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.

Quantitative Proteomics of the 2016 WHO Neisseria gonorrhoeae Reference Strains Surveys Vaccine Candidates and Antimicrobial Resistance Determinants

“…Proteomic approaches have an advantage over genomics in drug and vaccine discovery endeavors by delivering information pertaining to protein abundance, post-translational modification(s), structure-function relationships, and protein-protein interactions (40 -42). In addition, subcellular fractionation steps preceding proteomic applications reduce sample complexity, increase the likelihood of discovering low-abundance proteins, and aid in defining protein localization, all of which provide further insights into the proteins' functions and interactomes (43,44). For N. gonorrhoeae, proteomic approaches have begun to deliver proteinaceous vaccine candidates (29,30,39,45) and to support elucidation of AMR patterns (46,47).…”

Structural and functional insights into the role of BamD and BamE within the β-barrel assembly machinery in Neisseria gonorrhoeae

“…Using a quantitative proteomic approach, we have previously identified BamA GC , BamD GC , and BamE GC in both cell envelopes and naturally released membrane vesicles derived from four different N. gonorrhoeae strains (7,55). Intriguingly, differences in homologous proteins' subcellular localization between even closely related organisms such as N. meningitidis and N. gonorrhoeae have been reported (42).…”

“…Obtained genetic constructs were verified by Sanger sequencing at the Center for Genomic Research and Biocomputing at Oregon State University and USA Macrogen. Transformation of N. gonorrhoeae was performed as described previously (55).…”

Detection of bacterial-reactive natural IgM antibodies in desert bighorn sheep populations

“…After carefully decanting the supernatant, the cell pellet was frozen at -20°C. Cell envelope proteins were isolated following previously established methods based on French press cell lysis, followed by treatment with chaotropic reagent (sodium carbonate) and differential centrifugation [36–38]. In comparison to other sub-fractionation methods this protocol allows an enrichment of outer membrane proteins, yielding a sample compatible with mass spectrometry.…”