Abstract
Intracellular pathogens have evolved countless different and fascinating strategies to facilitate their multiplication and survival within host cells. Understanding the biology of intracellular pathogens requires a detailed knowledge of the molecular interactions with and contributions by the host cell. The focus of this thesis is on the role of the
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secretory pathway in the infection process of two intracellular pathogens from totally different classes: the mouse hepatitis coronavirus (MHV) and the bacterium Salmonella typhimurium. Both pathogens are known to require and to interact with the host secretory pathway during their replication, but detailed insights into these interactions are lacking. During the infection of host cells with intracellular pathogens, changes in the host cell proteome are likely to occur. The host cell will adapt its proteome in an attempt to eliminate the pathogen, while the pathogen on the other hand, alters the host cell proteome to promote its own replication and survival. In the research described in this thesis we applied a quantitative proteomics approach, called Stable Isotope Labeling by Amino acids in Cell culture (SILAC), to identify changes in the host cell proteome. We reasoned that proteins that are affected in their abundance or distribution upon infection are likely to be somehow involved in the pathogen life cycle. More specifically, we investigated changes in protein composition of Golgi-enriched fractions isolated from coronavirus- or Salmonella-infected cells. 116 and 105 proteins were identified, the abundance of which was changed significantly in the Golgi-enriched fraction upon infection of cells with either MHV infection or Salmonella typhimurium. Amongst these affected proteins, proteins involved in membrane traffic and cell signalling as well as nucleic acid binding proteins were overrepresented. Only little overlap was observed between proteins shown to be affected in MHV- and Salmonella-infected cells, indicating that the changes in the host cell proteome were pathogen specific. The functional relevance of a subset of the identified proteins in the infection process was studied using follow-up experiments such as RNA interference, biochemical assays and immunofluorescence studies. In this way, several proteins were shown to be critically involved in either the coronavirus or the Salmonella infection cycle. Identification of host proteins that play a role in infections of pathogens might ultimately lead to the development of new therapeutic intervention possibilities.
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