Outer membrane vesicles displaying engineered glycotopes elicit protective antibodies.

The O-antigen polysaccharide (O-PS) component of lipopolysaccharides on the surface of gram-negative bacteria is both a virulence factor and a B-cell antigen. Antibodies elicited by O-PS often confer protection against infection; therefore, O-PS glycoconjugate vaccines have proven useful against a number of different pathogenic bacteria. However, conventional methods for natural extraction or chemical synthesis of O-PS are technically demanding, inefficient, and expensive. Here, we describe an alternative methodology for producing glycoconjugate vaccines whereby recombinant O-PS biosynthesis is coordinated with vesiculation in laboratory strains of Escherichia coli to yield glycosylated outer membrane vesicles (glycOMVs) decorated with pathogen-mimetic glycotopes. Using this approach, glycOMVs corresponding to eight different pathogenic bacteria were generated. For example, expression of a 17-kb O-PS gene cluster from the highly virulent Francisella tularensis subsp. tularensis (type A) strain Schu S4 in hypervesiculating E. coli cells yielded glycOMVs that displayed F. tularensis O-PS. Immunization of BALB/c mice with glycOMVs elicited significant titers of O-PS-specific serum IgG antibodies as well as vaginal and bronchoalveolar IgA antibodies. Importantly, glycOMVs significantly prolonged survival upon subsequent challenge with F. tularensis Schu S4 and provided complete protection against challenge with two different F. tularensis subsp. holarctica (type B) live vaccine strains, thereby demonstrating the vaccine potential of glycOMVs. Given the ease with which recombinant glycotopes can be expressed on OMVs, the strategy described here could be readily adapted for developing vaccines against many other bacterial pathogens.

Production of secretory and extracellular N-linked glycoproteins in Escherichia coli.

The Campylobacter jejuni pgl gene cluster encodes a complete N-linked protein glycosylation pathway that can be functionally transferred into Escherichia coli. In this system, we analyzed the interplay between N-linked glycosylation, membrane translocation and folding of acceptor proteins in bacteria. We developed a recombinant N-glycan acceptor peptide tag that permits N-linked glycosylation of diverse recombinant proteins expressed in the periplasm of glycosylation-competent E. coli cells. With this "glycosylation tag," a clear difference was observed in the glycosylation patterns found on periplasmic proteins depending on their mode of inner membrane translocation (i.e., Sec, signal recognition particle [SRP], or twin-arginine translocation [Tat] export), indicating that the mode of protein export can influence N-glycosylation efficiency. We also established that engineered substrate proteins targeted to environments beyond the periplasm, such as the outer membrane, the membrane vesicles, and the extracellular medium, could serve as substrates for N-linked glycosylation. Taken together, our results demonstrate that the C. jejuni N-glycosylation machinery is compatible with distinct secretory mechanisms in E. coli, effectively expanding the N-linked glycome of recombinant E. coli. Moreover, this simple glycosylation tag strategy expands the glycoengineering toolbox and opens the door to bacterial synthesis of a wide array of recombinant glycoprotein conjugates.