Human pathogenic bacteria, such as Staphylococcus aureus and Streptococcus pneumoniae, that cause life-threatening diseases and are resistant to many antibiotics, pose a serious health risk and, thus, a global social challenge. Therefore, further elucidation of bacterial resistance mechanisms to antimicrobial substances and the host immune defense is urgently required to improve existing treatment options and to develop new therapeutic approaches. The stringent response, which confers a non-specific stress resistance and facilitates the survival of bacteria, is induced in response to unfavorable environmental influences, such as nutrient starvation, by the accumulation of the alarmones guanosine tetra- or pentaphosphate ((p)ppGpp). While it was demonstrated that the stringent response is associated with increased antibiotic resistance, virulence and persistence, the underlying molecular mechanisms, particularly in Gram-positive bacteria, are still incompletely understood. Within this dissertation, it could be shown that (p)ppGpp is also essential for the oxidative stress resistance of S. aureus in the stationary phase (chapter 1). Quantitative analyses revealed higher respiratory chain activity and elevated total and free iron levels, causing increased intracellular levels of reactive oxygen species (ROS) in the (p)ppGpp0 mutant. Accordingly, the addition of iron chelators and antioxidants restored these physiological changes and mitigated the increased sensitivity of the (p)ppGpp0 mutant to oxidative stress and antibiotics. Thus, the maintenance of the intracellular iron and redox homeostasis was identified as a key mechanism of (p)ppGpp to promote increased stress resistance and antibiotic tolerance in S. aureus. The host immune system produces ROS and reactive chlorine species (RCS), such as H2O2 and hypochlorous acid (HOCl), as a central defense strategy. Consequently, the resistance of human pathogens to these reactive species is essential for their survival in the host. Low molecular weight thiols, such as glutathione (GSH) and bacillithiol (BSH), represent an important defense mechanism. Despite their function as antioxidants, they also protect thiol groups of proteins from irreversible over-oxidation through post-translational modification via S-thiolations. While the redox pathway for the regeneration of S-glutathionylated proteins has been fully elucidated, the equivalent process in Gram-positive Firmicutes, which use BSH instead of GSH, was incompletely understood. As presented in chapter 2, this work contributed to elucidate this further. It was demonstrated that the BSH redox pathway, consisting of BSH, bacilliredoxin A (BrxA) and the NADPH-dependent flavin oxidoreductase YpdA, is essential for the survival of S. aureus in the presence of H2O2 and HOCl. In addition, pathogens, such as S. aureus, encode for redox-sensing transcription factors, which utilize conserved cysteine residues to sense and respond to redox stress conditions via post-translational thiol modifications (chapter 3). Since these regulators control regulons that represent important enzymatic and non-enzymatic resistance mechanisms in bacteria, the work in hand focused on the identification and characterization of further transcription factors of the bacterial thiol-stress defense. For that purpose, RNA-seq analyses were used to investigate the bacterial stress response of S. pneumoniae to HOCl (chapter 4). The NmlR regulon was most strongly induced by HOCl stress and identified as an important resistance mechanism against oxidative stress and for the survival inside human macrophages. It consists of the nmlR and adhC genes that encode for the MerR-family transcriptional regulator NmlR and the Zn2+-dependent class III alcohol dehydrogenase AdhC. While NmlR was characterized previously as an aldehyde sensor, within this work, it was shown that NmlR also activates the transcription of the nmlR-adhC operon in response to oxidants. Molecular analyses revealed that the conserved cysteine (Cys52) is required for redox sensing by intermolecular disulfide formation and S-glutathionylation of NmlR. Although quinones are applied as potent antimicrobials since centuries, the bacterial resistance mechanisms are still incompletely resolved. Therefore, this work focussed on the characterization of the quinone stress response of S. aureus. The transcription profiles of the methylhydroquinone (MHQ) and lapachol stress responses revealed a high overlap of the provoked expression changes (chapters 5 and 6). For example, both quinones induced the oxidative stress response and the quinone-specific MhqR and QsrR regulons. Further analysis showed that, in contrast to MHQ, lapachol does not act as an electrophile but exerts its toxicity through the production of ROS (chapter 5). The antimicrobial effect of lapachol was oxygen dependent and could be reduced significantly by microaerophilic growth conditions. Phenotype analyses identified the H2O2 detoxifying catalase (KatA) and the BrxA/BSH/YpdA/NADPH redox pathway as important resistance mechanisms against lapachol stress in S. aureus. In contrast, the MhqR regulon conferred protection against MHQ but not lapachol. This finding suggests a substrate specificity of the enzymes MhqE and MhqD (chapter 6). Since the single cysteine residue (Cys95) of MhqR is not required for DNA binding and quinone sensing, it can be concluded that the MhqR repressor is not inactivated by a thiol-based mechanism but probably through ligand binding. In contrast, QsrR, another quinone-specific regulator of S. aureus, was shown to be regulated by different thiol switches (chapter 7). Unlike the MhqR regulon, the QsrR regulon was not only strongly induced by quinones but also by various oxidants. While QsrR was shown to sense oxidants by an intermolecular disulfide formation between the redox-active Cys4 and Cys29’, allicin caused the S-thioallylation of all three cysteine residues (Cys4, Cys29, Cys32) in vitro. Northern blot analyses indicated that the S-thioallylation of Cys4 is sufficient for the QsrR inactivation, while Cys4 and either Cys29’ or Cys32’ are required for the induction of the QsrR regulon by oxidants in vivo. Further transcriptional analysis and functional characterization revealed that the MhqR and QsrR regulons are also implicated in the resistance to various antibiotics, including ciprofloxacin and rifampicin (chapters 6 and 7). In addition, the ΔmhqR mutant showed an increased survival rate in long-term infection experiments with murine macrophages compared to the wild type, indicating that the MhqR regulon might function in persistence. Additionally, within the present work, the function of the GbaA regulon in the thiol stress response was examined (chapter 8). While the deletion of the SACOL2592-nmrA-2590 operon caused an enhanced susceptibility to diamide, allicin and aldehydes, the ΔgbaB mutant was impaired in its survival upon MHQ stress. It was further revealed that the conserved cysteines Cys55 and Cys104 of GbaA are required for the stress resistance against electrophiles. Substances that interfere with the bacterial redox homeostasis, either through the production of ROS, their electrophilic properties, or the inhibition of proteins, are currently developed and tested as alternatives to antibiotics (chapter 9). Especially ROS-generating substances represent promising treatment options since their non-specific mode of action minimizes the likelihood of resistance evolution. However, based on the usually lower cytotoxicity, specific inhibitors, e.g., of the low molecular weight thiol biosynthesis, might be more suitable for clinical application. Incited by the current global problem of the pandemic caused by the SARS-CoV-2 virus, the question of the antiviral activity of redox-based substances arose. Since the antimicrobial effect of allicin has been studied in detail, and previous studies suggested that allicin exerts an immunomodulatory effect, the antiviral activity of allicin on SARS-CoV-2 was investigated in a cell culture model within a collaboration project with the working group of Prof. Dr. Drosten (chapter 10). It was demonstrated that the administration of biocompatible allicin doses to SARS-CoV-2 infected cells reduced the viral RNA amount and the number of infectious viral particles by up to 70%. Using label-free quantitative proteomics, it was shown that SARS-CoV-2 causes the profound reprogramming of several host pathways, including gene expression and metabolism. In addition, a strong induction of the interferon signaling pathway and the interferon-stimulated gene signature was detected. Allicin treatment reverted several SARS-CoV-2 induced changes, including the expression of the interferon pathways, to levels of uninfected cells and reduced the expression of viral proteins significantly. The demonstrated antimicrobial activity of thiol-based substances suggests that they can be used in a modified form to combat infectious diseases. Lapachol, whose mode of action in S. aureus was elucidated in the present work, but also allicin, could serve as lead compounds to create more stable and less cytotoxic derivates. In addition, by combining global transcriptome analyses with molecular and microbiological assays, the present work contributed to the elucidation of bacterial adaptation strategies. In particular, the transcription factors NmlR, MhqR and QsrR were identified as important resistance mechanisms of the human pathogenic bacteria S. pneumoniae and S. aureus. Since the expression of these regulons is associated with increased bacterial resistance to the immune system and antibiotics, these proteins represent promising molecular targets for drug development.
