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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   

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

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

  1. Grow L. pneumophila Lp01 strain on a charcoal-yeast extract (CYE) plate (Recipe No. 1) from a glycerol frozen stock for 3 days at 37 °C.
  2. Use a sterile swab to streak a L. pneumophila colony on the entire surface of CYE plates and cultivate for 2 days at 37 °C.
    Note: Two plates covered with a bacterial lawn are needed to obtain sufficient starting material to prepare 1 L culture.
  3. Take the whole bacteria using sterile cell scrapers and suspend it with ~5 ml ACES-buffered yeast extract (AYE) medium (Recipe No. 2) in a sterile tube. Measure OD600. Add the bacterial solution into 1 L AYE medium in a 2 L flask to make the suspension of OD600 0.2.
  4. Grow the bacteria for 12 h at 37 °C with rotary shaking (250 rpm).
  5. Chill the flask in icy water. Measure OD600. (It should be 2.0-3.0, indicating the late log phase of growth). Transfer the culture solution to chilled centrifuge tubes (2-3 tubes of 500 ml capacity).
  6. Harvest the bacteria by centrifugation at 12,000 x g (8,000 rpm) for 15 min at 4 °C (Refrigerated centrifuge: Kubota, model: 7780 or equivalent models; Rotor: Kubota, model: AG-5006).
  7. Resuspend the bacterial pellet with 140 ml of cold sucrose solution (Recipe No. 4) containing 1x cOmpleteTM protease inhibitor cocktail.
    Note: Thoroughly suspending bacterial pellets enhances the efficiency of the detergent lysis in step 11.
  8. Transfer the suspension to a 200 ml beaker with a magnetic stir bar on ice.
  9. Set the beaker on a magnetic stirrer at room temperature, and stir mildly until uniform suspension is achieved.
  10. Add PMSF (final 1 mM, Recipe No. 5), EDTA (final 1 mM, Recipe No. 6) and lysozyme (final 0.1 mg/ml, Recipe No. 7) in this order.
    Note: This is the process of spheroplast formation.
    Prepare a 20 mg/ml 200x stock solution. Add 0.7 ml of this stock solution to the 140 ml sucrose solution, yielding a final lysozyme concentration of approximately 0.1 mg/ml in the bacterial resuspension. Keep stirring for another 30 min at room temperature.
  11. Add slowly (drop by drop) 7 ml of 20% (w/v) Triton X-100 stock solution (final 1% w/v, Recipe No. 8) with stirring.
    Note: This is the most important step for detergent lysis of bacterial membranes. Carefully monitor the change of color and viscosity.
  12. Keep stirring until the solution becomes very clear (approximately for 30 min).
  13. Add 420 μl of 1 M MgSO4 stock solution (final 3 mM, Recipe No. 9) and 70 μl of 10 mg/ml DNase I stock solution (final 5 μg/ml, Recipe No. 10) in this order.
    Note: This step is required to digest DNA.
  14. Stir for 10 min.
  15. Add EDTA (final 10 mM).
  16. Monitoring pH using a pH meter, adjust pH to 10.0 by adding 1 N NaOH (Recipe No. 12).
    Note: During this step, large membrane vesicles and fragments that remain are disrupted. The core complex is stable at this high pH, while loosely associated contaminant proteins can be detached from the core complex. For the purpose of analyzing the loosely associated components of the T4SS, pH value can be modified to maintain these proteins in the complex.
  17. Move the beaker on ice.

Note: Procedure below should be done at 4 °C.

  1. Transfer the lysate to chilled 50 ml centrifuge tubes (divide into 3 tubes).
  2. Centrifuge the lysate at 12,000 x g for 20 min at 4 °C to remove non-lysed materials. (Refrigerated centrifuge: Kubota, model: 7780 or equivalent models; Rotor: Kubota, model: AG-6512C).
  3. Recover supernatant and transfer to ultracentrifuge tubes (Beckman Coulter).
  4. Apply ultracentrifugation at 100,000 x g for 30 min at 4 °C to precipitate protein complexes. (Ultracentrifuge: Beckman Coulter, model: Optima L-100 XP or equivalent models; Rotor: Beckman Coulter, model: Type 70Ti)
  5. Discard supernatant. Soak the pellet with 0.5 ml of cold TET solution (Recipe No. 13) including 1 mM PMSF per tube. To dissolve completely, leave the pellet in TET overnight at 4 °C.
  6. Merge the completely dissolved suspensions and transfer them in a new conical or centrifuge tube (total ~5 ml). Use extra 2-3 ml of cold TET solution to completely recover the dissolved proteins from the tubes and merge them into the new tubes.
  7. Centrifuge the suspension at 14,000 x g for 15 min at 4 °C to remove precipitate. (Refrigerated centrifuge: Kubota, model: 7780 or equivalent models; Rotor: Kubota, model: AG-6512C).
  8. Submit the supernatant to a second round of ultracentrifugation at 100,000 x g for 30 min at 4 °C. (Ultracentrifuge: Beckman Coulter, model: Optima L-100 XP or equivalent models; Rotor: Beckman Coulter, model: Type 70Ti)
  9. Resuspend the pellet in ~300 μl of cold TET. The sample is ready for biochemical and electron microscopic analyses (Figures 1A and 1B).
  10. (Optional) Further separation by Superose 6 10/300 column chromatography equilibrated with TET plus 50 mM NaCl can be used to remove large aggregates either made of T4SS or contaminant proteins. Collect the fractions and identify the fractions containing the core complex by gel electrophoresis and transmission electron microscopy (as shown in steps 28 and 29) (Figures 1C and 1D).
    Note: As TET solution contains Triton X-100 that disturbs UV monitoring, the UV peaks do not always accord with the presence of the protein complexes. Alternative approaches like sucrose density gradient ultracentrifugation can be applicable for further purification.
  11. For biochemical analysis, apply samples on 12.5% SDS-polyacrylamide gel (PAGE) and stain with ready-made Coomassie Brilliant Blue stain solution, CBB stain One (Nacarai).
  12. For transmission electron microscopy analysis, apply samples on glow-discharged carbon grids and negatively stained with 2% (w/v) PTA pH 7.0 (Recipe No. 14) or 2% (w/v) uranyl acetate (Recipe No. 15). Take micrographs at an accelerating voltage of 80 kV.

Data analysis

The second round of ultracentrifugation enhances the purity of the isolated complex, and the following column chromatography further removes the background contaminations (Figure 1).


Figure 1. Example of isolated Dot/Icm T4SS core complex. A. SDS-PAGE analysis of the isolated complex in comparison between 1st and 2nd rounds of ultracentrifugation (steps 21 and 25, respectively). Whole cell: Whole cell lysate (step 20); cfg1 ppt: 1st ultracentrifugation pellet (step 22); cfg2 ppt: 2nd ultracentrifugation pellet (step 26). As a negative control, a L. pneumophila strain lacking all dot/icm genes (∆T4SS) was also submitted to the isolation procedure (the rightmost lane). B. The electron micrograph of the fraction obtained by the 2nd ultracentrifugation; C. SDS-PAGE analysis of the fractions obtained by a size exclusion column chromatography (step 27); D. The electron micrograph of the fraction A10 of (C). Molecular weight markers are shown in kDa. MOMP: Major Outer membrane Protein. The images are adapted from Kubori et al. (2014).

Recipes

  1. CYE plate (for 24 plates, 1 L)
    10 g ACES
    10 g yeast extract
    Dissolve in ~0.9 L MilliQ water in a 1 L glass beaker, adjust pH by adding 1 N KOH (see Recipe No. 11; ~40 ml) and monitoring with pH meter
    Bring the volume up to 1 L
    Add to a 2 L autoclavable flask containing a magnetic stirrer bar and 2 g activated charcoal and 15 g agar
    Mix briefly by stirring
    Autoclave the media at 121 °C for 30 min
    Cool down at room temperature until the temperature reaches ~60 °C with gentle stirring. During the time, prepare L-cysteine (0.4 g solved in 10 ml sterile MilliQ water) and Fe(NO3)3 (0.135 g solved in 10 ml sterile MilliQ water) solutions in sterile 15 ml conical tubes, and filter them with Millex-GP filter unit with 10 ml syringe
    Add L-cysteine and Fe(NO3)3 solutions (10 ml each) to 1 L medium with stirring
    Keep stirring for another 5 min to mix homogeneously
    Pour ~40 ml per Petri dish and cool down to solidify
  2. AYE medium (1 L)
    Same as CYE except that agar is omitted
    Note: AYE is not good for use on the same day of preparation. For good result, AYE should be made one day before the Legionella culture. Old media (more than a week after preparation) is not recommended.
  3. Tris-Cl solution (pH 8.0)
    1 M solution
    Adjust pH to 8.0 with HCl
    Autoclave at 121 °C for 20 min
  4. Sucrose solution
    0.5 M sucrose
    150 mM Tris-Cl (pH 8.0)
  5. PMSF stock solution
    100 mM in isopropanol
    Storage at -20 °C
  6. EDTA stock solution
    0.5 M EDTA
    Adjust pH to 8.0 with NaOH
    Autoclave at 121 °C for 20 min
  7. Lysozyme solution
    20 mg/ml in sucrose solution
    Prepare just before use
  8. Triton X-100 stock solution
    20% (w/v) solution containing ~2 g of AG501-X8 Resin for deionizing
  9. MgSO4 stock solution
    1 M solution
    Autoclave at 121 °C for 20 min
  10. DNase I stock solution
    Dissolve the powder in sterile water to give 10 mg/ml solution
    Storage at -20 °C
  11. KOH solution
    1 N solution
  12. NaOH stock solution
    1 N solution
  13. TET solution
    10 mM Tris-Cl (pH 8.0)
    1 mM EDTA
    0.1% Triton X-100
  14. PTA solution
    2% (w/v) phosphotungstic acid. Adjust to pH 7.0
  15. Uranyl acetate solution
    2% (w/v) uranyl acetate
    Filter with 0.22 μm ultra-free MC

Acknowledgments

This work has been financially supported by MEXT/JSPS KAKENHI Grants 15H01322 (to TK). The protocol presented here has been adapted from Kubori et al. (2014).

References

  1. Berger, K. H. and Isberg, R. R. (1993). Two distinct defects in intracellular growth complemented by a single genetic locus in Legionella pneumophila. Mol Microbiol 7(1): 7-19.
  2. Fields, B. S., Benson, R. F. and Besser, R. E. (2002). Legionella and Legionnaires’ disease: 25 years of investigation. Clin Microbiol Rev 15(3): 506-526.
  3. Hubber, A. and Roy, C. R. (2010). Modulation of host cell function by Legionella pneumophila type IV effectors. Annu Rev Cell Dev Biol 26: 261-283.
  4. 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.
  5. Kubori, T., Matsushima, Y., Nakamura, D., Uralil, J., Lara-Tejero, M., Sukhan, A., Galan, J. E. and Aizawa, S. I. (1998). Supramolecular structure of the Salmonella typhimurium type III protein secretion system. Science 280(5363): 602-605.
  6. Kubori, T. and Nagai, H. (2016). The Type IVB secretion system: an enigmatic chimera. Curr Opin Microbiol 29: 22-29.
  7. Marlovits, T. C., Kubori, T., Sukhan, A., Thomas, D. R., Galan, J. E. and Unger, V. M. (2004). Structural insights into the assembly of the type III secretion needle complex. Science 306(5698): 1040-1042.
  8. Nagai, H. and Kubori, T. (2011). Type IVB secretion systems of legionella and other Gram-negative bacteria. Front Microbiol 2: 136.
  9. Vincent, C. D., Friedman, J. R., Jeong, K. C., Buford, E. C., Miller, J. L. and Vogel, J. P. (2006). Identification of the core transmembrane complex of the Legionella Dot/Icm type IV secretion system. Mol Microbiol 62(5): 1278-1291.

简介

军团菌具有关键的分泌机制,以将毒力因子递送至真核宿主细胞。在本协议中,我们描述了从L / L分离Dot / Icm IV型分泌系统的天然核心复合物的步骤。肺炎支原体旨在进行生物化学和透射电子显微镜分析。

嗜肺军团菌是革兰氏阴性细菌病原体,其导致被称为退伍军人病的肺部感染(Fields等人,2002)。 L。嗜肺杆菌利用由 dot / cm 基因编码的IV型分泌系统(T4SS)将大约300种细菌蛋白转运到其真核宿主细胞质中以劫持细胞过程(Hubber和Roy, 2010)。由超过20种蛋白组成,T4SS是建立在细菌内膜和外膜上的纳米机器(Nagai和Kubori 2011; Kubori和Nagai 2016)。 Dot / Icm T4SS的核心复合物是系统的生物化学稳定部分,并形成桥接内膜和外膜的输送导管(Kubori等人,2014)。核心复合物由至少五种蛋白质组成;三种外膜相关蛋白,DotC,DotD和DotH,以及两种内膜蛋白DotF和DotG(Vincent等人,2006)。基于来自鼠伤寒沙门氏菌的另一种细菌纳米机械的III型分泌系统的生物化学分离方法(Kubori等人,1998; Marlovits等人, ,2004),我们修改了方案,使其适应于LT的T4SS的纯化。气肺。在该方案中,我们提出了从洗涤剂裂解的野生型L中分离T4SS的天然核心复合物的过程。基于通过超速离心分离的嗜肺杆菌。使用该程序分离的T4SS可用于进行前述的生化和透射电子显微镜分析(Kubori等人,2014)。

关键字:军团菌, IV型分泌, 核心复合体, 分离, 电子显微镜技术, 细菌, 纳米机, 结构

材料和试剂

  1. 无菌拭子
  2. 无菌细胞刮刀(IWAKI,目录号:9000-220)
  3. 无菌锥形管(50毫升和15毫升)
  4. 无菌培养皿(直径100毫米)
  5. 分光光度计的比色杯(1.5ml)(BOECO,目录号:BRA 759017)
  6. Millex-GP过滤器单元(EMD Millipore,目录号:SLGP033RS)
  7. 无菌10ml注射器(Terumo,目录号:SS-10LZ)
  8. Ultrafree MC滤波器(EMD Millipore,目录号:UFC30GV00)
  9. 无菌移液管(10ml)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:170356)
  10. L。嗜肺杆菌Lp01菌株(Philadelphia-1 rpslL hsdR )(Berger和Isberg,1993)
  11. cOmplete TM蛋白酶抑制剂混合物(Roche Diagnostics,目录号:11697498001)
  12. 氯化钠(NaCl)(Nacarai Tesque,目录号:31320-05)
  13. 12.5%预制聚丙烯酰胺凝胶(ATTO,目录号:e-PAGEL E-R12.5L;或等同物)
  14. 辉光排放的碳网格(Nisshin EM,目录号:649)
  15. 考马斯亮蓝(CBB)染色One(Nacarai Tesque,目录号:04543-51)
  16. ACES(Sigma-Aldrich,目录号:A3594)
  17. Bacto TM酵母提取物(BD,Bacto TM,目录号:212750)
  18. MilliQ水
  19. 活性炭(Sigma-Aldrich,目录号:C5510)
  20. Bacto TM琼脂(BD,Bacto TM,目录号:214010)
  21. L-半胱氨酸盐酸盐一水合物(Nacarai Tesque,目录号:10313-55)
  22. 硝酸铁(III),水合铁(III),Fe(NO 3 3)3·9H 2 O(Nacarai Tesque,目录号:19514-55) br />
  23. 三羟甲基氨基甲烷(Tris)(Nacarai Tesque,目录号:35434-21)
  24. 盐酸(HCl)(Nacarai Tesque,目录号:18321-05)
  25. 蔗糖(Nacarai Tesque,目录号:30404-45)
  26. 苯甲基磺酰氟(PMSF)(Nacarai Tesque,目录号:27327-94)
  27. 异丙醇(Sigma-Aldrich,目录号:190764)
  28. EDTA·2Na(Nacarai Tesque,目录号:15130-95)
  29. 氢氧化钠(NaOH)(Nacarai Tesque,目录号:31511-05)
  30. 溶菌酶(Wako Pure Chemical Industries,目录号:120-02674)
  31. Triton X-100(Nacarai Tesque,目录号:35501-15)
  32. AG501-X8 Resin(Bio-Rad Laboratories,目录号:143-7425)
  33. 七水硫酸镁(MgSO 4·7H 2 O)(Nacarai Tesque,目录号:21003-75)
  34. DNase I(Sigma-Aldrich,目录号:DN25)
  35. 氢氧化钾(KOH)(Nacarai Tesque,目录号:28616-45)
  36. 乙酸异丙酯(UA)(Merck,目录号:8473)
  37. 磷钨酸(PTA)(TAAB,目录号:p013)
  38. CYE板(参见食谱)
  39. AYE培养基(见食谱)
  40. Tris-Cl溶液(pH 8.0)(参见食谱)
  41. 蔗糖溶液(参见食谱)
  42. PMSF储备溶液(见配方)
  43. EDTA储备溶液(参见食谱)
  44. 溶菌酶溶液(参见食谱)
  45. Triton X-100储备溶液(见配方)
  46. MgSO 4次要储备溶液(参见食谱)
  47. DNase I储备溶液(参见食谱)
  48. KOH溶液(参见食谱)
  49. NaOH储液(参见食谱)
  50. TET解决方案(请参阅食谱)
  51. PTA解决方案(见配方)
  52. 乙酸溶液溶液(见食谱)

设备

  1. 玻璃瓶(2升)
  2. 37℃振荡培养箱
  3. 37℃培养箱
  4. 分光光度计
  5. 冷藏式离心机(KUBOTA,型号:7780;等效型号)
  6. 转子用于离心(KUBOTA,型号:AG-5006,AG-6512C)
  7. 无菌离心管(500 ml容量,聚丙烯或聚碳酸酯)
  8. 无菌离心管(容量为50ml,多聚体或聚碳酸酯)
  9. 清洁烧杯(200毫升)
  10. 磁力搅拌棒
  11. 磁力搅拌器
  12. pH计
  13. 超速离心机(Beckman Coulter,型号:Optima< sup>> L-100 XP;或等效型号)
  14. 转子用于超速离心(Beckman Coulter,型号:70Ti型)
  15. 超速离心管(Beckman Coulter,目录号:355631)
  16. Microfuge(Eppendorf,型号:5415 R)
  17. 玻璃烧杯(1升)
  18. 高压灭菌瓶(2升)
  19. 高压灭菌器
  20. 电泳仪(ATTO,型号:AE-6530;等效型号)
  21. 电子显微镜(JEOL,型号:JEM-1011)
  22. ÄKTA净化器(GE Healthcare)
  23. 超级6 10/300 GL柱(GE Healthcare,目录号:17517201)

程序

  1. 长大嗜碱性杆菌Lp01菌株在木糖酵母提取物(CYE)板(Recipe No.1)上)从丙三醇冷冻原液中在37℃下培养3天。
  2. 使用无菌拭子连线。肺炎支原体菌落在CYE板的整个表面上,并在37℃下培养2天。
    注意:需要用细菌草坪覆盖的两块板才能获得足够的起始材料,以制备1升培养物。
  3. 使用无菌细胞刮刀取全细菌,并用无菌管中的5 ml ACES缓冲酵母提取物(AYE)培养基(Recipe No.2)悬浮。测量OD 600 。将细菌溶液加入2L烧瓶中的1L AYE培养基中,使OD 600的悬浮液为0.2。
  4. 用旋转振荡(250rpm)在37℃下培养细菌12小时
  5. 在冰冷的水中冷却烧瓶。测量OD 600 。 (应该是2.0-3.0,表明生长的延迟对数阶段)。将培养液转移到冷冻离心管(2-3管500ml容量)中。
  6. 在4℃(冷冻离心机:久保田,型号:7780或等效模型;转子:久保田,型号:AG-5006)以12,000xg(8,000rpm)离心收获细菌15分钟。
  7. 用含有1x cOmplete TM蛋白酶抑制剂混合物的140ml冷蔗糖溶液(配方4)重悬细菌沉淀。
    注意:彻底悬浮细菌颗粒可提高步骤11中洗涤剂溶解的效率。
  8. 将悬浮液用冰块上的磁力搅拌棒将悬浮液转移到200毫升烧杯中
  9. 在室温下将烧杯放在磁力搅拌器上,轻轻搅拌直到达到均匀的悬浮液
  10. 按顺序添加PMSF(最终1mM,配方5号),EDTA(最终1mM,配方6号)和溶菌酶(最终0.1mg/ml,配方7号)。
    注意:这是造粒过程。
    准备20毫克/毫升200倍的储备溶液。将0.7ml该储备溶液加入到140ml蔗糖溶液中,在细菌悬浮液中产生约0.1mg/ml的最终溶菌酶浓度。在室温下继续搅拌30分钟。
  11. 在搅拌下逐渐加入7ml 20%(w/v)Triton X-100储备溶液(最终1%w/v,食谱8号)。
    注意:这是细菌膜洗涤剂溶解最重要的一步。仔细监测颜色和粘度的变化。
  12. 继续搅拌直到溶液变得非常清洁(大约30分钟)。
  13. 加入420μl1M MgSO 4储备溶液(最终3mM,配方9号)和70μl10mg/ml DNase I储备溶液(最终5μg/ml,配方10号)。
    注意:此步骤是消化DNA所必需的。
  14. 搅拌10分钟
  15. 加入EDTA(最终10 mM)
  16. 使用pH计监测pH值,通过加入1N NaOH调节pH至10.0(配方12号) 注意:在此步骤中,残留的大膜囊泡和碎片被破坏。核心复合物在该高pH下是稳定的,而松散相关的污染蛋白质可以从核心复合物中分离出来。为了分析T4SS的松散相关成分,可以修改pH值以将这些蛋白质维持在复合物中。
  17. 将冰块放在冰上。

注意:以下程序应在4°C下进行。

  1. 将裂解液转移到冷冻的50 ml离心管中(分3管)
  2. 在4℃下以12,000×g离心裂解物20分钟以除去未裂解的物质。 (冷冻离心机:久保田,型号:7780或等效型号;转子:久保田,型号:AG-6512C)。
  3. 恢复上清液并转移到超速离心管(Beckman Coulter)
  4. 在10℃下,在4℃下超速离心30分钟以沉淀蛋白质复合物。 (超离心机:Beckman Coulter,型号:Optima L-100 XP或等效型号;转子:Beckman Coulter,型号:Type 70Ti)
  5. 丢弃上清液。用0.5ml冷TET溶液(配方13号)浸泡沉淀,包括每管1mM PMSF。要完全溶解,请在TET中将沉淀物在4℃下过夜。
  6. 合并完全溶解的悬浮液,并将其转移到新的锥形或离心管(总共〜5ml)中。使用额外的2-3毫升冷TET溶液,以完全从管中回收溶解的蛋白质,并将其合并到新的管中。
  7. 在4℃下以14,000×g离心悬浮液15分钟以除去沉淀物。 (冷冻离心机:久保田,型号:7780或等效型号;转子:久保田,型号:AG-6512C)。
  8. 在4℃下将上清液以100,000xg的速度进行第二轮超速离心30分钟。 (超离心机:Beckman Coulter,型号:Optima L-100 XP或等效型号;转子:Beckman Coulter,型号:Type 70Ti)
  9. 将沉淀重悬至〜300μl冷TET。样品准备进行生物化学和电子显微镜分析(图1A和1B)。
  10. (可选)通过Superose进一步分离6用TET加50mM NaCl平衡的10/300柱色谱可用于去除由T4SS或污染蛋白质构成的大聚集体。收集级分并通过凝胶电泳和透射电子显微镜鉴定含有核心复合物的级分(如步骤28和29所示)(图1C和1D)。
    注意:由于TET溶液含有干扰紫外线监测的Triton X-100,所以UV峰并不总是符合蛋白质复合物的存在。蔗糖密度梯度超速离心等替代方法可用于进一步纯化。
  11. 对于生化分析,应用12.5%SDS-聚丙烯酰胺凝胶(PAGE)上的样品,并用现成的考马斯亮蓝染色溶液,CBB染色一(Nacarai)染色。
  12. 对于透射电子显微镜分析,将样品应用于辉光排放的碳网上,并用2%(w/v)PTA pH7.0(配方14号)或2%(w/v)乙酸二烷基酯(15号方案) 。以80kV的加速电压拍摄显微照片。

数据分析

第二轮超速离心增强了分离的复合物的纯度,并且以下柱色谱法进一步消除了背景污染物(图1)。


图1.分离的Dot/Icm T4SS核心复合物的实施例A.分离复合物的SDS-PAGE分析比较第1轮和第2轮超速离心(分别为步骤21和25)。全细胞:全细胞裂解物(步骤20); cfg1 ppt:第一超速离心沉淀(步骤22); cfg2 ppt:第二超速离心沉淀(步骤26)。作为阴性对照,a。还缺少所有 dot/icm 基因(ΔT4SS)的肺炎支原体菌株分离程序(最右边的泳道)。 B.通过第二次超速离心获得的级分的电子显微照片; C.通过尺寸排阻柱色谱法(步骤27)获得的级分的SDS-PAGE分析; D.(C)的级分A10的电子显微镜照片。分子量标记以kDa表示。 MOMP:主要外膜蛋白。图像从Kubori等人改编而成。 (2014)。

食谱

  1. CYE板(24板,1升)
    10克ACES
    10克酵母提取物
    在1升烧杯中溶解〜0.9L MilliQ水,通过加入1N KOH调节pH(参见配方11;〜40ml)并用pH计监测
    使音量达到1 L
    加入到含有磁力搅拌棒和2g活性炭和15g琼脂的2L耐高压灭菌烧瓶中 搅拌即时混合
    在121℃高压灭菌30分钟 在室温下冷却直到温度达到〜60℃,轻轻搅拌。在此期间,将L-半胱氨酸(0.4克溶于10ml无菌MilliQ水中)和Fe(NO 3 3)3 N(0.135克)溶于10ml无菌MilliQ水)溶液在无菌15毫升锥形管中,并用10ml注射器
    用Millex-GP过滤器过滤 在搅拌下将L-半胱氨酸和Fe(NO 3 N 3)3种溶液(各10ml)加入到1L培养基中, 继续搅拌5分钟,均匀混匀 每道培养皿倒入40ml,冷却固化
  2. AYE中(1L)
    与CYE相同,除了省略了琼脂 注意:AYE在准备当天不好用。为了取得良好的效果,AYE应该在军团菌文化的一天之前。旧媒体(准备后一周以上)不推荐。
  3. Tris-Cl溶液(pH8.0)
    1 M解决方案
    用HCl调节pH至8.0 在121℃高压灭菌20分钟
  4. 蔗糖溶液
    0.5 M蔗糖
    150mM Tris-Cl(pH8.0)
  5. PMSF库存解决方案
    100 mM在异丙醇中 -20°C储存
  6. EDTA储备溶液
    0.5 M EDTA
    用NaOH调节pH至8.0 在121℃高压灭菌20分钟
  7. 溶菌酶溶液
    20毫克/毫升蔗糖溶液
    准备在使用之前
  8. Triton X-100储备溶液
    含有〜2g AG501-X8用于去离子的树脂的20%(w/v)溶液
  9. MgSO 4次要储备液
    1 M解决方案
    在121℃高压灭菌20分钟
  10. DNase I库存解决方案
    将粉末溶解在无菌水中,得到10mg/ml溶液 -20°C储存
  11. KOH溶液
    1 N解决方案
  12. NaOH储备溶液
    1 N解决方案
  13. TET解决方案
    10mM Tris-Cl(pH8.0)
    1 mM EDTA
    0.1%Triton X-100
  14. PTA解决方案
    2%(w/v)磷钨酸。调整至pH 7.0
  15. 乙酸铀溶液
    2%(w/v)乙酸二铀酸盐
    用0.22微米超滤MC滤波

致谢

这项工作得到MEXT/JSPS KAKENHI拨款15H01322(传统知识)的财政支持。本文介绍的协议已经从Kubori等人改编。 (2014)。

参考文献

  1. Berger,KH和Isberg,RR(1993)。  Two细胞内生长的明显缺陷由嗜肺军团菌中的单个遗传基因座相互补充.Mol Microbiol 7(1):7-19。
  2. 字段,BS,Benson,RF和Besser,RE(2002)。 军团菌和军团病症:25年的调查。 Clin Microbiol Rev 15(3):506-526。
  3. Hubber,A.和Roy,CR(2010)。  调制由嗜肺军团菌IV型效应物组成的宿主细胞功能。 Annu Rev Cell Dev Biol 26:261-283。
  4. Kubori,T.,Koike,M.,Bui,XT,Higaki,S.,Aizawa,S。和Nagai,H。(2014)。军团菌发病机制所必需的IV型分泌系统核心复合体的原生结构。 Proc Natl Acad Sci USA 111(32):11804-11809。
  5. Kubori,T.,Matsushima,Y.,Nakamura,D.,Uralil,J.,Lara-Tejero,M.,Sukhan,A.,Galan,JE和Aizawa,SI(1998)。< a class = ke-insertfile"href ="https://www.ncbi.nlm.nih.gov/pubmed/9554854"target ="_ blank">鼠伤寒沙门氏菌III型蛋白分泌系统的超分子结构。 科学 280(5363):602-605。
  6. Kubori,T.和Nagai,H。(2016)。 IVB型分泌系统:一种神秘的嵌合体。 Curr Opin Microbiol 29:22-29。
  7. Marlovits,TC,Kubori,T.,Sukhan,A.,Thomas,DR,Galan,JE and Unger,VM(2004)。< a class ="ke-insertfile"href ="https://www.ncbi .nlm.nih.gov/pubmed/15528446"target ="_ blank">对III型分泌针复合体的组装的结构洞察。 科学 306(5698):1040-1042 。
  8. Nagai,H。和Kubori,T。(2011)。类型IVB分泌系统的军团菌和其他革兰氏阴性细菌。前微生物 2:136.
  9. Vincent,CD,Friedman,JR,Jeong,KC,Buford,EC,Miller,JL和Vogel,JP(2006)。鉴定军团菌 Dot/Icm IV型分泌系统的核心跨膜复合物。 em> 62(5):1278-1291。
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
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.
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