Since the mid-1990s, there have been tremendous advances in our understanding of the roles that lipid-modifying enzymes play in various intracellular membrane trafficking events. Phospholipases represent the largest group of lipid-modifying enzymes and accordingly display a wide range of functions. The largest class of phospholipases are the phospholipase A( 2 ) (PLA 2 ) enzymes, and these have been most extensively studied for their roles in the generation lipid signaling molecules, e.g. arachidonic acid. In recent years, however, cytoplasmic PLA 2 enzymes have also become increasingly associated with various intracellular trafficking events, such as the formation of membrane tubules from the Golgi complex and endosomes, and membrane fusion events in the secretory and endocytic pathways. Moreover, the ability of cytoplasmic PLA 2 enzymes to directly affect the structure and function of membranes by altering membrane curvature suggests novel functional roles for these enzymes. This review will focus on the role of cytoplasmic PLA 2 enzymes in intracellular membrane trafficking and the mechanisms by which they influence membrane structure and function.

As new disease threats arise and existing pathogens grow resistant to conventional interventions, attention increasingly focuses on the development of vaccines to induce protective immune responses. Given their admirable safety records, protein subunit vaccines are attractive for widespread immunization, but their disadvantages include poor immunogenicity and expensive manufacture. We show here that engineered Escherichia coli outer membrane vesicles (OMVs) are an easily purified vaccine-delivery system capable of greatly enhancing the immunogenicity of a low-immunogenicity protein antigen without added adjuvants. Using green-fluorescent protein (GFP) as the model subunit antigen, genetic fusion of GFP with the bacterial hemolysin ClyA resulted in a chimeric protein that elicited strong anti-GFP antibody titers in immunized mice, whereas immunization with GFP alone did not elicit such titers. Harnessing the specific secretion of ClyA to OMVs, the ClyA-GFP fusion was found localized in OMVs, resulting in engineered recombinant OMVs. The anti-GFP humoral response in mice immunized with the engineered OMV formulations was indistinguishable from the response to the purified ClyA-GFP fusion protein alone and equal to purified proteins absorbed to aluminum hydroxide, a standard adjuvant. In a major improvement over current practice, engineered OMVs containing ClyA-GFP were easily isolated by ultracentrifugation, effectively eliminating the need for laborious antigen purification from cell-culture expression systems. With the diverse collection of heterologous proteins that can be functionally localized with OMVs when fused with ClyA, this work signals the possibility of OMVs as a robust and tunable technology platform for a new generation of prophylactic and therapeutic vaccines.protein subunit | adjuvant

We have engineered bacterial outer membrane vesicles (OMVs) with dramatically enhanced functionality by fusing several heterologous proteins to the vesicle-associated toxin ClyA of Escherichia coli. Similar to native unfused ClyA, chimeric ClyA fusion proteins were found localized in bacterial OMVs and retained activity of the fusion partners, demonstrating for the first time that ClyA can be used to co-localize fully functional heterologous proteins directly in bacterial OMVs. For instance, fusions of ClyA to the enzymes β-lactamase and organophosphorus hydrolase resulted in synthetic OMVs that were capable of hydrolyzing β-lactam antibiotics and paraoxon, respectively. Similarly, expression of an anti-digoxin single-chain Fv antibody fragment fused to the C-terminus of ClyA resulted in designer "immuno-MVs" that could bind tightly and specifically to the antibody's cognate antigen. Finally, OMVs displaying green fluorescent protein fused to the C-terminus of ClyA were highly fluorescent and, as a result of this new functionality, could be easily tracked during vesicle interaction with human epithelial cells. We expect that the relative plasticity exhibited by ClyA as a fusion partner should prove useful for: (i) further mechanistic studies to identify the vesiculation machinery that regulates OMV secretion and to map the intracellular routing of ClyA-containing OMVs during invasion of host cells; and (ii) biotechnology applications such as surface display of proteins and delivery of biologics.

The ability of PEI to transfer functionally active siRNA to cells in culture is surprisingly dependent on its biophysical and structural characteristics when compared to its relative success and ease of use for DNA delivery.

We report here that a broad spectrum of phospholipase A 2 (PLA 2 ) antagonists produce a concentrationdependent, differential block in the endocytic recycling pathway of transferrin (Tf) and Tf receptors (TfRs) but have no acute affect on Tf uptake from the cell surface. At low concentrations of antagonists (ϳ1 M), Tf and TfR accumulated in centrally located recycling endosomes, whereas at higher concentrations (ϳ10 M), Tf-TfR accumulated in peripheral sorting endosomes. Several independent lines of evidence suggest that this inhibition of recycling may result from the inhibition of tubule formation. First, BFA-stimulated endosome tubule formation was similarly inhibited by PLA 2 antagonists. Second, endocytosed tracers were found in larger spherical endosomes in the presence of PLA 2 antagonists. And third, endosome tubule formation in a cell-free, cytosoldependent reconstitution system was equally sensitive PLA 2 antagonists. These results are consistent with the conclusion that endosome membrane tubules are formed by the action of a cytoplasmic PLA 2 and that PLA 2 -dependent tubules are involved in intracellular recycling of Tf and TfR. When taken together with previous studies on the Golgi complex, these results also indicate that an intracellular PLA 2 activity provides a novel molecular mechanism for inducing tubule formation from multiple organelles.