Soutenance de thèse : Molecular bases of a multiprotein assembly linking membrane biology to stress adaptation in enterobacteria.

Par Moritz Kirchner (IBS/Groupe Imagerie microscopique d’assemblages complexes)

Enteric bacteria encounter a variety of stress factors when infecting their host. This includes but is not limited to acid stress, oxygen limitation and antibiotic stress. Bacteria counteract such stressors using various stress response systems. A central factor in the stress adaptation of enterobacteria is their cell envelope, in particular their cell membrane. A system of proteins which has been demonstrated to be involved in multiple stress adaptation pathways is the RavA-ViaA-LdcI triad in Escherichia coli. RavA and ViaA are two proteins with as of yet unknown function that have been shown to sensitise E. coli to aminoglycoside antibiotics under anaerobic conditions. RavA forms a large complex with LdcI, an inducible lysine decarboxylase which is one of the central enzymes involved in acid stress response of E. coli. Both RavA and ViaA have been shown to bind specific anionic lipids and their lipid binding capacity is strongly linked to their sensitising effect to aminoglycosides.

This thesis presents the exploration of the connections these three proteins have to each other and their other binding partners by optical and electron microscopy in order to elucidate how their action leads to antibiotic sensitisation and what role their connection to the cell membrane and acid stress might play in this regard. It is shown that LdcI prefers to localise to the cell periphery to efficiently counteract acid stress and that mutations of its active site lead to large domain movements which might play a crucial role in its enzymatic activity. Furthermore, an investigation of liposome decoration by RavA and ViaA is presented, demonstrating the effect they have on membrane shape. In addition, the first cryo-EM structure of ViaA is presented using helical repeat proteins to stabilise the protein. Finally, predictions of RavA and ViaA protein-protein interactions in the inner E. coli membrane reveal possible functional targets of the proteins and the implications of these new possible avenues for action are discussed with regard of the microscopy investigations.

Mots clés : Stress adaptation, Membrane modulation, SMLM, cryo-EM, RavA, ViaA, LdcI