Pathogen manipulation of intracellular membrane traffic

The use of in vitro assays to study membrane fusion events has been instrumental in discovering the molecular requirements of membrane fusion. The processes involved in phagosome-lysosome fusion are not clear. Some intracellular pathogens, such as Rhodococcus equi, are able to prevent phagosome-lysosome fusion. Thus, this study aimed to develop an in vitro phagosome-lysosome fusion assay to better understand the fusion of these two organelles and to understand the molecular mechanisms behind R. equi pathogenesis.
Transfer of biotin-conjugated horseradish peroxidase (biotin-HRP) from lysosomes to streptavidin-bead containing phagosomes (sBCPs) was used to measure organelle fusion. A protocol for the purification of lysosomes from J774.2 mouse macrophages was developed using superparamagnetic iron oxide nanoparticles (SPIONs). Lysosomes were enriched with LAMP1 and devoid of EEA1. Biotin-HRP from lysosomes could be detected and was able to bind to sBCPs in a binding assay. A protocol for the purification of sBCPs from J774.2 cells was also developed. Phagosomes were enriched with LAMP1 and contained Rab5, Rab7 and EEA1. Fusion between organelles, in cytosol and in the presence of an ATP regenerating system, was not detected and may have been disrupted by factors present in pig brain cytosol used to support the vesicle fusion.
R. equi virulence depends a 90 Kb plasmid harbouring several virulence-associated proteins (Vaps). Only one of these, VapA, is essential for virulence but R. equi may have additional virulence factors. To identify these potential additional R. equi genes, a R. equi gene library was expressed in yeast which were then screened for membrane trafficking defects. The screen failed to yield any potential R. equi virulence factors that disrupted membrane trafficking in yeast. In a targeted approach, recombinant VapA when fed to cells, induced swelling of mammalian cell lysosomes and late endosomes. This activity was conferred by the C-terminal core of VapA. VapA induced LAMP1 expression suggesting lysosome dysfunction. The effects of VapA were not seen with other R. equi Vap proteins.
This thesis presents the framework for further developing an in vitro phagosome-lysosome fusion assay. Secondly, this work builds on our understanding of how R. equi uses VapA to survive intracellularly.