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Michael Hensel (2011-2017)

Biogenesis of novel membrane compartments in Salmonella enterica-infected host cells

Intracellular Salmonella enterica inhabits a unique membrane bound compartment, termed Salmonella-containing vacuole (SCV). By means of the SPI2-encoded type III secretion system, Salmonella manipulate the host cell vesicular transport. These activities result in biogenesis of the SCV and induction of an extensive interconnected network of tubular membrane compartments, such as Salmonella-induced filaments of SIF.

In the first phase of SPP1580, we developed a live cell correlative light and electron microscopy approach to investigate SCV/SIF biogenesis. The data indicate that SIF develop as single membrane tubules, eventually enclosing cytosol and cytoskeleton as double membrane compartments. Based on these findings, in the second phase of SPP1580 we will investigate the physiological consequences of formation of the SCV/SIF network. The experiments will analyse interchange of membrane material and luminal content, access of intracellular Salmonella of endocytic cargo, and the pH with in the SCV. The CLEM approach should be used to study the intracellular habitat in other host cell types relevant for Salmonella, such as macrophages, dendritic cells or polarized epithelial cells.

As a further approach to understand the biogenesis of SCV/SIF, we developed a new approach for enrichment of host cell membranes modified by action of intracellular Salmonella. The proteome of Salmonella-modified membranes (SMM) was determined and indicated the presence of various small GTPases of the Rab family, cytoskeletal subunits and linkers of the F-actin cytoskeleton to membranes. We confirmed that presence of the several SMM proteins by live cell imaging and will further extend live cell analyses toward the dynamics of interaction. The functional relevance of SMM proteins will be determined by transfection with dominant-negative variants and siRNA-mediated knockdown. In order to understand the mechanism of double membrane formation, we will investigate the presence of components of vesicular fusion, as well as of Salmonella effectors shown to be required for double membrane formation.

Our combined approaches will shed light on the unique manipulation of the host cell endomembrane system by intracellular Salmonella.

 

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