Correlative electron microscopy studies of the intracellular lifestyle of Brucella

A number of pathogens have evolved strategies that allow them to survive and multiply inside eukaryotic cells. This lifestyle requires the ability to interact and influence different processes within the host cell. The bacteria need ways of avoiding detection and clearance. Moreover, the pathogen’s survival relies on its ability to establish a replicative niche, which is often a modified host cell compartment. A plethora of interactions between intracellular pathogens and host cell organelles have been described by different means. There is, however, a need to gain more knowledge about this subject. Our studies focus on Brucella, a zoonotic pathogen responsible for 500’000 infections annually. Upon host entry, Brucella follows a complex trafficking process that allows the bacterium to avoid lysosome degradation and establish a replicative niche inside the host’s endoplasmic reticulum. The molecular mechanisms responsible for this process remain largely unknown. In my work, we employed a combination of light and electron microscopy techniques that allowed the visualization of different stages of the intracellular lifestyle of Brucella inside host cells. The goal was to shed new light on the interactions of the bacteria with different host cell organelles and cellular processes. In research article I, we present a more detailed description of the Brucella replicative niche by using three-dimensional correlative light and electron microscopy. This approach allowed us to resolve the interactions of the pathogen with the endoplasmic reticulum to a great detail. We provide strong indications that the replicative niche of Brucella is in fact integrated with the organelle. Our initial results in HeLa cells were additionally recapitulated using an in vivo model. In research article II, we look in more detail at the role of host cell factors in the intermediate trafficking of Brucella. We focus on a number of components of the retromer machinery that have been identified in a siRNA screen. We were able to show that knockdown of the protein VPS35 blocks the transition of the Brucella vacuole into the endoplasmic reticulum by arresting it at the late endosome stage. This suggests the role of a previously unknown mechanism in the establishment of the Brucella replicative niche. Additionally, I present a series of unpublished results that are part of on-going research. They provide exciting new findings regarding the localization of Brucella during its intracellular trafficking towards the replicative niche. We have established new cell lines and reporter strains that were used in a combination of different imaging approaches to describe the occurrence of cytosolic Brucella at this stage. It remains to be determined if this novel observation represents a crucial step in Brucella biology or an off-pathway event. Together, the results improve our understanding of Brucella lifestyle inside host cells. We were able to identify new factors involved in the intermediate trafficking of Brucella and indicate previously unknown events that may occur in the process. We also provided a more detailed description of the replicative niche and improved the understanding of its interactions with the endoplasmic reticulum. Additionally, we explored the potential as well as limitations of combining different light and electron microscopy approaches for studying intracellular pathogens