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Isolation of the Dot/Icm Type IV Secretion System Core Complex from Legionella pneumophila for Negative Stain Electron Microscopy Studies

从嗜肺军团杆菌中分离适于电子显微镜负染色技术检测的Dot/Icm IV型分泌系统核心复合物

Tomoko Kubori Tomoko Kubori
HN Hiroki Nagai

发布: 2017年04月20日第7卷第8期 DOI: 10.21769/BioProtoc.2229 浏览次数: 8137

评审: Anastasia D GaziAnonymous reviewer(s)

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Abstract

Legionella possesses a pivotal secretion machinery to deliver virulence factors to eukaryotic host cells. In this protocol, we describe the procedure for isolation of the native core complex of the Dot/Icm type IV secretion system from L. pneumophila aiming to perform biochemical and transmission electron microscopy analyses.

Keywords: Legionella (军团菌) search Type IV secretion (IV型分泌) search Core complex (核心复合体) search Isolation (分离) search Electron microscopy (电子显微镜技术) search Bacteria (细菌) search Nanomachine (纳米机) search Structure (结构) search

Background

Legionella pneumophila is a Gram-negative bacterial pathogen that causes lung infection known as Legionnaires’ disease (Fields et al., 2002). L. pneumophila utilizes a type IV secretion system (T4SS) encoded by the dot/icm genes to transport about 300 bacterial proteins into the cytosol of their eukaryotic host to hijack cellular processes (Hubber and Roy, 2010). Composed of more than 20 proteins, the T4SS is a nanomachine built on the bacterial inner and outer membranes (Nagai and Kubori 2011; Kubori and Nagai 2016). The core complex of Dot/Icm T4SS is a biochemically stable part of the system and forms a transport conduit bridging the inner and outer membrane (Kubori et al. 2014). The core complex is composed of at least five proteins; three outer membrane-associated proteins, DotC, DotD and DotH, and two inner membrane proteins, DotF and DotG (Vincent et al., 2006). Based on the procedure for biochemical isolation of another bacterial nanomachine, the type III secretion system, from Salmonella typhimurium (Kubori et al., 1998; Marlovits et al., 2004), we modified the protocol to adapt it to the purification of the T4SS of L. pneumophla. In this protocol, we present the procedure to isolate the native core complex of the T4SS from detergent lysed wild-type L. pneumophila based on separation by ultracentrifugation. T4SS isolated using this procedure can be used to perform biochemical and transmission electron microscopy analyses described previously (Kubori et al., 2014).

Materials and Reagents

  1. Sterile swabs
  2. Sterile cell scrapers (IWAKI, catalog number: 9000-220 )
  3. Sterile conical tubes (50 ml and 15 ml)
  4. Sterile Petri dishes (100 mm in diameter)
  5. Cuvettes for spectrophotometer (1.5 ml) (BOECO, catalog number: BRA 759017 )
  6. Millex-GP filter units (EMD Millipore, catalog number: SLGP033RS )
  7. Sterile 10 ml syringe (Terumo, catalog number: SS-10LZ )
  8. Ultrafree MC filters (EMD Millipore, catalog number: UFC30GV00 )
  9. Sterile pipets (10 ml) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 170356 )
  10. L. pneumophila Lp01 strain (Philadelphia-1 rpslL hsdR) (Berger and Isberg, 1993)
  11. cOmpleteTM protease inhibitor cocktail (Roche Diagnostics, catalog number: 11697498001 )
  12. Sodium chloride (NaCl) (Nacarai Tesque, catalog number: 31320-05 )
  13. 12.5% precast polyacrylamide gels (ATTO, catalog number: e-PAGEL E-R12.5L ; or equivalent)
  14. Glow-discharged carbon grids (Nisshin EM, catalog number: 649 )
  15. Coomassie brilliant blue (CBB) stain One (Nacarai Tesque, catalog number: 04543-51 )
  16. ACES (Sigma-Aldrich, catalog number: A3594 )
  17. BactoTM yeast extract (BD, BactoTM, catalog number: 212750 )
  18. MilliQ water
  19. Activated charcoal (Sigma-Aldrich, catalog number: C5510 )
  20. BactoTM agar (BD, BactoTM, catalog number: 214010 )
  21. L-cysteine hydrochloride monohydrate (Nacarai Tesque, catalog number: 10313-55 )
  22. Iron(III) nitrate enneahydrate, Fe(NO3)3·9H2O (Nacarai Tesque, catalog number: 19514-55 )
  23. Tris(hydroxymethyl)aminomethane (Tris) (Nacarai Tesque, catalog number: 35434-21 )
  24. Hydrochloric acid (HCl) (Nacarai Tesque, catalog number: 18321-05 )
  25. Sucrose (Nacarai Tesque, catalog number: 30404-45 )
  26. Phenylmethylsulfonyl fluoride (PMSF) (Nacarai Tesque, catalog number: 27327-94 )
  27. Isopropanol (Sigma-Aldrich, catalog number: 190764 )
  28. EDTA·2Na (Nacarai Tesque, catalog number: 15130-95 )
  29. Sodium hydroxide (NaOH) (Nacarai Tesque, catalog number: 31511-05 )
  30. Lysozyme (Wako Pure Chemical Industries, catalog number: 120-02674 )
  31. Triton X-100 (Nacarai Tesque, catalog number: 35501-15 )
  32. AG501-X8 Resin (Bio-Rad Laboratories, catalog number: 143-7425 )
  33. Magnesium sulfate heptahydrate (MgSO4·7H2O) (Nacarai Tesque, catalog number: 21003-75 )
  34. DNase I (Sigma-Aldrich, catalog number: DN25 )
  35. Potassium hydroxide (KOH) (Nacarai Tesque, catalog number: 28616-45 )
  36. Uranyl acetate (UA) (Merck, catalog number: 8473 )
  37. Phosphotungstic acid (PTA) (TAAB, catalog number: p013 )
  38. CYE plate (see Recipes)
  39. AYE medium (see Recipes)
  40. Tris-Cl solution (pH 8.0) (see Recipes)
  41. Sucrose solution (see Recipes)
  42. PMSF stock solution (see Recipes)
  43. EDTA stock solution (see Recipes)
  44. Lysozyme solution (see Recipes)
  45. Triton X-100 stock solution (see Recipes)
  46. MgSO4 stock solution (see Recipes)
  47. DNase I stock solution (see Recipes)
  48. KOH solution (see Recipes)
  49. NaOH stock solution (see Recipes)
  50. TET solution (see Recipes)
  51. PTA solution (see Recipes)
  52. Uranyl acetate solution (see Recipes)

Equipment

  1. Glass flasks (2 L)
  2. 37 °C shaking incubator
  3. 37 °C incubator
  4. Spectrophotometer
  5. Refrigerated centrifuge (KUBOTA, model: 7780 ; or equivalent models)
  6. Rotor for centrifugation (KUBOTA, models: AG-5006 , AG-6512C )
  7. Sterile centrifuge tubes (500 ml capacity, polypropylene or polycarbonate)
  8. Sterile centrifuge tubes (50 ml capacity, polyallomer or polycarbonate)
  9. Clean grass beakers (200 ml)
  10. Magnetic stir bars
  11. Magnetic stirrer
  12. pH meter
  13. Ultracentrifugation (Beckman Coulter, model: OptimaTM L-100 XP ; or equivalent models)
  14. Rotor for ultracentrifugation (Beckman Coulter, model: Type 70Ti )
  15. Tubes for ultracentrifugation (Beckman Coulter, catalog number: 355631 )
  16. Microfuge (Eppendorf, model: 5415 R )
  17. Glass beakers (1 L)
  18. Autoclavable flasks (2 L)
  19. Autoclave
  20. Electrophoresis apparatus (ATTO, model: AE-6530 ; or equivalent model)
  21. Electron microscope (JEOL, model: JEM-1011 )
  22. ÄKTA purifier (GE Healthcare)
  23. Superose 6 10/300 GL column (GE Healthcare, catalog number: 17517201 )

Procedure

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文章信息

版权信息

© 2017 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

  1. Kubori, T. and Nagai, H. (2017). Isolation of the Dot/Icm Type IV Secretion System Core Complex from Legionella pneumophila for Negative Stain Electron Microscopy Studies. Bio-protocol 7(8): e2229. DOI: 10.21769/BioProtoc.2229.
  2. Kubori, T., Koike, M., Bui, X. T., Higaki, S., Aizawa, S. and Nagai, H. (2014). Native structure of a type IV secretion system core complex essential for Legionella pathogenesis. Proc Natl Acad Sci U S A 111(32): 11804-11809.

    3. ris ris Download Citation in RIS Format

分类

微生物学 > 微生物-宿主相互作用 > 细菌

生物化学 > 蛋白质 > 结构

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