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The intracellular pathogen R. equi manages to survive the phagosome maturation by arresting phagosomes in a prephagolysosomal state. The rhodococcal virulence-associated protein A (VapA) contributes to this process [1]. We propose a pore forming activity for VapA.Based on experiments with model membranes mimicking the phagosome membrane we suggest a five-step mode of action for VapA. Biosensor experiments indicated a binding of VapA to the bilayers (1) being accompanied by an increase of membrane rigidity. In contrast to common pore-forming molecules only a weak intercalation dependent on the lateral membrane pressure occured (2). AFM analysis revealed a strong aggregation of VapA on cholesterol-poor lipid domains (3). This induced a reorganization of the domain structure of the bilayer. Defined and undefined lesions increasing the permeability of the reconstituted membranes (4) and finally the induction of membrane disintegration by VapA (5) were observed using electrophysiological measurements on freestanding membranes and a fluorescence based nanopore dye release assay.We assume two different interaction states for VapA defined by different protein orientation which depend on lipid package, lipid composition and pH value. The potential relevance of these observations for understanding of R. equi virulence will be discussed.References[1] von Bargen, C. and Haas, A., 2009, FEMS Microbiol Rev 33: 870-891.

Mycobacterium tuberculosis is resistant against the antimicrobial activity of a large number of host defence peptides (HDPs) in the phagosome, which are active against other Gram-negative or -positive bacteria. One reason for this resistance is the unique structure of the mycobacterial cell wall. Mycobacteria produce a thick mycolate-rich outer covering which functions as an exceptionally efficient barrier. We demonstrated that the membrane forming properties of trehalose dimycolate (TDM), as one of the important components of the mycobacterial outer barrier, are unique. TDM, which is organized in domains, leads to an increase of the overall membrane stability by a factor of three (as obtained in force spectroscopic experiments using atomic force microscopy). The membrane structure was investigated by X-ray reflectivity and GISAXS.Moreover, we used reconstituted planar lipid bilayers composed of TDM and phospholipids to perform electro-physiological experiments for determining the membrane permeabilizing activity of different HDPs. Furthermore, we used atomic force microscopy to characterize the surface topography of reconstituted membranes and mycobacteria. The biophysical results were compared with results from killing assays. Interestingly LL32, which is the highly active fragment of human cathelicidin, induced dramatic changes in the membrane morphology without inducing a significant permeabilization. LL32 is able to kill other Gram positive or negative bacteria by induction of membrane lesions, but up to a concentration of 30 μg/ml LL32 could not kill M. tuberculosis. Surprisingly, LL32 even stimulated an increased growth of the mycobacteria. In contrast two synthetic peptides based on LL32 and a linear version of the human beta-defensin-3 (hBD-3) showed a pronounced killing and permeabilization.

Plasma membrane tension is not only responsible for a large variety of different cellular functions including cell migration, division, and growth but also for complex collective processes such as embryology, tumorgenesis or wound healing. Since membrane tension is largely dominated by the attachment of the actin cortex to the inner leaflet of the plasma membrane, we investigated the role of ezrin, a major cross-linker of the membrane-cytoskeleton interface, for tension homeostasis. For this purpose, we carried out ezrin depletion experiments and also enhanced the number of active ezrin molecules at the interface by injection of PIP2 into MDCK II cells. Cortical tension and membrane tension were assessed by force indentation experiments and subsequent membrane tether extraction. The linkage between the actin cortex and the plasma membrane was reinforced by injection of PIP2 micelles into individual living cells, while the opposite was achieved by adding siRNA against ezrin and administration of the chemical inhibitors such as neomycin and NSC 668394, respectively. A substantial stiffening of cells was observed accompanied by an increase in membrane tension after addition of PIP2 micelles. In contrast, ezrin depletion led to a decrease of membrane tension accompanied by loss of excess surface area, increase of cortical tension due to remodelling of the actin cytoskeleton. Interestingly, the cell morphology also changed upon ezrin depletion. We found a substantial reduction of cell height. The data confirms the importance of the ezrin-mediated connection between plasma membrane and cortex for cellular mechanics and cell morphology.