The foodborne, facultative intracellular pathogen Salmonella enterica serovar Typhimurium has the capability to remodel the host endosomal system and establish a network consisting of tubular membranous structures connected with the Salmonella-containing vacuole (SCV). However, despite its ability to form these Salmonella-modified membranes (SMM), the cellular origin and composition of SMM are mostly unknown.
Thus we developed a novel approach facilitating the isolation of SMM. Therefore, we combined differential fractionation and affinity-based enrichment using an epitope-tagged SPI2-T3SS effector SseF with liquid chromatography-tandem mass spectrometry (LC-MS/MS). This enabled us to probe the composition of SMM during infection of the epithelial cell line HeLa and mouse macrophage RAW264.7 cells. The identified SMM proteome consists of 247 host proteins in HeLa cells and 262 proteins in macrophages, respectively. The protein compositions revealed the importance of cytoskeletal and host trafficking proteins for SMM and indicated a redirection of host trafficking towards SMM. In addition our results suggested a contribution of the traffic between ER and Golgi apparatus, e.g. COPI and COPII vesicles, that has not been assumed as origin for SMM before. A selection of proteins involved in trafficking and cytoskeleton formation were validated by confocal light microscopy.
Furthermore we analysed the effect of IFNgamma activation on the SMM proteome in RAW264.7 macrophages and revealed a strong impact on the protein composition. The identification of IFNgamma-inducible proteins and a higher percentage of ER proteins implied that Salmonella is able to adjust its SMM according to the cell status.
Comparison of the SMM proteome with host compartments of other intracellular pathogens indicated communalities that might be important for the biogenesis of pathogen-containing vacuoles despite of their individual replication niches.
Furthermore, the methods were adopted for a non-membrane integral Salmonella effector SseJ. Proteome analyses were conducted using the sensitive Velos Orbitrap system. This data set revealed not only the impact of the bait protein during the SMM probing but also differences between MS platforms. Nevertheless, interesting host and Salmonella proteins were identified using this approach, which are starting points for further research.
Altogether, this work provides new insights into the origin of Salmonella-modified membranes and serves as a rich source for new theories that will help to understand the biogenesis and function of the Salmonella-containing vacuole and its connected tubular membrane network.
| Identifer | oai:union.ndltd.org:uni-osnabrueck.de/oai:repositorium.ub.uni-osnabrueck.de:urn:nbn:de:gbv:700-2016060114501 |
| Date | 01 June 2016 |
| Creators | Vorwerk, Stephanie |
| Contributors | Prof. Dr. Michael Hensel, Prof. Dr. Hubert Hilbi |
| Source Sets | Universität Osnabrück |
| Language | English |
| Detected Language | English |
| Type | doc-type:doctoralThesis |
| Format | application/zip, application/zip, application/pdf |
| Rights | http://rightsstatements.org/vocab/InC/1.0/ |
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