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- Nygård Skalman, Lars, 1985-
(author)
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Pathogen entry mechanisms and endocytic responses to plasma membrane damage
- 2017
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Doctoral thesis (other academic/artistic)abstract
- Endocytosis is a fundamental cellular process by which cells transport material from the outside to the inside of the cell through the formation of membrane invaginations that bud off from the plasma membrane. This process is important for nutrient uptake, regulating cell surface receptors and the overall plasma membrane composition. Cells have several different types of endocytic pathways where clathrin- mediated endocytosis is the most studied. Importantly, pathogens and secreted virulence factors bind to cell surface receptors and hijack the endocytic pathways in order to enter host cells. Depending on their size and molecular composition, pathogens and virulence factors are thought to make use of distinct endocytic pathways into the cell. This thesis focuses on early host cell interactions with virus, bacterial membrane vesicles and a pore-forming toxin, with a particular emphasis on endocytic mechanisms and plasma membrane repair.During entry of pathogens, it is thought that interactions with specific cell surface molecules drive the recruitment of endocytic proteins to the plasma membrane. Viruses possess a very defined molecular composition and architecture, which facilitate specificity to these interactions. We found that Adenovirus 37, a human ocular pathogen, binds to αVβ1 and α3β1 integrins on human corneal epithelial cells and that this interaction is important for infection. In contrast to viruses, membrane vesicles shed from Helicobacter pylori are heterogeneous in size and molecular composition. These vesicles harbour various adhesins and toxins that may facilitate binding to the cell surface and recruitment of different endocytic pathways. We developed a quantitative internalization assay and showed that the H. pylori vesicles were internalized mainly via clathrin-mediated endocytosis but were also capable of exploiting other endocytic pathways.Damage to the plasma membrane disrupts cellular homeostasis and can lead to cell death if not repaired immediately. Although endocytic mechanisms have been shown to be important for plasma membrane repair, little is known about their specific role. Listeriolysin O (LLO) is a bacterial toxin that can form pores in the plasma membrane and disrupt cellular homeostasis. We developed a reporter system for real-time imaging of the endocytic response to LLO pore formation. We found that two clathrin-independent endocytic pathways were important for plasma membrane repair. However, they were not directly involved in removing LLO pores from the plasma membrane. Our data suggests that these endocytic systems might rather influence membrane repair by their ability to regulate the plasma membrane composition, shape and tension.In conclusion, this thesis describes how pathogens and their virulence factors make use of specific mechanisms to enter host cells as well as revealing new insights on the role of the endocytic pathways in plasma membrane repair.
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| 3. |
- Lindmark, Barbro, 1963-
(author)
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Modulators of Vibrio cholerae predator interaction and virulence
- 2009
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Doctoral thesis (other academic/artistic)abstract
- Vibrio cholerae, the causal agent of cholera typically encodes two critical virulence factors: cholera toxin (CT), which is primarily responsible for the diarrhoeal purge, and toxin-co-regulated pilus (TCP), an essential colonisation factor. Nontoxigenic strains expressing TCP can efficiently acquire the CT gene through lysogenic conversion with CTXΦ, a filamentous phage that encodes CT and uses TCP as a receptor. V. cholerae is a Gram-negative bacterium and a natural inhabitant of estuarine and coastal waters throughout both temperate and tropical regions of the world. In the aquatic environment, V. cholerae encounters several environmental stresses, such as change in salinity, UV stress, nutrient limitation, temperature fluctuations, viral infections and protozoan predation. To fully understand the pathogenic and virulence potential of V. cholerae, knowledge is required of its interactions with, not only human, but also environmental factors. By using the nematode Caenorhabditis elegans as host model, we were able to identify a previously uncharacterised protein, the extracellular protease PrtV. PrtV was shown to be required for the killing of. elegans and also necessary for survival from grazing by the ciliate Tetrahymena pyriformis and the flagellate Cafeteria roenbergensis. The PrtV protein, which belongs to a M6 family of metallopeptidases was cloned and purified for further characterisations. The purified PrtV was cytotoxic against the human intestinal cell line HCT8. By using human blood plasma, fibrinogen, fibronectin and plasminogen were identified as candidate substrates for the PrtV protease. Outer membrane vesicles (OMVs) are released to the surroundings by most Gram-negative bacteria through “bulging and pinching” of the outer membrane. OMVs have been shown to contain many virulence factors important in pathogenesis. Therefore, we investigated the association of PrtV with OMVs. PrtV was not associated with OMVs from the wild type O1 strain. In contrast, in an LPS mutant lacking two sugar chains in the core oligosaccharide PrtV was found to be associated with the OMVs. The OMV-associated PrtV was shown to be proteolytically and cytotoxically active. V. cholerae strains are grouped into >200 serogroups. Only the O1 and O139 serogroups have been associated with pandemic cholera, a severe diarrhoeal disease. All other serogroups are collectively referred to as non-O1 non-O139 V. cholerae. Non-O1 non-O139 V. cholerae can cause gastroenteritis and extraintestinal infections, but unlike O1 and O139 strains of V. cholerae, little is known about the virulence gene content and their potential to become human pathogens. We analysed clinical and environmental non-O1 non-O139 isolates for their putative virulence traits. None of them carry the genes encoding CT or the TCP, but other putative virulence factors were present in these isolates. The incidence of serum resistance was found to vary considerably and was independent of encapsulation. Three strains were strongly serum-resistant, and these same strains could also kill C. elegans.
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