Isolation of Cell Envelopes and Naturally Released Membrane Vesicles of Neisseria gonorrhoeae

Ryszard A. Zielke1, Aleksandra E. Sikora1

1 Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, Oregon
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 4A.3
DOI:  10.1002/9780471729259.mc04a03s34
Online Posting Date:  August, 2014
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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. Curr. Protoc. Microbiol. 34:4A.3.1‐4A.3.17. © 2014 by John Wiley & Sons, Inc.

Keywords: Neisseria gonorrhoeae; cell envelopes; native membrane vesicles; fractionation

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Aerobic Growth of GC on Plates and in Liquid Media
  • Basic Protocol 2: Preparation of the Cell Envelope Fraction
  • Basic Protocol 3: Preparation of Naturally Released MVs
  • Support Protocol 1: Sample Preparation for Transmission Electron Microscopy
  • Support Protocol 2: SDS‐PAGE and Coomassie Staining
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Aerobic Growth of GC on Plates and in Liquid Media

  Materials
  • GC glycerol stock, frozen (clinical isolates; see unit 4.1)
  • GCB (plates; see recipe)
  • GCBL (liquid medium; see recipe)
  • 15‐cm microbiological loops or sterile wooden applicators (Puritan)
  • 37°C incubator with 3% to 10% CO 2 flow
  • Dacron swabs, sterile (Puritan)
  • 50‐ml conical polypropylene tubes with caps, sterile (VWR)
  • Vortexer
  • Spectrophotometer and 1‐ml disposable cuvettes (VWR)
  • 2800‐ml Fernbach shake flasks (e.g., Pyrex)
  • 37°C shaking incubator (Eppendorf)
  • Biosafety level 2 (BSL‐2) cabinet (Labconco)
  • 500‐ml centrifuge bottles or 250‐ml centrifuge bottles with adapters (Nalgene)
  • Refrigerated floor or countertop centrifuge with a rotor capable of accommodating 500 ml buckets (e.g., Beckman Avanti JE; see Biosafety Considerations in the Strategic Planning Section)
  • 1‐ or 5‐ml disposable pipets (Greiner Bio‐One) and pipet filler (e.g., Hirshmann)

Basic Protocol 2: Preparation of the Cell Envelope Fraction

  Materials
  • GC cell pellet ( protocol 1)
  • 1× phosphate‐buffered saline (PBS), pH 7.5 (see recipe) supplemented with Complete Protease Inhibitor Cocktail tablet, EDTA‐free (Roche), according to the manufacturer's directions
  • Protein quantification kit (e.g., 2‐D‐Quant Kit, GE Healthcare; DC Protein Assay Kit, Bio‐Rad)
  • 0.1 M sodium carbonate (pH 11), 4°C: 8.4 g Na 2CO 3/1 liter of water; do not adjust pH; store up to 6 months at 4°C
  • 10‐ml disposable pipets, sterile, and pipet filler (e.g., Hirshmann)
  • French laboratory press (high‐pressure homogenizer), cooled to 4°C
  • 50‐ml conical, polypropylene tubes, sterile
  • 250‐ml beaker
  • Refrigerated floor or countertop centrifuge with a rotor capable of accommodating 50‐ml conical tubes (e.g., Avanti JE with JA‐12 rotor, Beckman)
  • Sonicator (e.g., Qsonica Q700)
  • Rocking platform (VWR), rotisserie (VWR), or stir plate with sterile stir bar
  • Ultracentrifugation tubes (e.g., Beckman cat. no. 355642)
  • Ultracentrifuge (e.g., Beckman with Type 70 Ti rotor)
  • 1.5‐ml microcentrifuge tubes, sterile (e.g., USA Scientific)

Basic Protocol 3: Preparation of Naturally Released MVs

  Materials
  • Culture supernatants ( protocol 1, step 16b)
  • Complete Protease Inhibitor Cocktail Tablets, EDTA‐free (Roche)
  • 1× phosphate‐buffered saline (PBS), pH 7.5 (see recipe)
  • 1× PBS, pH 7.5/0.2% (w/v) SDS
  • Protein quantification kit (e.g., 2‐D‐Quant Kit, GE Healthcare; DC Protein Assay Kit, Bio‐Rad)
  • 1000‐ml vacuum filter units with 0.22‐µm polyethersulfone membranes, sterile
  • Ultracentrifuge (e.g., Beckman with Type 45 Ti rotor), 4°C
  • 70‐ml centrifuge bottle assembly (Beckman, cat. no. 355622)
  • 25‐ml disposable pipet, sterile, and pipet filler (e.g., Hirshmann)

Support Protocol 1: Sample Preparation for Transmission Electron Microscopy

  Materials
  • MVs ( protocol 3)
  • 1× phosphate‐buffered saline (PBS), pH 7.5 (see recipe), ice‐cold
  • 2% (w/v) ammonium molybdate (e.g., Alfa Aesar)
  • Parafilm M
  • Fine‐point forceps (e.g., Pelco)
  • Formvar/carbon‐coated copper grid (Electron Microscopy Sciences)
  • Whatman filter paper
  • Nitrogen cabinet (Electron Microscopy Sciences) or desiccator
  • Grid storage box (Electron Microscopy Sciences)
  • Transmission electron microscope (e.g., Titan 80‐200, FEI)
  • Additional reagents and equipment for carrying out sample preparation and sample staining for transmission electron microscopy (TEM; e.g., unit 2.1)

Support Protocol 2: SDS‐PAGE and Coomassie Staining

  Materials
  • Cell envelope or MV fractions ( protocol 2 or protocol 3)
  • 4× protein loading buffer (see recipe)
  • 4% to 12% (Life Technologies) or 4% to 20% (Bio‐Rad) precast gradient polyacrylamide gels
  • Protein standard (e.g., Bio‐Rad), prepared according to the manufacturer's directions
  • 1× Tris/glycine/SDS running buffer (see recipe)
  • Colloidal Coomassie Brilliant Blue stain (see recipe)
  • 1.5‐ml microcentrifuge tube (e.g., USA Scientific)
  • Thermoblock (VWR), set to 95°C
  • Microcentrifuge tube closures (USA Scientific)
  • Electrophoresis apparatus (e.g., Bio‐Rad)
  • Gel staining box (e.g., Tarsous)
  • Gel documentation system (e.g., ChemiDoc, Bio‐Rad)
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Figures

Videos

Literature Cited

Literature Cited
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