Figure 2. Expression of recombinant PSA glycotope on OMVs.
(a) Schematic of PSA biosynthesis pathway, which involves NeuABCD for the formation of CMP-NeuNAc and NeuS for its polymerization into PSA. (b) Positive-ion MALDI-TOF spectrum of permethylated PSA. Glycolipids were extracted from JC8033 cells carrying plasmids pPSA and pNeuD. The major signal at m/z 791.4, with additional significant peaks at m/z 1152.6, 1513.7, and 1874.9 corresponding to tri, tetra, and penta NeuNAc structures. The predominance of the dimer seen here is consistent with preliminary NMR analysis of the same material. (c) Dot blot analysis of non-denatured OMV fractions generated from plasmid-free JC8033 cells (−) or JC8033 cells carrying either pNeuD, pPSA, or pPSA/pNeuD together. Also shown are OMV fractions from JC8033 cells carrying PSA/pNeuD where the pPSA plasmid lacked cstII (pPΔC), lgtB (pPΔL), or both (pPΔCL). Strain EV36 served as a positive control. (d) Dot blot analysis of non-denatured OMV fractions generated from plasmid-free JC8033 cells (−) or the following strains each carrying pPSA/pNeuD: JC8033; JC8034, which lacked waaL; and JC8035, which lacked wecA. Also shown are OMV fractions from hypervesiculating versions of MG1655 and ClearColi (MG1655-ves and ClearColi-ves, respectively) carrying pPSA/pNeuD. Blots were probed with SEAM 12 antibody to confirm the presence or absence of PSA on exterior of OMVs.