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

Daniel C Stein,Avantika Dhabaria,Lenea H Stocker,Abigail E Powell,Nicholas Soto,Catherine Fenselau,Philip Deshong,Shweta Ganapati,Emma Williams,David Watts,Salsawi Kebede

doi:10.3390/pharmaceutics12090787

Daniel C Stein, Avantika Dhabaria + Show 9 more

Open Access

https://doi.org/10.3390/pharmaceutics12090787

Copy DOI

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.

Highlights

Cell surface receptors play a crucial role in cell–cell communication and recognition [1]
Methods that improve the extraction of bacterial antigens from membranes or simplify the reconstitution of cellular components into stable hydrophobic matrices would represent a major improvement in understanding how various bacterial surface components contribute to bacterial biology
Since most liposomes are thermodynamically stable as films rather than as a vesicle [21], production of the liposome requires significant manipulation for formation, usually by sonication or repeated passage through a membrane to produce the unstable vesicle morphology (this mechanical stress may denature the protein(s) incorporated into the leaflet)

Summary

Introduction

Cell surface receptors play a crucial role in cell–cell communication and recognition [1]. Despite the success of using liposomes to formulate cell surface components in the laboratory, they have significant limitations as vehicles for displaying cell surface proteins and lipids in a stable membrane-like environment for biochemical studies or in clinically relevant vaccine formulations (see Akbarzadeh et al for a review of liposomes) [5]. SVs have numerous advantages over their liposomal counterparts as a vehicle for extraction and presentation of cell surface components in vaccine formulations and for studies on membrane biology. The ability to create complex nanomaterials teamed with the robust character of SVs, in biological buffers, would make the resulting functionalized vesicles attractive for vaccine formulations and drug delivery due to facilitate preparation, ease of handling, and stability in long-term storage. Methods that improve the extraction of bacterial antigens from membranes or simplify the reconstitution of cellular components into stable hydrophobic matrices would represent a major improvement in understanding how various bacterial surface components contribute to bacterial biology

Methods

Results

Discussion

Conclusion