Investigation of the Mode of Action of the Protein VapA of Rhodococcus Equi on Phagosome Membranes

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.