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Kern, Beate (2015): Analysis of Helicobacter pylori VacA-containing vacuoles and VacA intracellular trafficking. Dissertation, LMU München: Medizinische Fakultät



The human pathogen Helicobacter pylori colonizes half of the global population. Residing at the stomach epithelium, it contributes to the development of diseases like gastritis, duodenal and gastric ulcers, and gastric cancer. It has evolved a range of mechanisms to aid in colonization and persistence, manipulating the host immune response to avoid clearance. A major factor in this is the secreted vacuolating cytotoxin VacA which has a variety of effects on host cells. VacA is endocytosed and forms anion-selective channels in the endosome membrane, causing the compartment to swell. The resulting VacA-containing vacuoles (VCVs) can take up most of the cellular cytoplasm. Even though vacuolation is VacA's most prominent and namesake effect, the purpose of the vacuoles is still unknown. VacA exerts influence on the host immune response in various ways, both pro- and anti- inflammatorily. Most importantly, it disrupts calcium signaling in T-lymphocytes, inhibiting T-cell activation and proliferation and thereby suppressing the host immune response. Furthermore, VacA is transported to mitochondria, where it activates the mitochondrial apoptosis pathway. Within the cell, VacA has only been shown to localize to endocytic compartments/VCVs and mitochondria. Considering its diverse effects, however, the existence of other cellular sites of action seems plausible. In this study, the VCV proteome was comprehensively analyzed for the first time in order to investigate VCV function. To this end, three different strategies for VCV purification from T-cells were devised and tested. Eventually, VCVs were successfully isolated via immunomagnetic separation, using a VacA-specific primary antibody and a secondary antibody coupled to magnetic beads. The purified vacuoles were then measured by mass spectrometry, revealing not only proteins of the endocytic system, but also proteins usually localized in other cellular compartments. This apparent recruitment of proteins involved in all kinds of cellular pathways indicates a central function of VCVs in VacA intoxication effects. In a global evaluation, the VCV proteome exhibited an enrichment of proteins implicated in immune response, cell death, and cellular signaling; all of these are processes that VacA is known to influence. One of the individual proteins contained in the sample was STIM1, a calcium sensor normally residing in the endoplasmic reticulum (ER) that is important in store- operated calcium entry (SOCE). This corroborates the findings of a concurrent report, in which VacA severely influenced SOCE and colocalized with STIM1. A direct interaction of STIM1 with VacA was examined in a pull-down assay, but could be neither shown nor excluded. Immunofluorescence experiments conducted in HeLa cells confirmed the presence of VacA in the ER and also found it to traffic to the Golgi apparatus, identifying these two cellular compartments as novel VacA target structures. The exact route of VacA transport remains unclear, but the involvement of both the ER and the Golgi suggests the possibility of retrograde trafficking, analogous to other bacterial toxins like shiga and cholera toxins. In summary, the elucidation of the VCV proteome and the discovery of the ER and the Golgi apparatus as VacA target structures have generated intriguing starting points for future studies. The detection of many proteins implicated in VacA intoxication effects in the VCV proteome leads to the proposal of VCVs as signaling hubs that may coordinate the complex meshwork of VacA effects. Further investigation of individual proteins is expected to help greatly in illuminating this matter.