Resumen de Dynamics of Listeria Monocytogenes Stressosome Proteins in Response to Osmotic Stress and the Eukaryotic Intracellular Niche
Listeria monocytogenes is a Gram-positive bacterial pathogen that withstands environmental stress efficiently, both in non-host and host environments. Sigma B (SigB), a stress-responsive transcriptional factor, is activated by a supra-macromolecular complex, the stressosome, to allow reprogramming of gene expression for stress adaptation. The stressosome structure, inferred from complexes assembled in vitro, consists of the core proteins RsbR (here renamed RsbR1), its paralogues, RsbS and, the kinase RsbT. The active complex is proposed to be tethered to the membrane and to support RsbR1/RsbS phosphorylation by RsbT and the subsequent release of RsbT following signal perception. Despite the information obtained from structural analyses and the protein-protein interactions characterized in vitro within the RsbR1-RsbS-RsbT complex, there remains much to be learnt about the dynamics of these interactions and how they are modulated by live cells in response to stress.
In this thesis, I have addressed in L. monocytogenes the subcellular location of the main stressosome components, their interactions and the phosphorylation status in the absence/presence of osmotic stress. Unexpectedly, some of these proteins were detected mostly in the cytosol and with phosphorylation states that remained unaltered in response to hyperosmolarity. The kinase activity of RsbT on RsbR1/RsbS and its requirement for maximal SigB activation in response to osmotic stress were also demonstrated in vivo. Cytosolic RsbR1 interacts with RsbT, while this interaction diminishes at the membrane when RsbR1 paralogues (RsbR2, RsbR3 and RsbL) are present. These findings suggest that in vivo the active RsbR1-RsbS-RsbT complex forms only transiently and that membrane-associated RsbR1 paralogues could modulate its assembly.
This study also provides insights into the distribution of stressosome proteins during the interaction of L. monocytogenes with the mammalian host. RsbR1, detected mostly in the cytosol of bacteria grown in laboratory media, mobilized however to the membrane during replication of the pathogen inside eukaryotic cells. Although in the context of infection the role of SigB was previously reported only in the invasion of epithelial cells, my data support activation of SigB in vivo during L. monocytogenes intracellular growth. This activation might be impeded by the RsbR1 paralogues during the entry of epithelial cells.
The last chapter aimed to extend in L. monocytogenes the characterization of cell wall proteome changes in response to osmotic stress and the role that SigB and the stressosome play in this surface remodeling. Higher levels of certain surface proteins regulated by SigB were observed in response to hyperosmolarity. These surface proteins as well as SigB and its regulators, RsbR1 and RsbX, had strong influence on modelling the envelope architecture of L. monocytogene
