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.

Delivery of foreign antigens by engineered outer membrane vesicle vaccines

“…Because expression of clyA in E. coli is strongly repressed under normal laboratory conditions (24), no free ClyA was detected in the engineered or empty OMVs preparations. Consistent with earlier observations (22), the association of ClyA-GFP in engineered OMVs had no apparent effect on the size of vesicles (Fig. 2D).…”

“…The prospects of using OMVs to deliver antigens that are nonnative to the parental bacteria are not yet realized, largely because of challenges in transporting heterologous proteins to vesicles. In a recent demonstration of the remarkable tunability of OMVs by our group, heterologous proteins fused with the native bacterial protein ClyA are efficiently transported in their native functional forms to vesicles (22). Previous work also points to the utility of ClyA in enhancing the immunogenicity of antigens secreted from a live attenuated Salmonella vector, further suggesting the possibility that antigens may be exported from live vectors in the form of OMVs (23).…”

“…In this first demonstration of an engineered OMV vaccine, delivery of a poorly immunogenic green-fluorescent protein (GFP) antigen fused with ClyA in engineered OMVs elicited strong and sustained humoral responses in immunized mice, which were equivalent to the responses elicited by the standard adjuvant aluminum hydroxide. With the diversity of heterologous proteins that are able to be functionally localized in OMVs (22), this report illustrates the potential of engineered OMVs to deliver poorly immunogenic subunit antigens without the lengthy and expensive antigenpurification schemes necessary for traditional subunit vaccines or antigen-delivery systems.…”

“…Recent work by our group showed that genetic fusion with ClyA results in the efficient translocation of heterologous proteins to OMVs (22). Additionally, Galen et al (23) showed that antigens fused to ClyA exhibit immunostimulatory activity when secreted from a live Salmonella vaccine.…”

Predatory activity of Myxococcus xanthus outer-membrane vesicles and properties of their hydrolase cargo

“…Bacterial OMVs are potential biomolecule delivery vehicles, vaccine components and antibiotics (Khandelwal & Banerjee-Bhatnagar, 2003;Kim et al, 2008;Sanders & Feavers, 2011). OMVs generally exhibit lytic activity against a wide range of bacteria, leading to their description as 'predatory', even though studied OMVs are produced by non-predatory bacteria (Li et al, 1998;Mashburn-Warren & Whiteley, 2006).…”