biofilm formation and under nutrient limitation during the stationary growth phase was investigated. Additionally, the influence of the stringent response and (p)ppGpp on antibiotic tolerance was examined under these conditions. In the first part of this thesis, it was demonstrated that biofilm formation during relaxed conditions is independent of the stringent response. However, under nutrient-limited conditions or antibiotic exposure, (p)ppGpp synthesis by the small alarmone synthetases RelP and RelQ was found to be necessary for biofilm formation and maintenance. In addition, it was shown that an anti-biofilm peptide, originally proposed to inhibit (p)ppGpp function, acts independent of (p)ppGpp but inhibits biofilm formation and synergistically eradicates biofilms in combination with the antibiotic vancomycin. In the second part of this thesis, it could be demonstrated that (p)ppGpp synthesis is required to avoid excessive GTP accumulation during stationary growth phase. Maintaining GTP homeostasis ensures the culturability of S. aureus under nutrient starvation by preserving the proton motive force (PMF) and cell membrane architecture. (p)ppGpp-deficient S. aureus enters a division-incompetent, dormant state characterized by low levels of ATP, reduced metabolic activity, and alterations in membrane potential and architecture. It was demonstrated that control of GTP levels in nucleotide-sensitive promoters leads to the downregulation of genes involved in the TCA cycle and electron transport chain. To partially restore the culturability of the (p)ppGpp-deficient mutant, the transcription of qoxABCD, a terminal oxidase of the respiratory chain, was increased through mutation of the transcriptional start site. In addition, it was shown that the loss of the PMF renders the stationary phase bacteria vulnerable to various classes of antibiotics. These findings suggest that targeting the stringent response could be an effective strategy to combat bacterial infections. In particular, by targeting the functional consequences mediated by stringent response in S. aureus, e.g. by the application of biofilm, PMF or (p)ppGpp inhibitors, clinical applications could also be found.
