Bacterial communities engage in social activities, exhibiting behaviors such as communicating with small signaling molecules (quorum sensing [QS]) and building antibiotic-resistant biofilms. The opportunistic human pathogen Pseudomonas aeruginosa produces both freely diffusible QS molecules, as well as a QS molecule that is packaged or transported across cell membranes via the production of outer membrane vesicles. Despite the ubiquity of vesicle production in bacteria, the mechanism of outer membrane vesicle production has not been fully elucidated. In addition, most of our understanding of QS and biofilm formation arises from in vitro studies of bacterial communities containing large numbers of cells, often with greater than 10⁸ bacteria. However, many bacterial communities are comprised of small, densely packed aggregates of cells (≤10⁵ bacteria), and it is unclear how group behaviors and chemical interactions take place in densely packed, small populations. This dissertation has two main goals: i) to provide insights into the mechanism of bacterial membrane vesicle production, and ii) to understand how population size and the spatial distribution of cells affect cell-cell interactions and the nutritional microenvironment within a small (≤10⁵ bacteria) prokaryotic community. / text

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.