| dc.description.abstracteng | The second messenger cyclic diadenosine monophosphate (c-di-AMP) is essential for most of the bacteria synthesizing the nucleotide. c-di-AMP is produced by diadenylate cyclases (DACs) and degraded by specific phosphodiesterases (PDEs). c-di-AMP is involved in the control of different cellular processes, such as cell wall metabolism, carbon metabolism and osmoregulation. Osmoregulation is the cause for the essentiality of c-di-AMP, because regulation of osmolyte uptake systems has been shown to be affected by the second messenger. Hence, c-di-AMP is a major regulator of osmotic homeostasis in bacteria. The Gram-positive human pathogen Listeria monocytogenes possesses the DAC CdaA and the PDEs PdeA and PgpH for c-di-AMP synthesis and degradation, respectively. The CdaA-type of DAC is widespread in firmicute pathogenic bacteria and therefore a prime target for the development of novel antibiotics. The gene encoding CdaA is conserved with the genes cdaR and glmM, encoding the protein CdaR and the phosphoglucosamine mutase GlmM, respectively. It is however unclear, how bacteria modulate c-di-AMP concentrations in response to osmotic changes and how c-di-AMP affects important osmotic adaptation processes, such as potassium transport, in L. monocytogenes. Here, novel insights on the regulation of DAC activity and the impact of c-di-AMP on cellular processes are elucidated. Synthesis and degradation of c-di-AMP have to be tightly adjusted, in dependency of the external osmolarity to maintain an isosmotic intracellular environment. In concert with modulations of the cell wall metabolism this subsequently results in maintaining a balanced turgor pressure. Hence, the proteins CdaR and GlmM are prime targets to modulate CdaA activity. CdaR consists of an N-terminal transmembrane domain and four YbbR domains of unknown function. It is shown that CdaR, like CdaA, is a membrane protein, with the four YbbR domains located outside of the cell. Moreover, a ΔcdaR mutant shows defects in the adaptation to osmotic stress and an altered intracellular c-di-AMP concentration. For normal regulation of CdaA activity upon osmotic shock, both, the membrane localization and the presence of the YbbR domains are required. It is furthermore shown that both CdaR and GlmM are able to interact with CdaA and inhibit its activity in vivo. This highlights that CdaA probably integrates clues, signaling changes in osmolarity and cell wall biosynthesis, via its two regulators CdaR and GlmM, respectively. Additionally, protein-protein interaction studies, show possible interactions between CdaAR and the PDEs, indicating cross-talk of synthesis and degradation machineries and local signaling of c-di-AMP. Moreover, using directed and undirected approaches, novel targets in L. monocytogenes that might be regulated by c-di-AMP are investigated. The proteins KtrCD and KimA of L. monocytogenes are shown to possess potassium transporter activity and both, KtrCD and KimA are demonstrated to be inhibited by c-di-AMP in vivo. KtrC, furthermore, binds the nucleotide in vitro. The analysis of changes in global gene expression and protein synthesis demonstrates a broad impact of c-di-AMP on L. monocytogenes. Important cellular processes, such as the central metabolism, the regulation of transport processes and motility, as well as cell wall remodeling are implicated. Investigations of the connection between c-di-AMP signaling and alterations of the cell wall, additionally, demonstrated that L. monocytogenes rapidly adapts to the muralytic enzyme lysozyme by acquiring mutations in the promoter of the sRNA rli31, which has been shown to affect expression of cell wall modifying enzymes. To conclude, in the present study regulatory processes affecting c-di-AMP synthesis by changes in the osmolarity and the impact of c-di-AMP on global gene expression, protein biosynthesis, cell wall modifications and especially its impact on osmohomeostasis in the human pathogen L. monocytogenes, are investigated. The gained knowledge will help to understand the mechanisms Firmicutes use to sense changes in osmolarity and highlights mechanisms they use to adapt. Understanding regulation of DAC activity eventually can lead to the development of novel antibiotic to treat infections by these increasingly multi antibiotic resistant pathogens. | de |