Anne M. Doody

Phospholipase A2 (PLA2) Enzymes in Membrane Trafficking: Mediators of Membrane Shape and Function

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) (PLA2) 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 PLA2 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 PLA2 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 PLA2 enzymes in intracellular membrane trafficking and the mechanisms by which they influence membrane structure and function.

Delivery of foreign antigens by engineered outer membrane vesicle vaccines

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.

The phospholipase complex PAFAH Ib regulates the functional organization of the Golgi complex

The PAFAH 1b complex links phospholipid remodeling and membrane tubulation within the Golgi to dynein-dependent transport.

Mechanistic insight into the TH1-biased immune response to recombinant subunit vaccines delivered by probiotic bacteria-derived outer membrane vesicles.

Recombinant subunit vaccine engineering increasingly focuses on the development of more effective delivery platforms. However, current recombinant vaccines fail to sufficiently stimulate protective adaptive immunity against a wide range of pathogens while remaining a cost effective solution to global health challenges. Taking an unorthodox approach to this fundamental immunological challenge, we isolated the TLR-targeting capability of the probiotic E. coli Nissle 1917 bacteria (EcN) by engineering bionanoparticlate antigen carriers derived from EcN outer membrane vesicles (OMVs). Exogenous model antigens expressed by these modified bacteria as protein fusions with the bacterial enterotoxin ClyA resulted in their display on the surface of the carrier OMVs. Vaccination with the engineered EcN OMVs in a BALB/c mouse model, and subsequent mechanism of action analysis, established the EcN OMV’s ability to induce self-adjuvanted robust and protective humoral and TH1-biased cellular immunity to model antigens. This finding appears to be strain-dependent, as OMV antigen carriers similarly engineered from a standard K12 E. coli strain derivative failed to generate a comparably robust antigen-specific TH1 bias. The results demonstrate that unlike traditional subunit vaccines, these biomolecularly engineered “pathogen-like particles” derived from traditionally overlooked, naturally potent immunomodulators have the potential to effectively couple recombinant antigens with meaningful immunity in a broadly applicable fashion.

The phospholipase A2 enzyme complex PAFAH Ib mediates endosomal membrane tubule formation and trafficking

For the first time, a cytoplasmic phospholipase A2 enzyme, platelet-activating factor acetylhydrolase (I)b, is described that is directly involved in the formation of membrane tubules from endosomes and trafficking through the endocytic recycling pathway.

Cytoplasmic phospholipase A2 antagonists inhibit multiple endocytic membrane trafficking pathways

Previous studies have suggested a role for cytosolic Ca(2+)-independent phospholipase A(2) (PLA(2)) activity in the formation of endosome membrane tubules that participate in the export of transferrin (Tf) and transferrin receptors (TfR) from sorting endosomes (SEs) and the endocytic recycling compartment (ERC). Here we show that the PLA(2) requirement is a general feature of endocytic trafficking. The reversible cytoplasmic PLA(2) antagonist ONO-RS-082 (ONO) produced a concentration-dependent, differential block in the endocytic recycling of both low-density lipoprotein receptor (LDLR) and TfRs, and in the degradative pathways of LDL and epidermal growth factor (EGF). These results are consistent with the model that a cytoplasmic PLA(2) plays a general role in the export of cargo from multiple endocytic compartments by mediating the formation of membrane tubules.

Site-specific labeling of surface proteins on living cells using genetically encoded peptides that bind fluorescent nanoparticle probes.

We report a highly specific, robust, and generic method for noncovalent labeling of cellular proteins with highly fluorescent core-shell silica nanoparticles termed C dots. Our approach uses short genetically engineered peptides with affinity for silica (GEPS) that are site-specifically introduced at the termini or in loops of cellular proteins. Because GEPS are absent from native cell surface proteins, GEPS-tagged recombinant proteins can be selectively and rapidly labeled with fluorescent C dots. To demonstrate the versatility of our method, we targeted 30 nm C dots to two structurally distinct integral outer membrane proteins in Escherichia coli, FhuA and OmpX. Efficient labeling was achieved in 15 min or less and was observed to be highly sensitive and specific. This strategy provides a powerful technique, comparable to other chemical and biological labeling strategies, for efficient and quantitative investigation of protein function in live biological cells.

Identification of compartments involved in mammalian subcellular trafficking pathways by indirect immunofluorescence.

A characteristic of a successful DNA vaccine is its trafficking to the nucleus where it can be transcribed. Plasmid DNA coupled to a delivery vector must enter the cell, navigate its way through endocytic compartments, and ultimately reach the nucleus. Currently, the precise pathway taken by plasmid DNA is not clear. Understanding how plasmid DNA interacts with the cell and which path it follows to reach the nucleus will aid in the rational design of improved delivery vectors. Achieving this goal requires a means by which to monitor the subcellular trafficking of plasmid DNA and delivery vectors. Presented here are methods for identifying various endocytic compartments involved in mammalian subcellular trafficking pathways using indirect immunofluorescence. Together with labeled delivery vectors and/or plasmid DNA, these methods can aid in the understanding of the trafficking pathways involved in DNA delivery, and contribute to the rational design of more efficient delivery vectors.