Previously, we discovered a protein O-glycosylation (ogc) cluster conserved in all Burkholderia species, which glycosylates proteins with a trisaccharide glycan. Sera from Burkholderia-infected patients produce anti-glycan antibodies, suggesting that the Burkholderia protein glycosylation pathway can be exploited for potential vaccine development. Here, we successfully produced two prototypes of anti-Burkholderia vaccines: a recombinant glycoprotein-based vaccine and an E. coli LPS-display vaccine. To generate the former, we constructed a plasmid carrying a chimeric gene encoding three glycosylation sequons fused to the cholera toxin B subunit. The presence of the glycan was observed in recombinant proteins expressed in B. cenocepacia parental strain, but not in proteins expressed by the glycosyltransferase-deficient ΔpglLstrain, as determined by SDS-PAGE and fluorescent lectin blots. For the development of an E. coli LPS-display vaccine, we constructed a plasmid expressing the ogc cluster, which was introduced into an E. coli strain unable to synthesize O-antigen but carrying the O-antigen ligase WaaL. Our results show that the LPS of this strain contained an additional moiety consistent with the B. cenocepacia trisaccharide glycan, as demonstrated by silver-stained LPS gels and lectin blot. This extra moiety was not detected in aΔwaaL mutant. These results suggest that the plasmid was able to provide the necessary functions for the synthesis and membrane translocation of the lipid-linked trisaccharide, which became a substrate for the WaaL ligase and incorporation into the E. coli LPS. Therefore, we demonstrate that the O-glycosylation pathway can be manipulated for the construction of potential anti-Burkholderia vaccines.


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