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Preparation of Amyloid Fibril Networks
淀粉样纤维网络的制备   

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Materials Science and Engineering C
Oct 2017

Abstract

Networks of amyloid nanofibrils fabricated from common globular proteins such as lysozyme and β-lactoglobulin have material properties that mimic the extracellular microenvironment of many cell types. Cells cultured on such amyloid fibril networks show improved attachment, spreading and in the case of mesenchymal stem cells improved differentiation. Here we describe a detailed protocol for fabricating amyloid fibril networks suitable for eukaryotic cell culture applications.

Keywords: Amyloid fibrils (淀粉样纤维), Self-assembly (自组装), Biomaterials (生物材料), Stem cell culture (干细胞培养), Cell attachment (细胞附着), Biomimetic materials (仿生材料), Protein aggregation (蛋白质聚集)

Background

A wide variety of proteins and peptides can adopt non-native structures and aggregate into amyloid fibrils possessing a common cross β-sheet secondary structure (Nelson et al., 2005). Amyloid fibrils are the pathological hallmarks of a number of neurodegenerative diseases (Chiti and Dobson, 2006), however, there is a growing consensus that mature amyloid fibrils are non-toxic by-products and the toxic species are soluble oligomers, pre-fibrillar aggregates (Kayed et al., 2003), or perhaps the process of aggregation itself (Reynolds et al., 2011). Additionally, a number of functional amyloids with essential physiological features have been discovered in a wide range of organisms (including humans) (Chiti and Dobson, 2006).

Non-toxic synthetic amyloid fibrils can be made from inexpensive, readily available, food grade proteins (Jung et al., 2008; Lara et al., 2011) and have a number of important applications in a wide range of technologies (Dharmadana et al., 2017; Wei et al., 2017). For example 2D and 3D networks of amyloid fibrils have physical and mechanical properties that mimic the local microenvironment of many eukaryotic cells, namely the extracellular matrix (ECM), thus are promising biomimetic materials for cell culture applications (Reynolds et al., 2014 and 2015; Gilbert et al., 2017). Here we describe in detail a protocol to fabricate aqueous suspensions of amyloid fibrils and their subsequent adsorption onto solid supports where they have been shown to control cell adhesion (Reynolds et al., 2014), spreading (Reynolds et al., 2015) and direct the differentiation of Mesenchymal Stem Cells (MSCs) (Gilbert et al., 2017).

Materials and Reagents

  1. Personal Protective Equipment (PPE): Gloves (latex or nitrile), lab coat and safety glasses
  2. 15 ml polypropylene centrifuge tubes (Corning, catalog number: 430791 )
  3. Millex-GP Syringe Filter, 0.22 μm, Polyethersulfone, 33 mm diameter, non-sterile (Merck, catalog number: SLGP033NB )
  4. Syringe PP/PE without needle, Luer slip tip, 5 ml (Sigma-Aldrich, catalog number: Z116866 )
  5. Spectra/Por 1 RC Dialysis Membrane Tubing (6-8 kDa MWCO, 25.5 mm diameter) (Fisher Scientific, catalog number: 08-670C)
    Manufacturer: Spectrum Medical Industries, catalog number: 132660 .
  6. Dialysis tubing clamps (50 mm) (Sigma-Aldrich, catalog number: Z371092 )
  7. Pyrex crystallizing dish (used as oil bath) (Capacity 2.5 L) (diameter x height = 190 x 100 mm) (Corning, PYREX®, catalog number: 3140-190 )
  8. BRAND pipette tips (volume 0.1-20 μl), non-sterile (BRAND, catalog number: 732222 )
  9. Corning universal fit pipette tips, non-sterile, volume 1-200 μl (Corning, catalog number: 4865 ) and volume 100-1,000 μl (Corning, catalog number: 4867 )
  10. Muscovite Mica disks, grade V-1 diameter 12.5 mm (ProSciTech, catalog number: G51-12 )
  11. Double sided tape (for cleaving mica) (Agar Scientific, catalog number: AGG263 )
  12. Headspace glass vials (20 ml) (Sigma-Aldrich, catalog number: 27306 )
  13. β-Lactoglobulin from bovine milk ≥ 90% (PAGE), freeze-dried powder (Sigma-Aldrich, catalog number: L3908 )
  14. Lysozyme from chicken egg white ≥ 90%, freeze-dried powder (Sigma-Aldrich, catalog number: L6876 )
  15. Hydrochloric acid, ACS Reagent, 37% (Sigma-Aldrich, catalog number: 258148 )
  16. Sodium hydroxide, BioXtra, ≥ 98% Anhydrous Pellets (Sigma-Aldrich, catalog number: S8045 )
  17. Silicone oil (Sigma-Aldrich, catalog number: 85409 )
  18. Hanna pH standard buffer solutions, pH 10 (Sigma-Aldrich, catalog number: Z655155), pH 7 (Sigma-Aldrich, catalog number: Z655139)
    Manufacturer: Hanna, catalog numbers: HI 6010 and HI 6007 .
  19. Ricca Chemical Buffer, Reference Standard pH 1.68 (Fisher Scientific, catalog number: 1492-16 )
  20. 10% lysozyme (or β-Lactoglobulin) solution for purification (see Recipes)
  21. 2% lysozyme (or β-Lactoglobulin) solution for fibril formation (see Recipes)

Equipment

  1. BRAND glass beaker with spout 600 ml (BRAND, catalog number: 90648 )
  2. Hanna bench pH/ISE meter HI4222 (Sigma-Aldrich, catalog number: Z655333EU)
    Manufacturer: Hanna Instruments, model: HI-4222 .
    Note: This product has been discontinued.
  3. Laboratory centrifuge (Sigma Laborzentrifugen, catalog number: Sigma 3-30KHS )
  4. 12159 rotor (Sigma Laborzentrifugen, catalog number: 12159 )
  5. Barnstead E-pure (MilliQ) Water Filtration Device (producing water with a resistivity of ≥ 18.2 MΩ) (Thermo Fisher Scientific, Thermo FisherTM, catalog number: D4631 )
  6. Labco digital hotplate stirrer (Labtek, catalog number: 400.100.105 )
    Note: This product has been discontinued.
  7. Spinbar magnetic stirrer bars PTFE coated polygon 60 x 8 mm (Sigma-Aldrich, catalog number: Z266353 ) and 12.7 x 3 mm (SP Scienceware - Bel-Art Products - H-B Instrument, catalog number: F37119-0127 )
  8. Aldrich Clamp Holder (Sigma-Aldrich, catalog number: Z243620 )
  9. Aldrich Benchclamp 3-prong (Sigma-Aldrich, catalog number: Z556645 )
  10. Support Stand with Rod (Sigma-Aldrich, catalog number: Z509442 )
  11. BRAND Ice bucket with lid 4.5 L (BRAND, catalog number: 156100 )
  12. Gilson PIPETMAN
    Classic P20 max volume 20 μl (Gilson, catalog number: F123600 )
    Classic P200 max volume 200 μl (Gilson, catalog number: F10005M )
    Classic P1000 max volume 1,000 μl (Gilson, catalog number: F123602 )
  13. Martin Christ Alpha 1-2LDplus Entry Laboratory Freeze Dryer (Martin Christ, model: Alpha 1-2LDplus , catalog number: 101530), equipped with 50 ml single-neck round-bottom flasks (Sigma-Aldrich, catalog number: Z414484 )
  14. An air or nitrogen sauce (for drying samples, post rinse)

Procedure

  1. Initial protein purification
    To ensure that the final morphology of the amyloid fibrils is as homogenous as possible, commercial grade protein (either hen egg white lysozyme or β-Lactoglobulin) must be further purified to remove trace impurities, such as ash or lipids that can affect fibril morphology. This initial purification is performed by dialysis as originally outlined in Jung et al. (2008). All steps in this protocol should be performed with appropriate Personal Protective Equipment (PPE) (gloves, lab coat and safety glasses).
    1. Calibrate the digital pH meter as per the manufactures instructions using pH 10, 7 and 1.68 calibration standards. Or just pH 7 and 1.68 if three point calibration is not available on the pH meter used.
    2. Prepare a 10% (weight/weight) solution of protein in fresh MilliQ water (Recipe 1) in 15 ml centrifuge tubes. Once the protein is completely dissolved into the solution, adjust the pH to 4.6 using the digital pH meter, by drop-wise addition of 1 N hydrochloric acid (HCl).
    3. To remove and precipitate impurities, centrifuge the solution at 20,400 x g (for the 1215-H rotor listed above this is equivalent to 15,000 rpm) for 15 min.
      Note: Ensure that the centrifuge is properly balanced before use. Discard the pelleted impurities and add 1 N HCl (dropwise) to the supernatant to adjust to pH 2.
    4. Pass the acidified supernatant through a 0.22 μm filter into a length of the dialysis tubing closed at one end using the dialysis tubing clamp.
      Note: Dialysis tubing should be cut to an appropriate length so that it is no more than ~70% full to prevent bursting.
    5. Seal the dialysis tube using a second clamp and immerse the filled tubing in a large beaker filled with an aqueous solution of 0.01 N HCl (dialysing solution), and containing a 60 x 8 mm magnetic stir bar.
      Note: The precise volume of the beaker is not important but it should be at least one order of magnitude larger than the volume of dialysate, we typically used beakers with a volume of at least 3 L.
    6. Dialysis should occur at 4 °C, with the dialyzing solution under constant stirring for 5 days. The dialyzing solution should be replaced at least every other day.
      Note: Ensure that the fresh dialyzing solution is pre-chilled to 4 °C before switching.
    7. Constant stirring at 4 °C will likely require a refrigerated room that can safely contain a stirrer plate. If this is not available then the dialysis tubing can be kept in a regular refrigerator with no stirring, but it may be necessary to increase the total dialysis time and replace the dialyzing solution more frequently to maintain osmotic pressure.
    8. After dialysis, the protein solution in the dialysis bag should be readjusted to pH 2 (1 N HCl) and freeze-dried.
      Note: Where possible an acid trap should be used with the freeze dryer as prolonged exposure to acidic solutions can lead to corrosion of the equipment.
    9. To freeze-dry the samples, first, to prevent the fibril suspension boiling under reduced vacuum, pre-freeze the fibril suspensions in a regular laboratory freezer. Once frozen switch on the vacuum pump connected to the freeze-dryer and allow it to warm up for 15 min. Turn on the freeze-dryer, select freezing mode and allow the instrument to cool to approximately -55 °C. Connect the round bottom flask containing the frozen fibril suspension to one of the outer ports of the freeze dryer, open the main valve on the vacuum pump and then slowly open the valve on the port with the attached round bottom flask. Switch the instrument to its ‘main drying’ mode, and leave the round bottom flask attached until completely dry (dependent on volume but overnight drying was typically used for 10-20 ml suspensions). At the end of the freeze-drying close the valve to the main pump and release the vacuum in the chamber of the freeze-dryer. If attached to the walls of the round bottom flask, then the freeze-dried fibrils can be gently scraped off with a spatula. The purified, freeze-dried powder can now be stored at -20 °C until it is required for fibril self-assembly.
      Note: Never turn off the pump when there is still a vacuum in the chamber as this could result in oil being sucked from the pump.

  2. Amyloid fibril self-assembly
    All steps in this protocol should be performed with appropriate Personal Protective Equipment (PPE) (gloves, lab coat and safety glasses). As the reaction occurs at 90 °C for 24 h or more, particular care should be taken to ensure the heated oil bath is properly set up and does not pose any danger to yourself or other users of the laboratory.
    1. Dissolve the purified protein in MilliQ water making a 2% solution (weight/weight), using the pH meter to adjust the acidity to pH 2 by dropwise addition of 1 N HCl (Recipe 2). Pass the resulting acidified solution through a 0.22 μm filter into a glass vial, with a heatproof sealable lid (Figure 1).
    2. Prepare the oil bath by filling an appropriate heat-proof glass beaker (e.g., a crystallization dish) containing a magnetic stirrer bar with clean silicon oil to a level where it will completely cover the protein solution in the glass vial but not totally submerge the vials (Figure 1). Heat the silicon oil in the crystallization dish to 90 °C with gentle stirring (60 rpm, using a digital stirrer hotplate). Stirring is required to ensure homogeneous heating of the oil. At this point, it is important to emphasize that the oil bath should be stable at 90 °C before addition of the protein solution, to ensure constant heating over the entire reaction.
      Note: If the hotplate is not equipped with a thermometer and thermostat, then the temperature of the oil bath should be checked and adjusted manually using an external thermometer (deviations in temperature will affect the morphology of the resultant fibrils).


      Figure 1. Heated (and stirred) silicon oil bath with a clamped glass vial containing soluble protein/amyloid fibrils

    3. Using a stand and clamp carefully secure the glass vial containing the protein solution in the stirred hot oil bath.
    4. Fibril formation occurs over time via a progressive denaturing and hydrolysis of the protein and the subsequent self-assembly of the resultant amyloidogenic peptide fragments in a mechanism outlined in Adamcik and Mezzenga (2012). Control over the resultant fibril diameters can be exerted by adjusting the reaction times (Lara et al., 2011; Reynolds et al., 2014). Single protofilaments start to appear after around 5 h; these protofilaments stack together in a modular way (Adamcik et al., 2010; Adamcik and Mezzenga, 2012) to form thicker multi-fibrillar mature fibres over time, before eventually closing up into nanotubes (Lara et al., 2013) or, with the addition of salt [20-50 mM (NaCl) at 2% wt protein], forming hydrogels (Bolisetty et al., 2012). It is worth noting that both the mean diameter and concentration of the fibrils increases over time, therefore it is non-trivial to obtain high concentrations of fibrils composed of only 1 or 2 protofilaments. For cell culture applications, we have found that incubation of lysozyme in the hot oil for between 24-30 h results in fibrils of an optimum concentration and morphology to promote cell growth after adsorption to a 2D substrate (Reynolds et al., 2014; Gilbert et al., 2017). For β-Lactoglobulin fibrils less extensive investigations on the effects of fibril diameter on cell responses have been performed, however fibrils formed after just 5 h incubation have been shown to support MSC culture (Gilbert et al., 2017). Upon removing the protein fibrils from the oil bath, the glass vial should immediately be placed in an ice bath to rapidly quench the reaction and prevent further assembly. Success of the reaction can be qualitatively assessed by observing the color and turbidity of the reaction products. Before heating, the dissolved protein solution should be completely transparent and colorless (Figure 2A), after fibril formation some cloudiness and a yellow hue should be apparent (due to the non-soluble suspension of fibrils) (Figure 2B). For a protocol describing how to quantitatively assess the morphology of the fabricated fibrils by Atomic Force Microscopy (AFM) see Charnley et al. (2018).
    5. Before use as a cell culture material, suspensions of lysozyme fibrils were dialyzed in order to neutralize the pH. The procedure in Step A4 can be repeated, however now pure MilliQ water should be used as a dialyzing solution and 24 h dialysis with no requirement to change the dialyzing solution is sufficient. This procedure shouldn’t be performed for β-Lactoglobulin fibrils due to concerns about a lack of long-term stability at pH values above its isoelectric point (Jones et al., 2011; Gilbert et al., 2014) instead additional washing steps are employed to remove the acidic solvent after fibril adsorption to the mica discs before incubation with the cells (see Step C2).


      Figure 2. Visual differences between dissolved monomeric protein and amyloid fibril suspension. Photographs of the protein solutions present before heating (A) and the resulting fibril suspension (B) after 24 h of heating. Note the fibril solution has become slightly opaque and has a yellowish hue due to the presence of a suspension of amyloid fibrils.

  3. Fabricating Amyloid Fibril Networks
    Many solid supports may be suitable for amyloid fibril deposition. However, in our experiments we exclusively use muscovite mica discs (diameter 12 mm). Mica was chosen for a number of reasons. First, at pH 7 lysozyme fibrils carry a net positive charge, and mica carries a net negative charge, thus electrostatic interactions ensure a good coverage of amyloid fibrils on the substrates. Second, mica is composed of many flat sheets held together by weak non-covalent interactions, these sheets can be easily cleaved producing a pristine, dust free surface. Third mica substrates are almost atomically smooth therefore make ideal substrates for characterization by Atomic Force Microscopy (AFM) (Charnley et al. [2018]).
    It is worth noting that the first listed benefit of mica (encouraging fibril binding through electrostatic interactions) is only appropriate for lysozyme fibrils, and not β-Lactoglobulin fibrils which have no net charge at neutral pH.
    1. Cleave mica disk. There are many methods of cleaving mica, we prefer to use double-sided tape to stick the disc down to a lab bench and then use a fine tipped pair of tweezers to cleave the top layer from the stuck down disk.
    2. Deposit 10-20 μl of the 2% (initial monomeric protein concentration) amyloid fibril suspension onto the center of the freshly cleaved mica disc. Incubate at room temperature for 10 min, before gently rinsing the surface of the mica disc with 1 ml of MilliQ water, and gently drying the disc using either a compressed air or nitrogen line. Care should be taken when drying laminated mica discs, to ensure that a homogenous drying is achieved and the dispersed amyloid suspensions are not concentrated at one end of the mica disc during the drying process. Whilst it is not strictly a part of the protocol for fabricating the amyloid fibril networks, it is important to note that immediately before using the fibril networks as substrates for cell culture they should be sterilized to remove any bacterial contamination. In our experiments, we did this by incubating the mica disks with the adsorbed networks in a solution of the antibiotic Pen-Strep (100 ngml-1) for 1 h see Gilbert et al. (2017) for details. Sterilization in an Autoclave was avoided due to concerns that it would damage the fibril network.

Data analysis

Qualitative analysis of the success of the reaction was performed by simply observing the color and opacity change in the solution before and after incubation in the hot oil bath (Figure 2). A more quantitative analysis of the quality of the fibril coatings produced by atomic force microscopy (AFM) is provided in an associated protocol (Charnley et al. [2018]); and an example AFM image of amyloid nanofibrils adsorbed onto a mica substrate that should be produced from a successful reaction is shown below (Figure 3).


Figure 3. Example of the morphology of amyloid nanofibrils. Produced by the above protocol and imaged by AFM.

Notes

The protocol outlined here is typically very repeatable. However, the final morphology is very sensitive to small variations in temperature throughout the reaction, therefore the hotplate used should maintain a very stable temperature over relatively long periods of time (> 24 h), even small fluctuations in temperature can significantly affect the repeatability of this reaction.

Recipes

  1. 10% lysozyme (or β-Lactoglobulin) solution for purification
    Add 1 part powdered protein to 9 parts MilliQ water (by weight/volume) and adjust to pH 4.6 by the addition of 1 N HCl
  2. 2% lysozyme (or β-Lactoglobulin) solution for fibril formation
    Add 1 part purified freeze-dried protein to 49 parts MilliQ water (by weight/volume) and adjust to pH 2 by the addition of 1 N HCl

Acknowledgments

This work was performed in part at the ANFF-Vic node of the Australian National Fabrication Facility, a company established under the National Collaborative Research Infrastructure Strategy to provide nano-and micro-fabrication facilities for Australia’s researchers. JG acknowledges the Australian Government Department of Education and Training for an Endeavour Scholarship and the National Science Foundation Graduate Research Fellowship Program under Grant No. DGE-1333468. NPR and JG acknowledge the ARC Training Centre for Biodevices at Swinburne University of Technology (IC140100023) for funding (NPR) and for hosting for the duration of his scholarship (JG). MC acknowledges support from the Swiss National Science Foundation (SNSF) (grants PA00P3_142120 and P300P3_154664). JG and OGJ acknowledge further funding support from USDA Hatch Act funds (IND0-1162). The protocols in this work were adapted from a protocol defined in Jung et al. (2008) and Lara et al. (2011). The authors declare no conflict of interest or competing interests.

References

  1. Adamcik, J., Jung, J. M., Flakowski, J., De Los Rios, P., Dietler, G. and Mezzenga, R. (2010). Understanding amyloid aggregation by statistical analysis of atomic force microscopy images. Nat Nanotechnol 5(6): 423-428.
  2. Adamcik, J. and Mezzenga, R. (2012). Proteins fibrils from a polymer physics perspective. Macromolecules 45: 1137-1150.
  3. Bolisetty, S., Harnau, L., Jung, J. M. and Mezzenga, R. (2012). Gelation, phase behavior, and dynamics of beta-lactoglobulin amyloid fibrils at varying concentrations and ionic strengths. Biomacromolecules 13(10): 3241-3252.
  4. Charnley, M., Gilbert, J., Jones, O. G. and Reynolds, N. P. (2018). Characterisation of amyloid fibril networks by atomic force microscopy. Bio-protocol 8(4): e2732.
  5. Chiti, F. and Dobson, C. M. (2006). Protein misfolding, functional amyloid, and human disease. Annu Rev Biochem 75: 333-366.
  6. Dharmadana, D., Reynolds, N. P., Conn, C. E. and Valery, C. (2017). Molecular interactions of amyloid nanofibrils with biological aggregation modifiers: implications for cytotoxicity mechanisms and biomaterial design. Interface Focus 7(4): 20160160.
  7. Gilbert, J., Campanella, O. and Jones, O. G. (2014). Electrostatic stabilization of beta-lactoglobulin fibrils at increased pH with cationic polymers. Biomacromolecules 15(8): 3119-3127.
  8. Gilbert, J., Reynolds, N. P., Russell, S. M., Haylock, D., McArthur, S., Charnley, M. and Jones, O. G. (2017). Chitosan-coated amyloid fibrils increase adipogenesis of mesenchymal stem cells. Mater Sci Eng C 79: 363-371.
  9. Jones, O. G., Handschin, S., Adamcik, J., Harnau, L., Bolisetty, S. and Mezzenga, R. (2011). Complexation of beta-lactoglobulin fibrils and sulfated polysaccharides. Biomacromolecules 12(8): 3056-3065.
  10. Jung, J. M., Savin, G., Pouzot, M., Schmitt, C. and Mezzenga, R. (2008). Structure of heat-induced beta-lactoglobulin aggregates and their complexes with sodium-dodecyl sulfate. Biomacromolecules 9(9): 2477-2486.
  11. Kayed, R., Head, E., Thompson, J. L., McIntire, T. M., Milton, S. C., Cotman, C. W. and Glabe, C. G. (2003). Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis. Science 300: 486-489.
  12. Lara, C., Adamcik, J., Jordens, S. and Mezzenga, R. (2011). General self-assembly mechanism converting hydrolyzed globular proteins into giant multistranded amyloid ribbons. Biomacromolecules 12(5): 1868-1875.
  13. Lara, C., Handschin, S. and Mezzenga, R. (2013). Towards lysozyme nanotube and 3D hybrid self-assembly. Nanoscale 5(16): 7197-7201.
  14. Nelson, R., Sawaya, M. R., Balbirnie, M., Madsen, A. O., Riekel, C., Grothe, R. and Eisenberg, D. (2005). Structure of the cross-[beta] spine of amyloid-like fibrils. Nature 435(7043): 773-778.
  15. Reynolds, N. P, Charnley, M., Bongiovanni M. N., Hartley, P. G. and Gras, S. L. (2015). Biomimetic topography and chemistry control cell attachment to amyloid fibrils. Biomacromolecules 16(5): 1556-1565.
  16. Reynolds, N. P., Charnley, M., Mezzenga, R. M. and Hartley, P. G. (2014). Engineered lysozyme amyloid fibril networks support cellular growth and spreading. Biomacromolecules 15(2): 599-608.
  17. Reynolds, N. P., Soragni, A. Michael, R., Verdes, D., Liverani, E., Handschin S., Riek, R. and Seeger, S. (2011). Mechanism of membrane interaction and disruption by α-synuclein. J Am Chem Soc 133(48): 19366-19375.
  18. Wei, G., Su, Z., Reynolds, N. P., Arosio, P., Hamley, I. W., Gazit, E. and Mezzenga, R. M. (2017). Self-assembling peptide and protein amyloids: from structure to tailored function in nanotechnology. Chem Soc Rev 46: 4661-4708.

简介

结核分枝杆菌(Mtb)已经发展为从其宿主同化脂肪酸。然而,直到最近,还没有可靠的方法来量化宿主细胞感染期间细菌对脂肪酸的摄取。在这里,我们描述了一种新的方法来量化细胞内的杆菌对脂肪酸的摄取。我们用Mtb组成性表达mCherry感染巨噬细胞,然后用Bodipy-palmitate代谢标记它们。在标记步骤之后,我们在蔗糖垫上分离含有Mtb的吞噬体,并用洗涤剂破坏吞噬体。大量洗涤后,通过流式细胞术分析分离的细菌以确定与细菌相关的Bodipy-棕榈酸酯信号的水平。使用液体培养物中脂肪酸摄取缺陷的Mtb突变体菌株,我们确定该突变体在巨噬细胞感染期间同化比野生型菌株少10倍的Bodipy-棕榈酸酯。这种脂肪酸摄取的定量方法可以用于进一步鉴定参与细胞内Mtb和可能的其他细菌的脂质摄取的途径。

【背景】结核分枝杆菌(Mtb)吸收宿主来源的脂质(脂肪酸和胆固醇)的能力使病原体能够在其宿主内存活(Russell等人,2010; Lovewell 等,2016)。在小鼠感染期间和在人肺组织中,通过巨噬细胞内Mtb上调胆固醇和脂肪酸代谢相关基因来支持这个想法(Schnappinger等人,2003; Rachman等人。2006; Rohde等人,2007;Fontán等人,2008; Tailleux等人,2008; Homolka et al。,2010; Rohde et al。,2012)。在感染期间胆固醇代谢对Mtb的重要性得到了基因研究和通过鉴定靶向胆固醇代谢的新的抗结核化合物的支持(Pandey和Sassetti,2008; Wipperman等人,2014; VanderVen等人,2015)。然而,发现Mtb中脂肪酸摄取的具体机制不仅受到专用基因的明显冗余(Cole等人,1998)的阻碍,而且受到缺乏可靠的测定的阻碍。使用放射性底物进行代谢标记已经成为评估肉汤培养物中细菌摄入脂肪酸的效率的可接受方法(Forrellad等人,2014)。这种方法对于应用胞内Mtb是非常具有挑战性的,并且很少有团队报告成功使用这种方法(Daniel等人,2011)。或者,TEM和用亲脂性染料如Bodipy 493/503,尼罗红,油红染色可以促进在感染期间检测分枝杆菌内的脂质(Daniel等人,2011; Podinovskaia等人,2013; Caire-Brändli等,2014)。然而,这些标记方法都不直接评估底物的主动输入,而是指示积累的脂质的总量。因此,非常需要在感染期间通过Mtb进行活性脂肪酸代谢标记的手段,所述感染易于下游表征。

最近,显示荧光脂肪酸可以有效地递送到细胞内,并且可以通过显微镜检测(Podinovskaia等人,2013)。这些观察结果使我们开发了一种基于流式细胞计量术的基于Mtb在其宿主细胞内的荧光脂肪酸摄取量的新方法。该测定使我们能够证明在巨噬细胞感染过程中,ΔuMa:: hyg Mtb菌株在脂肪酸摄取方面是有缺陷的(Nazarova等人,2017)(图1) 。我们相信这种方法打开了基因筛选的大门,以进一步了解在宿主细胞感染过程中Mtb和可能的其他细胞内病原体吸收脂肪酸所涉及的机制。

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图1.在巨噬细胞感染期间用于定量结核分枝杆菌的脂肪酸摄取的方法的概述

关键字:淀粉样纤维, 自组装, 生物材料, 干细胞培养, 细胞附着, 仿生材料, 蛋白质聚集

材料和试剂

  1. 血清移液管,5ml,10ml,25ml和50ml(Corning,Costar的产品目录号:4487,4488,4489和4490)
  2. (Biotix,Neptune目录号:BT20,BT100,BT200; Thermo Fisher Scientific,Thermo Scientific,产品目录号:2079-HR),移液管头20μl,100μl,200μl,
  3. T25(具有过滤盖的无菌25cm2组织培养瓶)(TPP,目录号:90026)。
  4. T75或T150(无菌75cm2或150cm2具有过滤盖的组织培养瓶)(TPP,目录号:90076或90151)。
  5. 无菌1毫升结核菌素注射器与25号针(BD,目录号:309626)
  6. 细胞刮刀,25厘米(SARSTEDT,目录号:83.1830)

  7. 15毫升锥形管(SARSTEDT,目录号:62.554.100)
  8. 50毫升锥形管(SARSTEDT,目录号:62.547.100)
  9. Glasstic 与网格幻灯片(KOVA国际,目录号码:87144)
  10. 150×15毫米培养皿(VWR,目录号:25384-326)
  11. 2毫升螺帽管(VWR,目录号:16466-042)
  12. FACS管(VWR,目录号:60818-496)
  13. 3毫升注射器(带Luer-Lok TM尖端,BD,目录号:309657)
  14. (Fisher Scientific,Fisherbrand TM TM目录号:14-955-128和14-385-999)
    一次性塑料OD比色杯
  15. 结核分枝杆菌组成型表达mCherry(pMV306 smyc':: mCherry (Kan r ))来源:Russell和VanderVen实验室(Nazarova ,2017)
  16. 骨髓来源的鼠巨噬细胞(BALB / c小鼠,THE JACKSON LABORATORIES,目录号:000651)
    注意:分化在Nazarova et al。 (2017)。
  17. L细胞(NCTC克隆929 [L细胞,L-929,L株的衍生物])(ATCC,目录号:CCL-1)
  18. 磷酸盐缓冲盐水(PBS)1x(Mediatech,目录号:21-040)
  19. Tyloxapol(Acros Organics,目录号:422370050)
  20. Middlebrook 7H9肉汤培养基(BD,Difco TM,产品目录号:271310)
  21. 蒸馏水
  22. 甘油(VWR,目录号:97062-452)
  23. Middlebrook OADC富集(BD,BBL TM,目录号:212351)
  24. 硫酸卡那霉素(IBI Scientific,目录号:IB02120)
  25. 加热灭活的胎牛血清(Thermo Fisher Scientific,Gibco TM,目录号:10437028)
    注意:56°C热灭活30分钟
  26. 200mM L-谷氨酰胺(100x)(Mediatech,目录号:25-005)
  27. 100mM丙酮酸钠(Mediatech,目录号:25-000)
  28. 青霉素 - 链霉素溶液100倍(Mediatech,目录号:30-002)
  29. Dulbecco对Eagle培养基(DMEM)1x(Mediatech,目录号:10-017)的修改
  30. 葡萄糖(葡萄糖)(Fisher Scientific,目录号:BP350-1)
  31. 牛血清白蛋白(BSA)(Roche Diagnostics,目录号:03116964001)
  32. 来自冷水鱼皮的明胶(Sigma-Aldrich,目录号:G7765)
  33. 氯化钙(CaCl 2)(Sigma-Aldrich,目录号:C4901)
  34. 氯化镁(MgCl 2)(AMRESCO,目录号:J364)
  35. 不含脂肪酸的BSA(Roche Diagnostics,目录号:03117057001)
  36. 100%乙醇,200份证明(Decon Labs,产品目录号:V1016)
  37. Bodipy TM FL C16(Bodipy-棕榈酸酯)(Thermo Fisher Scientific,Invitrogen TM,目录号:D3821)
  38. Bodipy TM FL C12(Thermo Fisher Scientific,Invitrogen TM,目录号:D3822)
  39. Bodipy TM 558/568 C12(Thermo Fisher Scientific,Invitrogen TM,目录号:D3835)
  40. 氯化钾(KCl)(Mallinckrodt Chemicals或Avantor Performance Materials,MACRON,目录号:6858-04)
  41. 蔗糖(Avantor Performance Materials,J.T.Baker,目录号:4097)
  42. 亚乙基双(氧乙烯基)四乙酸(EGTA)(Avantor Performance Materials,J.T.Baker,目录号:L657)
  43. HEPES(VWR,目录号:BDH4162)
  44. Tween TM 80(Fisher Chemical,目录号:T164-500)
  45. 多聚甲醛(PFA)(Sigma-Aldrich,目录号:158127)
  46. 20%tyloxapol(见食谱)
  47. 7H9 OADC媒体(见食谱)
  48. 卡那霉素25毫克/毫升(见食谱)
  49. D10媒体(见食谱)
  50. L细胞条件培养基(见食谱)
  51. BMDM媒体(见食谱)
  52. 基础摄取缓冲液(BUB)(见食谱)
  53. 1%无脂肪酸BSA(见食谱)
  54. 4毫米Bodipy棕榈酸酯(见食谱)
  55. 比色杯缓冲液(见食谱)
  56. 匀浆缓冲液(见食谱)
  57. 20%吐温80(见食谱)
  58. 0.05%tyloxapol(见食谱)
  59. 4%PFA(见食谱)

设备

  1. 移液器控制器(Drummond Scientific,型号:Pipet-Aid ,目录号:4-000-101)
  2. 移液器(MIDSCI,Alphapette,型号:A-20,A-100,A-200,A-1000)
  3. 37℃,6%CO 2培养箱
  4. 56°C水浴
  5. 冰箱(4°C)
  6. -20°C冷冻机
  7. 分光光度计兼容600nm处的吸光度测量(Cole-Parmer,Jenway,型号:6320D)
  8. Beckman Allegra 6KR离心机(Beckman Coulter,型号:Allegra 6KR Kneewell TM))
  9. GH-3.8转子(Beckman Coulter,型号:GH-3.8转子)
  10. 具有足够分辨率的倒置显微镜检测巨噬细胞大小的细胞(奥林巴斯,模型:10倍物镜的IMT-2)
  11. Beckman Microfuge 18离心机(Beckman Coulter,型号:Microfuge 18)
  12. 流式细胞仪(BD,型号:FACS LSR II)

软件

  1. FlowJo(BD)

程序

  1. 细菌培养
    结核分枝杆菌菌株在37℃在7H9 OADC培养基(参见食谱)中在T25瓶中静置培养直至对数中期(OD 600-6〜0.6)生长。细菌培养物从冷冻库中开始并保持不超过5代。 Mtb荧光蛋白的组成型表达是进一步检测所必需的。我们使用来自smyc启动子( smyc ':: mCherry )的表达来自mCherry 的整合pMV306质粒的Mtb Erdman菌株。该质粒赋予卡那霉素抗性,因此生长培养基含有最终浓度为25μg/ ml的卡那霉素(参见食谱)。
    注意:
    1. 在T25烧瓶中不要生长超过10毫升的细菌培养物以确保Mtb有足够的氧气供应量。为了估计细菌培养生长的时间,考虑到在长时间培养的情况下,Mtb大约在两天内分裂一次。关于巨噬细胞感染的细菌培养生长的更多细节可以在(Nazarova和Russell,2017)中找到。
    2. 我们建议测试你的野生型菌株组成型表达荧光蛋白的能力,有效地摄入巨噬细胞感染期间显微镜Bodipy棕榈酸。我们注意到Mtb Erdman菌株在强启动子(smyc'或hsp60')下的mCherry表达来自复制质粒影响Bodipy-棕榈酸酯摄取。然而,具有相同质粒的CDC1551菌株表现出高水平的脂肪酸摄取。另外,应该注意通过共聚焦显微镜来确定棕榈酸Bodipy在细菌内与其表面上积累的关系。

  2. 巨噬细胞分离和培养
    可以使用各种来源的巨噬细胞。在感染前,我们从BALB / c小鼠分化来自骨髓细胞的巨噬细胞,并在37℃和6.0%CO 2下用抗生素维持在BMDM培养基(参见食谱)中10天。
    的 感染
    1. 在感染前一天,将无抗生素的BMDM培养基中的巨噬细胞种入T150组织培养瓶(3×10 7细胞和每瓶40-50ml BMDM培养基)以获得融合单层。如果巨噬细胞的数量是有限的,则一个含有1×10 7个细胞的T75培养瓶将提供足够的结果,然而,两个T150培养瓶(6×10 7细胞)可以容易地检测到
      。实验结束时,细菌团块
    2. 在感染当天测量中对数Mtb培养物的光密度。假定0.6的OD 600等于10 8细菌/ ml,则以3300rpm(〜2500×gg)离心所需的量的培养物12分钟在Beckman Allegra 6KR离心机中,GH-3.8转子。我们以4:1的MOI感染巨噬细胞,因此感染6×10 7个细胞需要2.4×10 8个细菌。为了确保足够数量的细菌沉淀,我们常规地在OD 600 = 0.6下离心3ml细菌。
      注:由于细菌数量的估计可能会有所不同,可能需要自行确定。但是,为了在MOI方面获得更高的可复制性,我们建议坚持我们的估计。巨噬细胞感染的更多细节可以在(Nazarova and Russell,2017)中找到。
    3. 离心后,取出上清液,重新悬浮在1.5毫升BUB的细菌颗粒(见食谱)。通过1毫升结核菌素注射器与25号针12-20倍相同的注射器和针头的细菌悬液。
      加入3.5毫升BUB到悬浮液中,共5毫升,并彻底搅拌
    4. 向每个含有3×10 7个细胞的T150烧瓶中加入2.4ml细菌悬浮液。充分混合,但温和。
      在37°C和6%CO 2培养4小时。
    5. 用新鲜的预热无抗生素BMDM培养基代替细胞外细菌。受感染的巨噬细胞在37℃和6%CO 2下在BMDM培养基中维持3天。在感染的第二天更换媒体是可选的。

    的 标签
    1. 在标记的当天(感染的第三天)预热的无菌1%无脂肪酸BSA(参见食谱)在37℃PBS中30-60分钟。加入4mM Bodipy-palmitate储备液(参见食谱)以获得100μM的浓度。为了标记一个T75烧瓶,将50μl的4mM Bodipy-棕榈酸酯储备液添加到1.95ml的不含1%无脂肪酸的BSA中。通过涡旋混合,直到溶液变成绿色。保持在37°C,避光。
      注意:
      1. 标签的日期可以考虑你正试图解决的问题来选择。我们在巨噬细胞感染的不同阶段测试了Bodipy-palmitate的吸收,并注意到在感染的第三天之前摄取水平不足以被检测到。
      2. 除了Bodipy棕榈酸酯,我们还测试了Bodipy FL C12和Bodipy 558/568 C12。 Bodipy FL C12在胞内细菌中积累,与Bodipy-palmitate一样高效,而Bodipy 558/568 C12则被Mtb弱化同化。我们选择使用棕榈酸Bodipy作为脂肪酸的长度(16碳)更常见于感染Mtb的巨噬细胞的膜。
    2. 为了标记一个T75烧瓶,将1.6ml在1%不含脂肪酸的BSA(来自标记步骤1)中的100μMBodipy-棕榈酸酯加入18.4ml预热比色杯缓冲液(参见配方),使标记的脂质的最终浓度为8μM,充分混合。使用15毫升标记一个T75烧瓶,30毫升标记一个T150烧瓶。第1步和第2步( 标签 )的体积相应增加,以适应更大的感染。
    3. 从感染的巨噬细胞中除去培养基,并用含有Bodipy-棕榈酸酯的比色杯缓冲液代替(体积描述于 Labeling 的步骤2)。在37℃和6.0%CO 2下孵育感染的巨噬细胞1小时。
    4. 1小时标记期后,取出含有Bodipy棕榈酸盐的比色杯缓冲液,加入新鲜预热的无标记的比色杯缓冲液1小时。一个T75烧瓶使用15毫升,一个T150烧瓶使用30毫升。
      在孵化1小时后立即进入下一步 注意:另外,1%不含脂肪酸的BSA中的Bodipy-palmitate可直接加入在BMDM培养基中培养的感染的巨噬细胞中。标签追逐也可以在新鲜预热的BMDM培养基中进行。在这种情况下不需要比色皿缓冲液。根据我们的经验,任何一种标签都可以得出相似的结果。

    胞内细菌的分离
    注意:该方案的这一部分基于(Pethe等人,2004)中描述的吞噬体分离。
    1. 从标记的感染细胞中取出比色杯缓冲液,并用10毫升匀浆缓冲液快速冲洗(见食谱)。
    2. 加入15 ml冰冷匀浆缓冲液,4℃孵育10-15分钟,用细胞刮刀刮取每个培养瓶中的巨噬细胞。将细胞转移到50ml锥形管中,并通过Beckman Allegra 6KR离心机GH-3.8转子以1,500rpm(514×g)离心10分钟使细胞沉淀。这个和所有以下的离心是在4°C完成,以阻止细菌进一步吸收脂质。
    3. 取出上清液,用1.5 ml匀浆缓冲液重新悬浮沉淀,移取细胞到15 ml锥形管中。通过用25号针头的1ml结核菌素注射器将细胞溶解25-70次。在显微镜下用100格栅玻片监测细胞裂解。继续直到>
      。当完整的细胞被细胞碎片取代时,95%的细胞被裂解 注意:为了安全起见,将感染材料的载玻片置于150 x 15 mm培养皿中。
    4. 加入均化缓冲液使体积增加到5毫升,重新悬浮。在Beckman Allegra 6KR离心机,GH-3.8转子中以800rpm离心细胞裂解物(〜146xg)10分钟。
    5. 将上清液(吞噬体悬浮液)转移到新的15ml锥形管中。颗粒主要由核和未经处理的细胞组成,被丢弃。
    6. 向悬浮液中加入20%吐温80(见配方)至终浓度为0.1%,充分混匀,4℃放置15分钟以裂解含有空泡的Mtb。
    7. 在Beckman Allegra 6KR离心机(GH-3.8转子)中,以2,500rpm(1,430×g)离心15分钟,通过摇动和分离细菌快速搅拌。
    8. 去除上清液并重新悬浮在PBS中的0.05ml泰洛沙泊(10ml)中的细菌沉淀(参见食谱)。在Beckman Allegra 6KR离心机,GH-3.8转子中,以3,300rpm(〜2,500×g g)离心细菌15分钟。
    9. 可选:重复上一步,进一步去除附着在细菌细胞表面的标记脂肪酸。
      注意:bodipy-palmitate与细菌细胞表面的非特异性结合产生背景信号。然而,如果测试的菌株/条件在标记物的特定摄取方面产生显着差异,则这种背景噪音不会大大影响结果。
    10. 除去上清液,并在2毫升螺帽管中将细菌固定在4%PFA中(参见食谱)24小时。
      注意:如果您在BSL3设备中使用流式细胞仪,可以在分离后立即分析细菌,而不需要固定。

数据分析

分离的细菌应该在收集后几天内分析,最好是在第二天。

  1. 在Beckman Microfuge 18离心机中以10,000rpm(9,000xg)离心固定样品5分钟。
  2. 去除上清液,重悬在1-2毫升PBS中的0.05%tyloxapol颗粒,并转移到FACS管。
  3. 通过1毫升结核菌素注射器与25号针12-20次,以获得单细胞菌悬液。
  4. 使用所描述的门控策略(图2)立即在流式细胞仪(BD FACS LSR II)上分析。选择正向和侧向散布的中等大小的群体以排除团块和小碎片,关注mCherry(PE-德克萨斯红)阳性群体(细菌),并比较样品之间的Bodipy-棕榈酸酯的FITC信号。由于mCherry和Bodipy信号之间的重叠很小,所以不需要补偿。作为阴性对照使用mCherry阳性细菌没有暴露在标签。
    注意:
    1. 由于它们是mCherry阴性,所以Bodipy-palmitate也积累在细胞器的细胞膜中,这些细胞器被排除在分析之外。
    2. 收集尽可能多的事件,最少50,000人。对于图2中的分析,我们收集了1,000,000个事件。
  5. 通过测定来自mCherry阳性细菌的Bodipy信号的平均荧光,使用FlowJo量化获得的数据。


    图2.用于分析胞内Mtb对Bodipy-棕榈酸酯摄取的门控策略在正向和侧向散射中选择中等大小的群体,并进一步分析mCherry信号的水平。对于代表细菌的mCherry阳性群体确定了Bodipy棕榈酸酯信号。图右侧是与三种不同菌株相关的检测到的Bodipy-棕榈酸酯信号的代表:野生型(黑色),△uAc :: hyg(红色)和互补菌株(蓝色)。灰色直方图表示未暴露于标记的mCherry阳性细菌。 (改编自Nazarova等人,2017)

食谱

  1. 20%泰洛沙泊(20毫升)
    1. 使用3毫升注射器添加4毫升tyloxapol到16毫升蒸馏水在50毫升锥形管
    2. 在56°C加热,偶尔涡旋,直到泰洛沙泊进入粘稠而清澈的溶液。
    3. 过滤消毒(0.22微米),在室温下储存长达12个月。
  2. 7H9 OADC介质(1 L)
    1. 将4.7克7H9 Difco TM Middlebrook 7H9肉汤基料和2毫升甘油溶于900毫升蒸馏水中
    2. 无菌加入2.5ml无菌20%泰洛沙泊,终浓度为0.05%,加入100ml BBL TM Middlebrook OADC Enrichment,混匀。
    3. 过滤消毒(0.22微米),在室温下保存
  3. 卡那霉素25mg / ml(10ml)
    1. 加入250毫克硫酸卡那霉素硫酸盐到10毫升蒸馏水,通过涡旋混合
    2. 过滤消毒(0.22微米),分装并储存在-20°C
  4. D10媒体(1 L)
    1. 加入100ml加热灭活的胎牛血清(终浓度10%),10ml 200mM L-谷氨酰胺(终浓度2mM),10ml 100mM丙酮酸钠(终浓度1mM),10ml青霉素,100x链霉素溶液至Dulbecco改良Eagle's培养基DMEM,使总体积为1L
    2. 过滤消毒(0.22μm),在4°C下保存不超过2个月
    3. 使用前在37°C预热
  5. L-细胞条件培养基
    1. 将冷冻的细胞融化到D10培养基中并在T150组织培养瓶中于37℃和6%CO 2下生长12-14天。
    2. 收集条件培养基,在Beckman Allegra 6KR离心机GH-3.8转子上以1,500rpm(514×g)离心10分钟除去细胞碎片。
    3. 上清液分装并储存在-20°C
  6. BMDM培养基(1 L)
    1. 加入100ml热灭活的胎牛血清(终浓度10%),10ml 200mM L-谷氨酰胺(终浓度2mM),10ml 100mM丙酮酸钠(终浓度1mM),100ml L-细胞条件培养基终浓度%)到达尔伯克氏改良伊格尔培养基DMEM中,使总体积为1L。在感染前加入10ml青霉素 - 链霉素溶液(100x)用于培养巨噬细胞的培养基
    2. 过滤消毒(0.22μm),在4°C下保存不超过2个月

    3. 使用前预热于37°C
  7. 基础摄取缓冲液(BUB)
    1. 加入2.25克葡萄糖,2.5克牛血清白蛋白,0.5毫升明胶,50毫克氯化钙,50毫克氯化镁到500毫升的PBS,混合
    2. 过滤消毒(0.22微米),在4°C储存不超过6个月
  8. 1%不含脂肪酸的BSA(50毫升)
    1. 将500毫克不含脂肪酸的牛血清白蛋白溶于50毫升PBS中
    2. 过滤消毒(0.22微米),分装并储存在-20°C
  9. 4mM Bodipy-棕榈酸酯
    1. 添加526微升的100%乙醇到1毫克的Bodipy棕榈酸酯,通过涡旋混合
    2. 在-20°C储存,避光
  10. 比色杯缓冲液(1L)
    1. 在1L PBS中溶解101mg CaCl 2 2,200mg KCl,102mg MgCl 2,901mg葡萄糖。
    2. 过滤消毒(0.22微米)并在室温下保存
    3. 使用前数天加入热灭活的胎牛血清至终浓度为10%,以获得所需体积,过滤灭菌(0.22μm)并在4℃下保存。
    4. 使用前在37°C预热
  11. 均化缓冲液(500ml)
    1. 将42.75克蔗糖,95毫克EGTA,2.38克HEPES,250微升明胶溶于400毫升蒸馏水中
    2. 调整pH值到7.0
    3. 用蒸馏水装满500毫升
    4. 过滤消毒(0.22微米)。
      在4°C储存和使用
  12. 20%吐温80(20ml)
    1. 使用3毫升注射器添加4毫升吐温80至16毫升蒸馏水在50毫升锥形管
    2. 在37°C加热,偶尔涡旋,直到吐温80进入溶液
    3. 过滤消毒(0.22微米),在室温下保存
  13. 0.05%泰洛沙泊(250毫升)

    1. 无菌添加625微升无菌20%泰洛沙泊至250毫升PBS
    2. 过滤消毒(0.22微米),在室温下保存
  14. 4%PFA(500毫升)
    1. 在加热的同时将20g PFA在PBS中混合。避免沸腾,否则会形成甲醛
    2. 分装并储存在-20°C。使用前在室温下解冻

致谢

我们感谢Linda Bennett提供了出色的技术支持。这项工作是由国家卫生研究院拨款(AI099569和AI119122)支持BCV和(AI080651和AI134183)DGR。该协议在之前的出版物中已经开发和报道(Nazarova等人,2017)。作者声明不存在利益冲突或利益冲突。

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引用:Charnley, M., Gilbert, J., Jones, O. G. and Reynolds, N. P. (2018). Preparation of Amyloid Fibril Networks. Bio-protocol 8(4): e2733. DOI: 10.21769/BioProtoc.2733.
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