| The formation and trafficking of membrane vesicles is an essential process in eukaryotes. These structures are formed to store, traffic, or digest cellular components and virtually all the organelles of the eukaryotic cell are able to form vesicles. In prokaryotes, vesicle formation was reported several decades ago; first in Gram negative bacteria and recently in Gram positive bacteria and in archaea. In contrast to the well established multiple cellular roles of membrane vesicles in eukaryotic cell biology, outer membrane vesicles (OMV) produced via blebbing of Gram negative outer membranes (OM) have frequently been regarded as cell debris or microscopy artifacts. It has only relatively recently been acknowledged that OMV possess multiple functional roles, including response to diverse forms of stress, intra-species communication, long distance virulence factor delivery and contribution to immune system evasion. Although the importance of OMV is now established, it remains to be determined whether OMV result from a selective process or by passive disintegration of the OM as well as the mechanism(s) involved in their biogenesis and secretion.;In this thesis the most abundant proteins from purified OM and OMV of Porphyromonas gingivalis and Salmonella typhimurium were identified by tandem mass spectrometry. This analysis has shown that the human oral pathogen P. gingivalis has a mechanism to selectively sort OM proteins into OMV. This process results in the preferential packaging of gingipains, a group of proteases that constitute a major virulence factor of P. gingivalis, and in the exclusion of abundant OM proteins from the protein cargo. Similar results were observed in S. typhimurium. Moreover in P. gingivalis, OM and OMV also differed in the lipopolysaccharide (LPS) composition, the OMV being enriched with tri-acylated high molecular weight LPS molecules. Furthermore, it has been shown that mutations affecting the LPS result in aberrant protein sorting into the OMV. These results assign a critical role to surface glycans in directing this sorting mechanism, equivalent to the role of galectin in eukaryotic endosomal vesicle formation. The existence of a process to pack specific virulence factors into OMV may significantly alter our current understanding of host-pathogen interactions. |
