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

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 assa...

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Autores principales: Stein, Daniel C., H. Stocker, Lenea, Powell, Abigail E., Kebede, Salsawi, Watts, David, Williams, Emma, Soto, Nicholas, Dhabaria, Avantika, Fenselau, Catherine, Ganapati, Shweta, DeShong, Philip
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7559012/
https://www.ncbi.nlm.nih.gov/pubmed/32825235
http://dx.doi.org/10.3390/pharmaceutics12090787
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author Stein, Daniel C.
H. Stocker, Lenea
Powell, Abigail E.
Kebede, Salsawi
Watts, David
Williams, Emma
Soto, Nicholas
Dhabaria, Avantika
Fenselau, Catherine
Ganapati, Shweta
DeShong, Philip
author_facet Stein, Daniel C.
H. Stocker, Lenea
Powell, Abigail E.
Kebede, Salsawi
Watts, David
Williams, Emma
Soto, Nicholas
Dhabaria, Avantika
Fenselau, Catherine
Ganapati, Shweta
DeShong, Philip
author_sort Stein, Daniel C.
collection PubMed
description 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.
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spelling pubmed-75590122020-10-26 Extraction of Membrane Components from Neisseria gonorrhoeae Using Catanionic Surfactant Vesicles: A New Approach for the Study of Bacterial Surface Molecules Stein, Daniel C. H. Stocker, Lenea Powell, Abigail E. Kebede, Salsawi Watts, David Williams, Emma Soto, Nicholas Dhabaria, Avantika Fenselau, Catherine Ganapati, Shweta DeShong, Philip Pharmaceutics Article 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. MDPI 2020-08-20 /pmc/articles/PMC7559012/ /pubmed/32825235 http://dx.doi.org/10.3390/pharmaceutics12090787 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Stein, Daniel C.
H. Stocker, Lenea
Powell, Abigail E.
Kebede, Salsawi
Watts, David
Williams, Emma
Soto, Nicholas
Dhabaria, Avantika
Fenselau, Catherine
Ganapati, Shweta
DeShong, Philip
Extraction of Membrane Components from Neisseria gonorrhoeae Using Catanionic Surfactant Vesicles: A New Approach for the Study of Bacterial Surface Molecules
title Extraction of Membrane Components from Neisseria gonorrhoeae Using Catanionic Surfactant Vesicles: A New Approach for the Study of Bacterial Surface Molecules
title_full Extraction of Membrane Components from Neisseria gonorrhoeae Using Catanionic Surfactant Vesicles: A New Approach for the Study of Bacterial Surface Molecules
title_fullStr Extraction of Membrane Components from Neisseria gonorrhoeae Using Catanionic Surfactant Vesicles: A New Approach for the Study of Bacterial Surface Molecules
title_full_unstemmed Extraction of Membrane Components from Neisseria gonorrhoeae Using Catanionic Surfactant Vesicles: A New Approach for the Study of Bacterial Surface Molecules
title_short Extraction of Membrane Components from Neisseria gonorrhoeae Using Catanionic Surfactant Vesicles: A New Approach for the Study of Bacterial Surface Molecules
title_sort extraction of membrane components from neisseria gonorrhoeae using catanionic surfactant vesicles: a new approach for the study of bacterial surface molecules
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7559012/
https://www.ncbi.nlm.nih.gov/pubmed/32825235
http://dx.doi.org/10.3390/pharmaceutics12090787
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