Inside out – The role of mycobacterial ESX secretion systems in phagosome escape

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The aim of the project is to elucidate the role of type VII secretion systems (T7SS) in phagosome escape of pathogenic mycobacteria. In particular, our focus will be on the inter- and intraspecies genetic diversity of ESX secretion systems and on the structural and functional variability of T7SS substrates belonging to the WXG100 superfamily. Based on the results of a comprehensive in silico screening, a selection of WXG100 proteins representing the genetic variation across the genus will be recombinantly expressed to study the impact of structural changes on protein function. The role of ESX substrates in phagosome escape will be analyzed using human and bovine blood derived macrophages. To decipher the complex interaction of ESX substrates with biological membranes we will use high resolution microscopy (EM, AFM, STORM) on intracellular bacteria in macrophages and various biophysical techniques (spectroscopy, electrophysiology, binding assays) on natural and reconstituted well-defined lipid membrane systems. The specific role of the ESX molecules for the exit of mycobacteria will be mimicked by addition of the purified proteins, of proteins encapsulated in reconstituted liposomal containers, and inactivated mycobacteria into different cell culture systems. The structure of WXG100 monomers and heterodimers will be characterized, as well as the molecular basis of protein-membrane interactions between WXG100 proteins and biological membranes with a particular focus on pH-, lipid-, voltage-, and protein-structure-specificities.