Extraction of Membrane Components from Neisseria gonorrhoeae Using Catanionic Surfactant Vesicles: A New Approach for the Study of Bacterial Surface Molecules

Abstract

Identification of antigens is important for vaccine production. We tested extraction protocols using cetyltrimethylammonium tosylate (CTAT) and sodium dodecylbenzenesulfonate (SDBS) to formulate surfactant vesicles (SVs) containing components from Neisseria gonorrhoeae. Carbohydrate and protein assays demonstrated that protein and carbohydrates were incorporated into the vesicle leaflet. Depending on the extraction protocol utilized, 100–400 µg of protein/mL of SVs solution was obtained. Gel electrophoresis followed by silver staining demonstrated that SV extracts contained lipooligosaccharide and a subset of bacterial proteins and lipoproteins. Western blotting and mass spectral analysis indicated that the majority of the proteins were derived from the outer membrane. Mass spectrometric and bioinformatics analysis of SVs identified 29 membrane proteins, including porin and opacity-associated protein. Proteins embedded in the SVs leaflet could be degraded by the addition of trypsin or proteinase K. Our data showed that the incorporation of CTAT and SDBS into vesicles eliminated their toxicity as measured by a THP-1 killing assay. Incorporation of gonococcal cell surface components into SVs reduced toxicity as compared to the whole cell extracts, as measured by cytokine induction, while retaining the immunogenicity. This process constitutes a general method for extracting bacterial surface components and identification of antigens that might be included in vaccines.

Evaluating methods to create protein functionalized catanionic vesicles

“…We are interested in anionic SDBS/CTAT SVs due to their potential as drug delivery vehicles, vaccine platforms, 6 and systems used to investigate carbohydrate-protein interactions 7–9 and bacterial surface molecules. 10…”

“…We are interested in anionic SDBS/CTAT SVs due to their potential as drug delivery vehicles, vaccine platforms, 6 and systems used to investigate carbohydrate-protein interactions [7][8][9] and bacterial surface molecules. 10 Anionic SVs are attractive for drug delivery and vaccine applications due to their ease of preparation and functionalization, and payload capacity. It has been demonstrated that small molecules can be incorporated into vesicles, 3,11,12 and vesicles have been functionalized with carbohydrates, 6,8 biotin, 13 folic acid, and peptides.…”