title: Isolation of Cell Envelopes and Naturally Released Membrane Vesicles of Neisseria gonorrhoeae.
abstract: Neisseria gonorrhoeae (GC) is a strict human pathogen and the agent of the sexually transmitted disease gonorrhea. Gonococcal infections have been successfully treated with antibiotics; however, GC has repeatedly developed resistance to each new antibiotic used. Currently, third-generation cephalosporins are recommended, and resistance to these antimicrobials is emerging worldwide. Additionally, no vaccine is available to prevent GC infections. With the dire possibility of untreatable gonorrhea, there is a critical need to identify new therapeutic targets. Cell envelope and membrane vesicle proteins are key factors in pathogenesis, antibiotic resistance, biofilm formation, and general bacterial fitness. Here we describe methods for isolation and purification of GC cell envelopes and spontaneously released membrane vesicles. The isolated proteome fractions can be used in multiple downstream applications, including gel-based and gel-free quantitative proteomics, studies focused on subcellular localization of proteins, transmission electron microscopy, or strain characterization. Presented methods may be easily adapted to other bacterial species.
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, Oregon.
authors.forename: Ryszard A
authors.initials: RA
authors.lastname: Zielke
authors.forename: Aleksandra E
authors.initials: AE
authors.lastname: Sikora
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001431:Bacteriological Techniques; D002462:Cell Membrane; D002498:Centrifugation / Q000295:instrumentation / Q000379:methods; D009344:Neisseria gonorrhoeae / Q000166:cytology* / Q000502:physiology*
chemical_list: D001426:Bacterial Proteins
keywords: Neisseria gonorrhoeae; cell envelopes; fractionation; native membrane vesicles