Engineered Bacterial Outer Membrane Vesicles with Enhanced Functionality

Abstract

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.

Introduction

Extracellular secretion of virulence factors is a strategy utilized by invading bacteria to establish a colonization niche, communicate with host cells, and modulate host defense and response. With few exceptions, bacterial protein secretion systems are characterized by the membrane translocation of a single protein or else small protein complexes.1, 2, 3, 4, 5 Recently, however, the production and release of outer membrane vesicles (OMVs) has been demonstrated as a novel secretion mechanism for the transmission of a diverse group of proteins and lipids to mammalian cells.⁶ OMVs are small proteoliposomes with an average diameter of 50–200 nm that are constitutively released from the outer membrane of pathogenic and non-pathogenic species of Gram-negative bacteria during growth.⁷ Biochemical analysis has demonstrated that OMVs are composed of outer membrane proteins, lipopolysaccharide, phospholipids and soluble periplasmic proteins,8, 9 the latter of which become trapped in the vesicle lumen during release from the cell surface. OMVs are largely devoid of inner membrane and cytoplasm components, although several studies indicate that chromosomal, phage and plasmid DNA can infiltrate OMVs as a means of OMV-mediated transfer of genetic information between bacteria.10, 11, 12, 13

An intriguing yet poorly understood phenomenon pertaining to OMVs is the observation that certain membrane and/or soluble periplasmic proteins are enriched in vesicles while others are preferentially excluded. The majority of these enriched proteins happen to be virulence factors including, for example, Escherichia coli cytolysin A (ClyA),¹⁴ enterotoxigenic E. coli heat labile enterotoxin (LT),⁸ and Actinobacillus actinomycetemcomitans leukotoxin,¹⁵ whereas proteins that are excluded from OMVs include numerous unidentified outer membrane (OM) proteins¹⁵ as well as E. coli DsbA.¹⁴ The preferential exclusion of proteins raises the interesting possibility that a sorting mechanism exists in the bacterial periplasm for discriminatory loading of a highly specific subset of proteins into OMVs.14, 16 Moreover, the observation that certain virulence factors are enriched in vesicles suggests that OMVs may have a key role in bacterial pathogenesis by mediating transmission of active virulence factors and other bacterial envelope components to host cells. Indeed, numerous vesicle-associated virulence factors (e.g., adhesins, immunomodulatory compounds, proteases and toxins) have been shown to induce cytotoxicity, confer vesicle binding to and invasion of host cells, and modulate the host immune response.8, 17, 18, 19, 20

To date, one of the best studied vesicle-associated virulence factors is the 34 kDa cytotoxin ClyA (also called HlyE or SheA) found in pathogenic and non-pathogenic E. coli strains,14, 21 and in Salmonella enterica serovars Typhi and Paratyphi A.²² Structural studies indicate that the water-soluble form of ClyA is a bundle of four major α-helices, with a small surface-exposed hydrophobic β-hairpin at the “head” end of the structure, and the N and C termini at the “tail” end,²³ while lipid-associated ClyA forms an oligomeric pore complex composed of either eight or 13 ClyA subunits.24, 25 Expression of the clyA gene is silenced in non-pathogenic E. coli K-12 laboratory strains by the nucleoid protein H-NS,²⁶ but is derepressed in H-NS-deficient E. coli, thereby inducing cytotoxicity towards cultured mammalian cells.²⁷ More recent evidence indicates that ClyA is exported from E. coli in OMVs and retains a cytolytically active, oligomeric conformation in the vesicles.¹⁴ However, the route by which ClyA crosses the bacterial inner membrane and assembles in OMVs remains a mystery, as it carries no canonical signal peptide,²¹ nor is it N-terminally processed.²⁸ Also undetermined is the role that ClyA has in vesicle-mediated interactions with mammalian cells. Thus, in the present study, we sought to engineer synthetic membrane vesicles (s-MVs) with non-native functions that could be used for a wide range of applications, including the analysis of the complete ClyA translocation process. Specifically, we have programmed s-MVs with enhanced functionality by creating chimeras between heterologous proteins such as green fluorescent protein (GFP) or β-lactamase (Bla) and ClyA. Using these engineered vesicles, we have determined that ClyA is capable of co-localizing a variety of structurally diverse fusion partners to the surface of E. coli and their released vesicles, but only when the periplasmic disulfide bond-forming machinery was present. Importantly, these cell- and OMV-associated proteins retained their biological activity, suggesting that the functionality of natural OMVs can be expanded easily via the expression of ClyA chimeras.