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Jul 2020

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Purification of the Bacterial Amyloid “Curli” from Salmonella enterica Serovar Typhimurium and Detection of Curli from Infected Host Tissues
从沙门氏菌鼠伤寒血清中纯化细菌淀粉样蛋白“Curli”并检测受感染宿主组织中的 Curli   

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Abstract

Microbiologists are learning to appreciate the importance of “functional amyloids” that are produced by numerous bacterial species and have impacts beyond the microbial world. These structures are used by bacteria to link together, presumably to increase survival, protect against harsh conditions, and perhaps to influence cell-cell communication. Bacterial functional amyloids are also beginning to be appreciated in the context of host-pathogen interactions, where there is evidence that they can trigger the innate immune system and are recognized as non-self-molecular patterns. The characteristic three-dimensional fold of amyloids renders them similar across the bacterial kingdom and into the eukaryotic world, where amyloid proteins can be undesirable and have pathological consequences. The bacterial protein curli, produced by pathogenic Salmonella enterica and Escherichia coli strains, was one of the first functional amyloids discovered. Curli have since been well characterized in terms of function, and we are just starting to scratch the surface about their potential impact on eukaryotic hosts. In this manuscript, we present step-by-step protocols with pictures showing how to purify these bacterial surface structures. We have described the purification process from S. enterica, acknowledging that the same method can be applied to E. coli. In addition, we describe methods for detection of curli within animal tissues (i.e., GI tract) and discuss purifying curli intermediates in a S. enterica msbB mutant strain as they are more cytotoxic than mature curli fibrils. Some of these methods were first described elsewhere, but we wanted to assemble them together in more detail to make it easier for researchers who want to purify curli for use in biological experiments. Our aim is to provide methods that are useful for specialists and non-specialists as bacterial amyloids become of increasing importance.

Keywords: Curli (Curli), Salmonella (沙门氏菌), Bacterial Amyloid (细菌淀粉样蛋白), Purification (净化), Immunoblotting (免疫印迹)

Background

The bacterial cell surface appendages termed curli were first discovered in an E. coli strain that was isolated from horse manure. The authors named the surface structures “curli” because of their curved appearance (Olsén et al., 1989). At nearly the same time, similar surface structures were observed in Salmonella enterica serovar Enteritidis, where they were termed thin aggregative fimbriae (Collinson et al., 1991). The curli and thin aggregative fimbriae had many unique properties, including a requirement for treatment with 90% formic acid (FA) to break the fibers apart into monomeric proteins that could be resolved by SDS-PAGE gel. A landmark study published in 1998 revealed that curli and thin aggregative fimbriae, including the genes required for biosynthesis, were virtually interchangeable between S. enterica and E. coli (Romling et al., 1998). With the publication of the genome sequence of S. Typhimurium LT2, the “curli” nomenclature was chosen for both species. In terms of function, curli are key proteins in the formation of S. enterica and E. coli biofilms, with a primary role in “short-range” cell-cell interactions leading to aggregation (Romling et al., 1998). This aggregation and biofilm formation has been linked to improved persistence and survival in the face of environmental insults (Anriany et al., 2001; Solano et al., 2002; Scher et al., 2005; Ryu and Beuchat, 2005; White et al., 2006). It is quite unique for a surface structure like curli to be conserved between these related, but long diverged species; we have proposed that curli and biofilm formation plays a key role in survival of these enteric bacteria in the environment as they cycle between their hosts. Their conservation throughout S. enterica and E. coli speaks to their importance in the lifecycle of both bacterial species.


Curli are now known to be ‘functional amyloids’. Another landmark paper showed that CsgA, the curli monomer, has self-polymerization properties that are hallmark of amyloids (Chapman et al., 2002). This explained some of the unique, biochemically resistant physical properties of curli that were observed upon their initial purification. The characteristic 3-D structure of amyloids has been termed cross-beta with regular, repeated β-sheets or strands that run perpendicular to the fiber axis. The speculation with curli is that when CsgA monomers are stacked upon each other to form fibers, there is a central non-polar core that would run along the length of curli fibers, leading to extreme stability (Collinson et al., 1999). Assembly of fibers occurs outside of the cell, where unfolded CsgA monomers pass through the outer membrane through the CsgG pore and then snap into their more rigid cross-beta structure (Evans and Chapman, 2014). Although amyloids are notoriously difficult to work with and are not easily amenable to protein crystallography because of the non-uniform fiber-fiber interactions, some structural features have been determined through the use of CsgA-specific peptides (Szulc et al., 2021). Perhaps the most striking analysis performed to date revealed that CsgA “curli” have structural similarity to the amyloid-beta fibrils that are characteristic of Alzheimer’s disease, with both sharing a characteristic structural fold called the steric β-zipper (Perov et al., 2019).


The role of curli in host-pathogen interactions has recently been clarified. Several early studies described curli in the context of host expression (Bian et al., 2000; Olsén et al., 1998; Sjobring et al., 1994), but for a long period, we thought them to be purely an “environmental” factor. Pioneering work by Çagla Tükel, Andreas Baumler, and colleagues identified purified curli fibers as potent stimulators of the innate immune system, where they interact with Toll-like receptor 2 (TLR-2) (Tükel et al., 2005), TLR1/2 and TLR1/2/9 due to the presence of extracellular DNA in complex with curli (Tükel et al., 2010; Tursi and Tükel, 2018). Systemic presentation of curli had a profound impact on autoimmunity in host species, leading to increased presence of circulating anti-dsDNA antibodies, stimulation of nod-like receptors (i.e., NLRP3), and the inflammasome (Gallo et al., 2015; Rapsinski et al., 2015). Most recently, we showed that curli are expressed by S. enterica after oral infection of mice, inside the large intestine, in both acute and long-term infection models (Miller et al., 2020). In these experiments, production of curli by S. enterica cells inside the host led to increased levels of autoimmunity and early signatures of arthritis in the knee joints of infected mice. The impact of curli was tied to the ability of S. enterica to invade host cell epithelium and cause inflammation, indicating that curli must access the lymphoid tissues surrounding the intestine to have negative effects. Many aspects of curli expression in vivo still need to be clarified.


In this manuscript, we present a series of step-by-step protocols with pictures showing how to purify these important bacterial surface structures.

Materials and Reagents

  1. Disposable Culture Tubes 16 × 100 mm (Fisherbrand, catalog number: 14-961-29)

  2. DifcoTM Luria-Bertani broth (BD Biosciences, catalog number: 244620)

  3. Polystyrene disposable Petri dishes 150 mm × 15 mm (VMR International, catalog number: 25384-326).

  4. Cotton tipped applicator, 6 inch (Puritan, catalog number: 1495992B)

  5. Precleaned Microscope Slides (Fisherbrand, catalog number: 12-550-343)

  6. 2 mL Safe Lock Tubes (Eppendorf, catalog number: 054027)

  7. 5 mm stainless steel bead (Qiagen, catalog number: 69989)

  8. 30 mL Oak Ridge round-bottom tubes (Thermo Scientific Nalgene, catalog number: 3115-0030)

  9. Disposable 5ml syringe (BD Biosciences, catalog number: 309647)

  10. Precision Glide Needle (BD Biosciences, catalog number: B305196)

  11. 0.22 µm Nitrocellulose membrane (FroggaBio, catalog number: TM300)

  12. Filter paper (Bio-Rad, catalog number: 1703965)

  13. 1 ply Kimwipes 11 × 21 cm (KimTech Science Brand, Kimberly-Clark, Code: 34155)

  14. BactoTM Tryptone (BD Biosciences, catalog number: 211705)

  15. DifcoTM Agar, Bacteriological (BD Biosciences, catalog number: 214530)

  16. Tris-HCl pH 8 (Invitrogen, catalog number: 15-568-025)

  17. Dimethyl sulfoxide (DMSO, Sigma-Aldrich, catalog number: D-1435, CAS number: 67-68-5)

  18. Trizma base (Sigma-Aldrich, CAS number: 77-86-1)

  19. Hydrochloric Acid (Bio Basic Canada Inc. CAS number: 7647-01-0)

  20. RNase A, Protease-Free, Highly Purified, Bovine Pancreas (Sigma-Aldrich, CAS number: 9001-99-4)

  21. Deoxyribonuclease I from Bovine Pancreas (Sigma-Aldrich, CAS number: 9003-98-9)

  22. MgCl2·6H2O (Bio Basic Canada Inc. CAS number: 7791-18-6)

  23. Lysozyme from Chicken Egg White (Sigma-Aldrich, CAS number: 12650-88-3)

  24. Sodium dodecyl sulfate (Bio Basic Canada Inc. catalog number: 151-21-3)

  25. 40% Acrylamide/Bis Solution 29:1 (Fisher Bioreagents, catalog number: BP1408-1)

  26. Ammonium persulfate (Sigma-Aldrich, CAS number: 7727-54-0)

  27. TEMED (Thermo Scientific, catalog number: 17919)

  28. Sterile distilled water

  29. β- mercaptoethanol (Sigma-Aldrich, LOT number: SHBJ8714; catalog number M6250)

  30. Ethyl Alcohol 95% Vol (Commercial Alcohols by Greenfield Global, Item number: P016EA95)

  31. Glycine (Bio Basic Canada Inc. catalog number: 56-40-6)

  32. BLUelf Prestained Protein Ladder (FroggaBio, catalog number: PM008-0500)

  33. Glycerol (Bio Basic, GB0232, CAS number: 56-81-5)

  34. Bromophenol Blue (Sigma-Aldrich, CAS number: 34725-61-6)

  35. NaCl (Bio Basic, DB0483, CAS number: 7647-14-5)

  36. Tween 20 (Sigma-Aldrich, CAS number: 9005-64-5)

  37. Methanol (Fisher Bioreagents, catalog no: BP1105-4)

  38. IRDye 680RD goat anti-rabbit IgG (Mandel Scientific, ERP No: LIC-926-68071)

  39. Nitro-BT (NBT) (Fisher BioReagentsTM, CAS number: 298-83-9)

  40. 5-Bromo-4-chloro-3-indolyl phosphate (BCIP) (Fisher BioReagentsTM, CAS number: 298-83-9)

  41. Formic Acid 98-100% (Millipore Sigma, CAS number: 64-18-6)

  42. 1,1,1,3,3,3-Hexafluoro-2-propanol; Hexafluoroisopropanol (HFIP) (Sigma-Aldrich, CAS number: 920-6611)

  43. InvitrogenTM NovexTM WedgeWellTM 4 to 20%, Tris-Glycine, 1.0 mm, Mini Protein Gel, 10-well (Thermo Fisher Scientific, catalog number: XP04200PK2)

  44. InvitrogenTM iBlotTM 2 Transfer Stacks, nitrocellulose, mini (Thermo Fisher Scientific, catalog number: IB23002)

  45. Infected tissue samples

  46. N-cetyl-N,N,N,-trimethyl ammonium bromide (CTAB) (Sigma-Aldrich, CAS number: 57-09-0)

  47. Phenol/chloroform/isoamyl alcohol (pH 7.7–8.3) (49.5:49.5:1) (Sigma-Aldrich, catalog number 77618)

  48. Chloroform (Sigma-Aldrich, CAS number: 67-66-3)

  49. MSB broth (see Recipes)

  50. YESCA broth (see Recipes)

  51. T agar (see Recipes)

  52. 2× SDS-PAGE sample buffer (100 mL) (see Recipes)

  53. 10× SDS-PAGE running buffer (1,000 mL) (see Recipes)

  54. 10× Towbin buffer or Transfer buffer (1,000 mL) (see Recipes)

  55. 10× TBS (1,000 mL) (see Recipes)

  56. 1× TBST (see Recipes)

  57. 12% separating gel (2 gels) (see Recipes)

  58. 5% stacking gel (2 gels) (see Recipes)

  59. AP buffer (Alkaline Phosphatase) (500 mL) (see Recipes)

  60. NBT/BCIP developing solution (see Recipes)

Equipment

  1. Water bath

  2. Autoclave vacuum steam sterilizer (GETINGE, model: 533LS)

  3. Biological Safety Cabinet (Therma Electron Corporation, model: 1284)

  4. Water Jacketed CO2 Incubator (Therma Electron Corporation, model: 3110)

  5. Sonicator (BRANSON, DIGITAL SONIFIER 450)

  6. Tissue Homogenizer (Retsch, high-speed mixer mill MM400)

  7. Superspeed Centrifuge (Thermo Scientific, SORVALL EVOLUTION RC)

  8. SDS-PAGE gel big apparatus (Bio-Rad, PROTEAN II xi Cell, model: 1651814)

  9. InvitrogenTM Mini Gel Tank (Thermo Fisher Scientific, model: A25977)

  10. Lyophilizer (FTS SystemTM, Duro-DryTM MP, model: FD2085C0000)

  11. Freezer (Panasonic, SANYO, model: MDF-U76VC)

  12. Weighing balance (METTLER TOLEDO, model: B2002-S)

  13. Trans-Blot SD semi-dry transfer cell (Bio-Rad Laboratories)

  14. InvitrogenTM iBlotTM 2 Gel Transfer Device (Thermo Fisher Scientific, model: IB21001)

  15. Odyssey CLx imaging system and Image Studio 4.0 software package (Li-Cor Biosciences, model: 9140)

  16. DeNovix DS-11 FX Microvolume spectrophotometer/fluorometer

Procedure

A mutant strain used for curli purification was S. enterica serovar Typhimurium 14028-3b ∆bcsA. This is a strain of wild-type S. Typhimurium 14028 that we engineered to produce more curli by introducing a csgD promoter mutation from S. enterica serovar Enteritidis 27655-3b (White and Surette, 2006). csgD encodes the master biofilm regulator in S. enterica and E. coli (Gerstel et al., 2003; Brombacher et al., 2006). The other modification was to introduce a single-gene knockout of bcsA (White et al., 2006), which encodes cellulose synthase, responsible for making cellulose polymers at the cell surface of S. enterica and E. coli biofilm cells (Zogaj et al., 2001; Solano et al., 2002). The presence of cellulose causes other substances to become trapped in the extracellular matrix during the purification process. Furthermore, because curli and cellulose are difficult to separate (White et al., 2003), these carbohydrates can represent a significant proportion of the final purified product. The use of a cellulose-negative ∆bcsA strain avoids purification of these unnecessary contaminants.

The same procedure can be used to purify curli from strains of S. enterica or E. coli that produce cellulose; it is just important to note that cellulose will represent a proportion of the final weight of the “mature curli”. The tight association between curli and cellulose (White et al., 2003) will make it impossible to separate them.


  1. Mature Curli Extraction and Purification


    Bacterial Strain and culture condition
    1. Grow strain S. Typhimurium 14028-3b ∆bcsA in 5 mL of Luria broth (1% salt) overnight at 37°C with shaking at 200 rpm.

    2. Dip sterile swabs in this overnight culture and spread evenly onto 120 large (150 mm × 15 mm) T agar plates to ensure that a homogenous lawn of bacteria covers the entirety of each plate.

    3. Incubate the inoculated plates at 28°C for 48 h.


    Mature curli extraction
    1. Collect the bacterial cells and extracellular material by scraping the agar surfaces using microscope slides (Video 1) or a sterile cell scraper.


      Video 1. Scraping Plates


    2. Suspend the cell materials from each set of 10 plates in 20 mL of 10 mM Tris-HCl (pH 8) supplemented with RNase A and DNase I (to 0.1 mg/mL final concentration).

    3. Vortex the suspended cell material until there are no visible clumps.

    4. Transfer 1 mL of cell slurry into 2 mL Safe-Lock tubes containing a 5 mm stainless steel bead (Qiagen) (approximately 20 tubes per 10 Petri plates).

    5. Place tubes in the mixer mill and homogenize at 30 Hz for 5 min in room temperature to break up the extracellular matrix.

    6. Transfer and combine the homogenized cell materials into 30 mL Oak Ridge centrifuge tubes.

    7. Lyse bacterial cells by sonication using the 3 mm probe with 10 × 30 s pulses at 30% amplitude with 2 min cooling on ice between pulses (Figure 1).



      Figure 1. Reference image of sonication apparatus used for bacterial cell lysis.

      Cells are suspended in a 30 mL Oak Ridge centrifuge tube, which is embedded in ice within a 500 mL beaker. The sonicator probe is placed within the centrifuge tube approximately 5 mm beneath the surface of the liquid cell slurry.


    8. After sonication, add 2 M MgCl2 to a final concentration of 1 mM and incubate the mixture at 37°C for 20 min without shaking.

    9. After incubation, add lysozyme to a final concentration of 1 mg/mL and incubate the mixture at 37°C for 40 min without shaking.

    10. Add SDS to a final concentration of 1%, along with 0.1 mg/mL DNase I, and incubate this mixture overnight at 37°C without shaking.

    11. The next day, centrifuge the mixture in 30 mL Oak Ridge centrifuge tubes at 25,000 × g and 4°C for 25 min.

    12. For each tube, remove supernatant and resuspend the pellet in 10 mL of 10 mM Tris-HCl (pH 8), boil for 10 min, and centrifuge the mixture at 25,000 × g for 25 min. Repeat this step twice.

    13. Resuspend the pellet with 10 mL of 10 mM Tris-HCl (pH 8) and add RNase A (0.1 mg/mL final conc.), DNase I (0.1 mg/mL final conc.) and Lysozyme (1 mg/mL final conc.) and incubate the mixture overnight at 37°C.

      Note: This step can be repeated several times, if the mixture is highly viscous (i.e., looks like a clump of mucus), without any reduction in amount or quality of the final, purified material.

    14. After the mixture has achieved a lower viscosity (i.e., becomes watery), centrifuge the mixture at 12,100 × g for 15 min.

    15. Wash the pellet twice with 10 mL of 10 mM Tris-HCl (pH 8) by centrifugation at 12,100 × g for 15 min. Reduced viscosity is necessary for this step to work efficiently and can slightly vary from batch to batch.

    16. Resuspend the pellet in 3 ml of 2× SDS-PAGE sample buffer and boil for 15 min (Crude protein sample).


    Mature Curli Purification in SDS-PAGE Gel.

    1. Prepare a 4 mm thick SDS-PAGE gel with 5% stacking gel and 12% separating gel (Figure 2A).

    2. Load 3 mL of Crude protein sample from step 16 (see above) into the well of preparative SDS-PAGE gel (Figure 2B) and run continuously at 100V until all the dye in the sample runs through the bottom (Figure 2C). Pack around the bottom of the electrophoresis apparatus with ice to prevent the gel from over-heating (Figure 2B).



      Figure 2. SDS-PAGE gel apparatus for curli purification from bacterial cells.

      (A) Reference image of gel casting system. (B) Three milliliters of crude protein sample loaded into the well, prior to SDS-PAGE. Note: pack the SDS-PAGE gel apparatus with ice or run the experiment in a 4°C cold room to prevent gel overheating. (C) Electrophoresis is performed until the sample loading dye reaches the bottom.


    3. Collect the insoluble material retained on the top of the well (Figure 3; arrow) using a 5 mL syringe with 18-gauge needle. Purified curli fibers will not enter the SDS-PAGE gel unless they are depolymerized with 90% FA (Collinson et al. 1991) or 100% HFIP (Zhou et al., 2013).



      Figure 3. Collection of curli aggregates from the top of the gel after electrophoresis.

      The red arrow denotes the purified curli fibrils on the top of the well before collection. Using a syringe and an 18-gauge needle, the researcher can collect the white material from the top of the gel.


    4. Wash recovered insoluble material with 10 mL of sterile distilled water and sediment by centrifugation at 16,000 × g and 4°C for 10 min. Repeat this wash step three times.

    5. Dissolve the pellet with 5 ml of 95% ethanol and centrifuge at 16,000 × g and 4°C for 10 min. Repeat this step twice.

    6. Resuspend the pellet with 10 mL of sterile distilled water, transfer to suitable container (e.g., 30 mL Oakridge centrifuge tubes; Figure 4A), and freeze the mixture at -80°C.

    7. Wrap the frozen tubes with 1 ply kimwipes, secure them with rubber band, and lyophilize for 24 h. White, flocculent material should be present at the bottom of each tube (Figure 4B).

      Note: Tubes can be either wrapped with 1 ply kimwipes or with aluminum foil. If the tubes are wrapped with aluminum foil, make few holes for the proper lyophilization of frozen material. These holes allow the change of frozen material from solid to vapor state.



      Figure 4. Lyophilization of purified curli aggregates.

      (A) The solution of purified curli in water was frozen at -80°C in 30 mL Oakridge centrifuge tubes, wrapped with lint free tissue, and lyophilized for 24 h. (B) White, flocculent material (red arrow) present in the bottom of each tube after lyophilization represents curli fibrils.


    8. Dissolve the lyophilized material in 2 mL of 0.2 M glycine (pH 1.5) and boil this mixture for 10 min to solubilize any Type I fimbriae that might be present (Müller et al., 1991).

    9. Centrifuge the sample at 27,500 × g for 25 min.

    10. Wash the pellets five times with sterile distilled water with centrifugation at 25,000 × g for 25 min.

    11. Resuspend the pellet with 5 mL of distilled water and transfer the mixture into a pre-weighed glass vial.

    12. Freeze the mixture at -80°C and lyophilize for 24 h.

    13. After lyophilization, weigh the glass vial containing sample to obtain the rough sample weight. The final purified material (Figure 5) should be white to off-white in color and “more powdery” than the material described in step 6.



      Figure 5. Quantitation and long-term storage of purified curli.

      All material collected after the final lyophilization step was transferred into pre-weighed, sterile glass vials, and the rough weight was calculated. This purified material can be stored at -20°C.


    14. Store the lyophilized material (Curli standard) at -20°C. Stock solutions are best prepared by weighing out a desired amount of purified curli into a glass vial, and resuspending in distilled water to achieve the desired concentration, such as 1 mg/mL. Once a stock solution is prepared, aliquot the mixture into Eppendorf tubes. Because purified curli fibers are insoluble in water, make sure to evenly mix the stock solution and use a wide-bore pipette to transfer the aliquot.

    15. To prepare depolymerized curli, add 90% FA or 100% HFIP to the mature curli (purified curli from step 13).

      Note: HFIP and FA are irritating and corrosive chemicals. Always use them under a chemical fume hood and wear appropriate protective equipment while handling. Store them in a well-ventilated, cool place.

    16. For HFIP treatment, weigh 1 mg of curli, dissolve it in 1 mL of HFIP, and aliquot them into 0.5 mL Eppendorf tubes. For FA treatment, prepare 1 mg/mL of curli in sterile water, aliquot 10 µL of stock into 0.5 mL Eppendorf tubes, and add 92 µL of FA.

    17. Immediately freeze the mixtures for 1 h at -80°C, make a hole in the lids (i.e., using a sterile needle), and lyophilize for 24 h.

      Note: Samples containing acid may damage some lyophilizers.

    18. After lyophilization, dissolve mature (from step 13) and FA/HFIP-treated or depolymerized curli (from step 15–16) with 30 µl of 1× SDS-PAGE sample buffer and load them into each SDS-PAGE gel lane.

    19. Check the purity of mature and depolymerized curli by immunoblot analysis (Figure 6A and 6C).



    Figure 6. Comparison of different forms of curli as detected by immunoblot analysis.

    (A and B) Samples were resolved by SDS-PAGE consisting of a 5% acrylamide stacking gel and 12% acrylamide resolving gel. Proteins were transferred to nitrocellulose membranes using the iBlot system. For all immunoblots, monomer (M), dimer (D), and higher molecular weight oligomers of CsgA were detected using rabbit anti-curli polyclonal serum, followed by IRDye 680RD goat anti-rabbit IgG and detection using the Odyssey CLx imaging system (Li-Cor Biosciences). (A) 20 µg and 10 µg of purified curli were depolymerized with 90% FA prior to SDS-PAGE. (B) 50 mg samples of homogenized tissues from mice infected with S. Typhimurium were treated three successive times with 90% FA prior to SDS-PAGE, as previously described (Miller et al., 2020). (C) 10 µg of purified full-length curli (Mature), curli intermediates (Interm) and FA or HFIP treated curli were loaded and resolved directly on pre-cast NovexTM 4–12% Tris-Glycine Mini Gels. The white star denotes the 25 kDa protein standard.


    1. Purification of Curli Intermediates

      Purification of curli intermediates relies on generating a ∆msbB mutant strain (Nicastro et al., 2019).

      Studies on human amyloids have proved that different types of intermediate structures formed during the multistep process of amyloid polymerization. Wherein, soluble human amyloid monomers first form oligomers, which then polymerize into protofibrillar structures and then cross-assemble them into a stable, mature fibrils. Even though the process of curli fibrillar assembly in Enterobacteriaceae has been well-studied, the intermediate oligomeric structures of curli have not been identified or studied until recently by Nicastro et al. (2019). The authors have used the ∆msbB strain to understand the fibrillization kinetics of curli, their intermediates, as well as the role of mature curli fibrillar aggregates in the assembly of bacterial extracellular Matrix. The ∆msbB gene in Salmonella encodes the enzyme that catalyzes one of the two secondary acylation reactions that completes lipid A biosynthesis. It synthesizes a full-length O-antigen-containing LPS molecule that lacks only the expected secondary acyl chain, and is less able to induce cytokine and inducible nitric oxide synthase responses in both in vitro and in vivo conditions. They found that intermediate protofibrillar structures of bacterial amyloid (Curli intermediates) are more cytotoxic, and the addition of bacterial DNA accelerates them to form a mature fibrillar structure, limiting cytotoxic effects. The more detailed information regarding the kinetics of curli fibrilization and its stability can be found elsewhere (Nicastro et al., 2019). We used the lambda red recombinase knockout procedure (Datsenko and Wanner, 2000) to generate S. Typhimurium 14028s ∆msbB. Our first attempts to generate the ∆msbB strain were unsuccessful because we had difficulties recovering the antibiotic resistant transformants on LB agar. It has been reported that a Salmonella msbB mutant strain shows poor growth on LB agar and that growth can be restored to near wild-type levels by switching to MSB agar (LB with no NaCl supplemented with Mg2+ and Ca2+) (Murray et al., 2001). Once the procedure was changed to recover CMR (Chloramphenicol-resistant) transformants on MSB agar instead of LB agar, the lambda red procedure worked efficiently.

      1. Grow strain S. Typhimurium 14028s ∆msbB in 5 mL of MSB broth with 34 µg/mL chloramphenicol overnight at 37°C with shaking at 200 rpm.

      2. Add 5 mL of overnight culture to 500 mL of YESCA broth supplemented with 4% DMSO in a 1-L flask and incubate at 26°C for 72 h with shaking at 200 rpm.

      3. After incubation, collect bacterial pellet by centrifugation at 10,000 × g for 10 min.

      4. Resuspend the pellet in 30 mL of 10 mM Tris-HCl (pH 8.0) and treat with RNase A (0.1 mg/mL final conc.), DNase I (0.1 mg/mL final conc.), and MgCl2 (1 mM final conc.) for 30 min at 37°C.

      5. After enzyme treatment, sonicate the suspension to break open the bacteria with 3 × 30 s pulses at 30% amplitude with 1 min cooling on ice between pulses.

      6. To the mixture, add lysozyme to a final concentration of 1 mg/mL and incubate for 40 min at 37°C.

      7. Add SDS to a final concentration of 1% to the mixture and incubate for 20 min at 37°C with shaking at 200 rpm.

      8. After incubation, pellet curli by centrifugation (10,000 × g for 10 min at 4°C).

      9. Resuspend the curli pellet in 10 mL of 10 mM Tris-HCl (pH 8.0) and boil for 10 min.

      10. Cool down the sample on ice before adding RNase A (0.1 mg/mL final conc.), DNase I (0.1 mg/mL final conc.), lysozyme (1 mg/mL final conc.) and MgCl2 (1 mM final conc.). Incubate this mixture at 37°C for 2 h. Repeat this step until the desired viscosity is reached (see section A steps 13 and 14).

      11. Centrifuge the mixture at 10,000 × g and 4°C for 10 min.

      12. Wash the pellet three times with 10 mL 10 mM Tris-HCl (pH 8.0) and resuspend in 3 mL of 2× SDS-PAGE sample buffer and boil for 10 min.

      13. Load the 3 mL of crude protein sample (from previous step) into a 4 mm-thick SDS-PAGE gel with 5% stacking gel and 12% separating gel and run continuously at 100 V until all the dye in the sample has run through the bottom.

      14. After electrophoresis, use a 5 mL syringe with 18-gauge needle to collect the curli aggregates from the top of the gel, resuspend in 5–10 mL of sterile water, pellet by centrifugation (10,000 × g for 10 min) and wash two times with sterile water.

      15. Resuspend the curli in 5 mL of 95% ethanol, pellet by centrifugation at 13,500 × g for 10 min, and wash two more times with ethanol.

      16. Resuspend the curli in sterile water and transfer the mixture into a pre-weighed glass vial.

      17. Freeze the mixture at -80°C and lyophilize for 24 h.

      18. After lyophilization, weigh the glass vial containing sample to obtain the rough sample weight and store the lyophilized material at -20°C. The purity of curli intermediates can be checked by immunoblot analysis (Figure 6C).


    2. Curli detection in host tissues/samples

      The procedure for analysis of mouse tissues was adapted from Miller et al. (2020). The same procedure could be used to analyze tissues from other animal species or for other types of samples.

      1. Place tissue samples (i.e., liver, cecum, colon, and small intestine) in tinfoil packets, snap freeze in liquid nitrogen, and store at -80°C.

      2. Pre-chill a mortar and pestle by storing at -20°C. Grind the frozen tissue samples into a fine powder under liquid nitrogen. The powdered samples can be scraped out of the mortar and pestle using a metal spatula onto weigh paper and transferred in 2 mL screwcap freezer vials, and stored at -80°C.

      3. Weigh 50 mg samples of powdered tissue, transfer into Eppendorf tubes and resuspend in 500 μL of 1× SDS-PAGE sample buffer and boil for 10 min. This step will dissolve most of the soluble cellular proteins.

      4. Cool down the solution on ice, and pellet the cell debris by centrifugation at 25,000 × g for 5 min.

      5. Resuspend the pellet in 500 μL of sterile distilled water and centrifuge at 25,000 × g for 5 min. Repeat this step twice. This washes away any remaining residues of SDS-PAGE sample buffer.

      6. Resuspend the pellet with 500 μL of 90% FA and freeze the sample at -80°C for at least 1 h.

      7. Lyophilize the mixture for 16–24 h. Repeat steps 6 and 7 three times. For purified curli standards, FA treatment only needs to be performed once.

        Note: Samples containing acid may damage some lyophilizers.

      8. After FA treatment, resuspend the lyophilized samples in 50 μL of 1× SDS-PAGE sample buffer and centrifuge the samples at 25,000 × g for 2 min.

        Note: Do not boil the samples, because it will cause the curli monomers to aggregate.

      9. Load a 20 μL aliquot of supernatant into each SDS-PAGE gel lane. We have used self-prepared 5% acrylamide stacking and 12% acrylamide resolving gels or commercial 4–12% acrylamide gradient gels. Load at least one gel lane with a commercial prestained protein ladder.

      10. Perform electrophoresis at 110 V. Carefully remove and place the gel on the nitrocellulose membrane and perform protein transfer for 40 min at 25V in Trans-Blot SD semi-dry transfer cell. Alternatively, proteins can be transferred to nitrocellulose membranes using the automated iBlot system.

      11. Detect curli proteins present in the tissue samples using any standard immunoblotting procedure. We commonly use rabbit anti-curli polyclonal serum as the primary antibody at 1:500 dilution in 5% skim milk in TBST and incubate at 4°C overnight or at 37°C for one hour. The secondary antibody used is either IRDye 680RD goat anti-rabbit IgG at 1:10,000 dilution (for fluorescent applications) or Goat-anti-rabbit IgG alkaline phosphatase conjugate at 1:2,000 dilution in 5% skim milk in TBST. The incubation is for 1 h 30 min at room temperature in the dark for fluorescence or 1 h at 37°C on a tilted platform shaker for colorimetric detection.

      12. Antibody binding is visualized using fluorescence with the Odyssey CLX imaging system and Image Studio 4.0 software package (Figure 6B). For colorimetric detection of curli, soluble BCIP/NBT are used as substrates. Alkaline phosphatase will produce a stable blue-purple product that will not fade upon exposure to light, and you do not need special equipment to visualize.


    3. DNA extraction from curli

      Previous work from the Tükel lab has shown that extracellular DNA (eDNA) associates with curli fibrils during the development of the mature biofilm and purified curli contains eDNA (Gallo et al., 2015). Since the presence of eDNA can influence immune recognition as well as TLR2/9 stimulation (Tursi and Tükel, 2018), it is good practice to determine just how much eDNA is associated with purified curli each time a batch is purified.

      1. Resuspend 500 μg of purified curli in 550 μL of TE buffer, 30 μL of 10% SDS, and 70 μL of proteinase K (20 mg/mL).

      2. Mix thoroughly and incubate at 37°C for 1 h.

      3. Add 100 μL of 5 M NaCl and mix by pipetting.

      4. Add 80 μL of CTAB/NaCl solution and mix thoroughly by pipetting using wide bore tips.

      5. Incubate at 37°C for 10 min.

      6. Add 300–400 μL phenol/chloroform/isoamyl alcohol to samples and mix by inverting the tubes

      7. Centrifuge at 15,682 × g and 4°C for 5 min.

      8. Transfer upper phase to a new Eppendorf tube.

      9. Add 700 μL of chloroform and mix by pipetting.

      10. Centrifuge at 15,682 × g and 4°C for 5 min.

      11. Transfer upper phase to a new Eppendorf tube and add equal volume isopropanol and shake the tube by hand.

      12. Incubate at -20°C for 30 min.

      13. Centrifuge at 13,362 × g and 4°C for 5 min.

      14. Discard the liquid and rinse the DNA pellet with 1 mL of 70% EtOH and centrifuge for 5 min at 5,220 × g.

      15. Resuspend the final DNA pellet in 30 μL of TE buffer or water.

      16. Measure the DNA content using a DeNovix DS-11 FX spectrophotomer/fluorometer.

    Data analysis

    For detection of bacterial produced curli from animal tissues, it is recommended to test at least two 50 mg samples from each tissue type, and to screen all individual mice from infected groups. In our previous work, we did not detect the presence of curli in each mouse that was infected with Salmonella (Miller et al., 2020). We are unsure if this was because of stochastic expression of curli (i.e., it is not produced in each mouse) or due to technical difficulties with detection. For this reason, the authors recommend that each individual animal within a group be tested.

    Recipes

    1. MSB broth (1,000 mL)

      Tryptone 10 g

      Yeast Extract 2.5 g

      1 mL of 1 M CaCl2

      1 mL of 1 M MgSO4

      Autoclave the solution on a standard liquid cycle (20 min at 15 psi).

    2. YESCA broth (1,000 mL)

      Casamino acid 10 g

      Yeast Extract 1 g

      Autoclave the solution on a standard liquid cycle (20 min at 15 psi) and add DMSO (final concentration 4%).

    3. T Agar (1,000 mL)

      Tryptone 10 g

      Agar 15 g

      Autoclave the solution on a standard liquid cycle (20 min at 15 psi).

    4. 2× SDS-PAGE sample buffer (100 mL)

      Tris (1 M, pH 6.8) 8 mL

      SDS (20%) 10 mL

      Glycerol 10 mL

      Bromophenol blue (0.1%) 600 μL

      β-mercaptoethanol (add fresh) 4 mL

      Distilled water 67.4 mL

    5. 10× SDS-PAGE running buffer (1,000 mL)

      Tris base 30.3 g

      Glycine 144.4 g

      SDS 10 g

    6. 10× Towbin buffer or Transfer buffer (1,000 mL)

      Tris base 30.3 g

      Glycine 144.4 g

      Prepare 1× transfer buffer by adding 100 mL of 10× transfer buffer with 200 mL of methanol and add 700 mL of distilled water.

    7. 10× TBS (1,000 mL)

      Tris base 24 g

      NaCl 88 g

      Dissolve the components in 900 mL of distilled water. Adjust the pH to 7.6 with HCl and bring up the final volume with distilled water. Prepare 1× TBS by adding 100 mL of 10× TBS with 900 mL of distilled water.

    8. 1× TBST

      Add 0.5 mL of Tween 20 to 1,000 mL of 1× TBS.

    9. 12% separating gel (2 gels)

      Distilled water 82.56 mL

      1.5 M Tris-HCl pH 8.8 48 mL

      Acrylamide/bis (40%) 57.6 mL

      10% SDS 1.92 mL

      10% APS 1.92 mL

      TEMED 96 μL

    10. 5% stacking gel (2 gels)

      Distilled water 23.84 mL

      0.5 M Tris-HCl pH 6.8 9.6 mL

      Acrylamide/bis (40%) 4.5 mL

      10% SDS 400 μL

      10% APS 480 μL

      TEMED 40 μL

    11. AP buffer (500 mL)

      100 mM Tris 6.06 g

      100 mM NaCl 2.92 g

      5 mM MgCl2·6H2O 0.51 g

      Dissolve the components in 450 mL of distilled water. Adjust the pH to 9.5 with 3 M HCl and bring up to 500 mL with distilled water and autoclave.

    12. NBT/BCIP developing solution

      AP buffer 10 mL

      NBT 66 μL

      BCIP 33 μL

    Acknowledgments

    This research was supported by a Natural Sciences and Engineering Research Council (NSERC) Discovery grant (Grant #2017-05737), the Jarislowsky Chair in Biotechnology, and a College of Medicine Research Award from the University of Saskatchewan to A.P.W. CT was supported by NIH grants AI153325, AI151893, and AI148770. E.G.H., was supported by an NSERC undergraduate research award. We thank Neil Rawlyk at VIDO for general technical assistance. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The mature curli purification protocol and immunodetection of curli from animal tissues are used in our recent publication (Miller et al., 2020). The protocols for purification of curli intermediates and DNA extraction were published in Nicastro et al. (2019). Published as VIDO manuscript series No. 968.

    Competing interests

    The authors have no competing interests to report.

    Ethics

    Mice were cared for and used in accordance with the Guidelines of the Canadian Council on Animal Care and the Regulations of the University of Saskatchewan Committee on Animal Care and Supply, following Animal Use Protocols #20110057 or 20170080, which were approved by the University of Saskatchewan’s Animal Research Ethics Board.

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简介

微生物学家正在学习理解“功能性淀粉样蛋白”的重要性,这种淀粉样蛋白由众多细菌种类产生,并具有超越微生物世界的影响。这些结构被细菌用来连接在一起,可能是为了提高存活率,防止恶劣的条件,也许是为了影响细胞间的交流。细菌功能性淀粉样蛋白也开始在宿主-病原体相互作用的背景下受到重视,有证据表明它们可以触发先天免疫系统并被认为是非自身分子模式。淀粉样蛋白的特征性三维折叠使它们在整个细菌界和真核世界中相似,在真核世界中,淀粉样蛋白可能是不受欢迎的并具有病理后果。由致病性肠沙门氏菌和大肠杆菌菌株产生的细菌蛋白 curli 是最早发现的功能性淀粉样蛋白之一。此后,Curli 在功能方面得到了很好的表征,我们才刚刚开始了解它们对真核宿主的潜在影响。在这份手稿中,我们通过图片展示了如何净化这些细菌表面结构的分步协议。我们已经描述了 S. enterica 的纯化过程,承认同样的方法可以应用于大肠杆菌。此外,我们描述了在动物组织(即胃肠道)中检测 curli 的方法,并讨论了在 S. enterica msbB 突变株中纯化 curli 中间体,因为它们比成熟的 curli 原纤维更具细胞毒性。其中一些方法首先在其他地方进行了描述,但我们希望更详细地将它们组合在一起,以便希望纯化 curli 用于生物实验的研究人员更容易。随着细菌淀粉样蛋白变得越来越重要,我们的目标是提供对专家和非专家有用的方法。
Wéishēngwù xué jiā zhèng


背景

称为 curli 的细菌细胞表面附属物最初是在从马粪中分离出来的大肠杆菌菌株中发现的。作者将表面结构命名为“curli”,因为它们具有弯曲的外观( Olsén 等。 , 1989)。几乎同时,在沙门氏菌中观察到相似的表面结构 enterica serovar Enteritidis,它们被称为细聚集菌毛(Collinson等,1991)。 curli 和薄的聚集菌毛具有许多独特的特性,包括需要用 90% 的甲酸 (FA) 处理以将纤维分解成可以通过 SDS-PAGE 凝胶解析的单体蛋白。 1998 年发表的一项具有里程碑意义的研究表明,卷曲菌和细长的聚集菌毛,包括生物合成所需的基因,在肠道链球菌和大肠杆菌之间几乎可以互换( Römling 等。 , 1998)。随着S基因组序列的公布。 Typhimurium LT2,“curli”命名法被选用于这两个物种。就功能而言,curli 是形成S. enterica和E. coli生物膜的关键蛋白质,在导致聚集的“短程”细胞 - 细胞相互作用中起主要作用( Römling 等。 , 1998)。这种聚集和生物膜的形成与在面对环境损害时提高持久性和生存率有关( Anriany 等。 , 2001;索拉诺 等。 , 2002;舍尔 等。 , 2005; Ryu 和Beuchat ,2005 年;怀特等人。 , 2006)。在这些相关但长期分化的物种之间保存像 curli 这样的表面结构是非常独特的。我们已经提出,当它们在宿主之间循环时,curli 和生物膜的形成在这些肠道细菌在环境中的生存中起着关键作用。它们在肠杆菌和大肠杆菌中的保护说明了它们在两种细菌生命周期中的重要性。
Curli 现在被称为“功能性淀粉样蛋白” 。另一篇具有里程碑意义的论文表明,卷曲单体CsgA具有自聚合特性,这是淀粉样蛋白的标志(Chapman等,2002)。这解释了在最初纯化时观察到的 curli 的一些独特的、耐生化的物理特性。淀粉样蛋白的特征性 3-D 结构被称为交叉 β,具有规则的、重复的β折叠或垂直于纤维轴延伸的链。 curli 的推测是,当CsgA单体相互堆叠形成纤维时,有一个中央非极性核心会沿着 curli 纤维的长度延伸,从而导致极高的稳定性(Collinson等,1999)。纤维的组装发生在细胞外,未折叠的CsgA单体通过CsgG孔穿过外膜,然后卡入其更刚性的交叉 beta 结构(Evans 和 Chapman,2014)。尽管众所周知,淀粉样蛋白难以使用,并且由于纤维-纤维相互作用不均匀,因此不容易适应蛋白质晶体学,但一些结构特征已通过使用 CsgA特异性肽( Szulc 等。 , 2021)。也许迄今为止进行的最引人注目的分析表明, CsgA “curli”与阿尔茨海默病特征的淀粉样蛋白-β原纤维具有结构相似性,两者都具有称为空间β-拉链的特征性结构折叠( Perov 等。 , 2019)。
curli 在宿主-病原体相互作用中的作用最近得到了澄清。一些早期的研究在宿主表达的背景下描述了curli(卞 等。 , 2000;奥尔森 等。 , 1998;工作环 等。 , 1994),但长期以来,我们认为它们纯粹是一个“环境”因素。 Çagla的开创性工作 Tükel 、Andreas Baumler 及其同事将纯化的 curli 纤维鉴定为先天免疫系统的有效刺激物,它们与 Toll 样受体 2 (TLR-2) 相互作用 ( Tükel 等。 , 2005), TLR1/2 和 TLR1/2/9 由于存在与 curli ( Tükel ) 复合物的细胞外 DNA 等。 , 2010; Tursi和Tükel ,2018 年)。 curli 的全身性表现对宿主物种的自身免疫产生了深远的影响,导致循环抗 dsDNA 抗体的存在增加、nod 样受体(即NLRP3)和炎性体的刺激(Gallo等,2015; Rapsinski 等。 , 2015 年)。最近,我们在急性和长期感染模型中发现,在小鼠口腔感染大肠后,肠链球菌会表达 curli(Miller等人,2020)。在这些实验中,宿主体内S. enterica细胞产生的 curli导致受感染小鼠膝关节的自身免疫水平和关节炎的早期特征增加。 curli 的影响与S. enterica侵入宿主细胞上皮并引起炎症的能力有关,这表明 curli 必须进入肠道周围的淋巴组织才能产生负面影响。体内curli 表达的许多方面仍有待阐明。
在这份手稿中,我们提出了一系列分步协议,并附有图片,展示了如何净化这些重要的细菌表面结构。

关键字:Curli, 沙门氏菌, 细菌淀粉样蛋白, 净化, 免疫印迹

材料和试剂
1.一次性培养管16×100 mm( Fisherbrand ,目录号:14-961-29)
2.Difco TM Luria-Bertani 肉汤(BD Biosciences,目录号:244620)
3.聚苯乙烯一次性培养皿150 mm×15 mm(VMR International,目录号:25384-326)
4.棉尖涂抹器,6英寸(Puritan,目录号:1495992B)
5.显微镜载玻片( Fisherbrand ,目录号:12-550-343)
6.2 mL Safe Lock 管(Eppendorf,目录号:054027)
7.5 mm不锈钢珠(Qiagen ,目录号: 69989)
8.30 mL Oak Ridge 圆底管(Thermo Scientific Nalgene,目录号: 3115-0030)
9.一次性5ml注射器(BD Biosciences,目录号: 309647)
10.Precision Glide Needle(BD Biosciences,目录号:B305196)
11.0.22μm硝酸纤维素膜( FroggaBio ,目录号: TM300 )
12.滤纸(Bio-Rad,目录号:1703965)
13.1 层Kimwipes 11 × 21 cm( KimTech Science Brand,Kimberly-Clark,代码:34155)
14.Bacto TM胰蛋白胨(BD Biosciences,目录号:211705)
15.Difco TM琼脂,细菌学(BD Biosciences,目录号:214530)
16.Tris-HCl pH 8(Invitrogen,目录号:15-568-025)
17.二甲基亚砜(DMSO,Sigma-Aldrich,目录号: D-1435,CAS 号:67-68-5)
18.Trizma碱基(Sigma-Aldrich,CAS 编号:77-86-1)
19.盐酸(Bio Basic Canada Inc. CAS 编号:7647-01-0)
20.RNase A,无蛋白酶,高度纯化,牛胰腺(Sigma-Aldrich,CAS 编号:9001-99-4)
21.来自牛胰腺的脱氧核糖核酸酶 I(Sigma-Aldrich,CAS 号:9003-98-9)
22.MgCl 2 · 6H 2 O(Bio Basic Canada Inc. CAS 编号:7791-18-6)
23.来自鸡蛋清的溶菌酶(Sigma-Aldrich, CAS 号:12650-88-3)
24.十二烷基硫酸钠(Bio Basic Canada Inc. 目录号:151-21-3)
25.40%丙烯酰胺/双溶液29:1(Fisher Bioreagents,目录号:BP1408-1)
26.过硫酸铵(Sigma-Aldrich,CAS 编号:7727-54-0)
27.TEMED(Thermo Scientific,目录号:17919)
28.无菌蒸馏水
29.β-巯基乙醇(Sigma-Aldrich,批号:SHBJ8714;目录号 M6250)
30.乙醇 95% Vol(Greenfield Global 的商业酒精,货号:P016EA95)
31.甘氨酸(Bio Basic Canada Inc. 目录号:56-40-6)
32.蓝精灵 预染蛋白梯( FroggaBio ,目录号:PM008-0500)
33.甘油(Bio Basic,GB0232,CAS 编号:56-81-5)
34.溴酚蓝(Sigma-Aldrich,CAS 号:34725-61-6)
35.NaCl(Bio Basic,DB0483,CAS 编号:7647-14-5)
36.吐温 20(Sigma-Aldrich,CAS 编号:9005-64-5)
37.甲醇(Fisher Bioreagents,目录号:BP1105-4)
38.IRDye 680RD 山羊抗兔 IgG(Mandel Scientific,ERP 编号:LIC-926-68071)
39.Nitro-BT (NBT)(Fisher BioReagents TM ,CAS 编号:298-83-9)
40.5-Bromo-4-chloro-3-indolyl phosphate (BCIP)(Fisher BioReagents TM ,CAS 编号:298-83-9)
41.甲酸 98-100%(Millipore Sigma,CAS 编号:64-18-6)
42.1,1,1,3,3,3-六氟-2-丙醇;六氟异丙醇 (HFIP)(Sigma-Aldrich,CAS 编号:920-6611)
43.英杰公司 Novex TM WedgeWell TM 4 至 20%,Tris-甘氨酸,1.0 mm,迷你蛋白凝胶,10 孔( Thermo Fisher Scientific, 目录号:XP04200PK2)
44.英杰公司 iBlot TM 2 Transfer Stacks,硝酸纤维素,迷你( Thermo Fisher Scientific,目录号:IB23002)
45.受感染的组织样本
46.N-鲸蜡基- N,N ,N,-三甲基溴化铵 (CTAB)(Sigma-Aldrich,CAS 号:57-09-0)
47.苯酚/氯仿/异戊醇(pH 7.7 - 8.3)(49.5:49.5:1)(Sigma-Aldrich,目录号:77618)
48.氯仿(Sigma-Aldrich,CAS 编号:67-66-3)
49.MSB肉汤(见食谱)
50.YESCA 肉汤(见食谱)
51.琼脂(见食谱)
52.2 × SDS-PAGE 样品缓冲液 (100 mL)(参见配方)
53.10 × SDS-PAGE 运行缓冲液 (1,000 mL)(参见配方)
54.10 × Towbin缓冲液或传输缓冲液 (1,000 mL)(参见配方)
55.10 × TBS (1,000 mL)(见配方)
56.1 × TBST(见配方)
57.12% 分离凝胶(2 个凝胶)(参见配方)
58.5% 浓缩凝胶(2 块凝胶)(参见食谱)
59.AP 缓冲液(碱性磷酸酶)(500 mL)(参见食谱)
60.NBT/BCIP 开发解决方案(见配方)




设备


1.水浴
2.高压灭菌器真空蒸汽灭菌器(GETINGE,型号:533LS)
3.生物安全柜(Therma Electron Corporation,型号:1284)
4.水套式 CO 2培养箱(Therma Electron Corporation,型号:3110)
5.声波器(布兰森,数字声波 450)
6.组织均质机( Retsch ,高速搅拌机MM400)
7.超高速离心机(Thermo Scientific,SORVALL EVOLUTION RC)
8.SDS-PAGE凝胶大仪(Bio-Rad,PROTEAN II xi Cell,型号:1651814)
9.Invitrogen TM迷你凝胶槽( Thermo Fisher Scientific,型号:A25977)
10.冻干机(FTS System TM , Duro-Dry TM MP,型号:FD2085C0000)
11.冰柜(松下、三洋、型号:MDF-U76VC)
12.天平(梅特勒-托利多,型号:B2002-S)
13.Trans-Blot SD 半干转移细胞(Bio-Rad Laboratories)
14.英杰公司 iBlot TM 2 凝胶转移装置(Thermo Fisher Scientific,型号:IB21001)
15.Odyssey CLx成像系统和 Image Studio 4.0 软件包(Li-Cor Biosciences,型号:9140)
16.DeNovix DS-11 FX 微量分光光度计/荧光计




程序


用于 curli 纯化的突变菌株是S 。 enterica serovar Typhimurium 14028-3b Δ bcsA 。这是一种野生型鼠伤寒沙门氏菌 14028 菌株,我们通过引入来自肠炎沙门氏菌血清型肠炎 27655-3b的csgD启动子突变来设计产生更多卷曲(White 和 Surette,2006)。 csgD编码肠杆菌和大肠杆菌中的主要生物膜调节剂(Gerstel等,2003; Brombacher 等。 , 2006)。另一种修改是引入bcsA的单基因敲除(White et al. , 2006),它编码纤维素合酶,负责在S. enterica和E. coli生物膜细胞的细胞表面制造纤维素聚合物 ( Zogaj 等。 , 2001;索拉诺 等。 , 2002)。纤维素的存在导致其他物质在纯化过程中被困在细胞外基质中。此外,由于卷曲和纤维素难以分离(White等人,2003),这些碳水化合物可以占最终纯化产品的很大一部分。使用纤维素阴性Δ bcsA菌株可避免纯化这些不必要的污染物。
相同的程序可用于从产生纤维素的肠杆菌或大肠杆菌菌株中纯化卷曲;重要的是要注意纤维素将代表“成熟卷曲”最终重量的一部分。 curli 和纤维素之间的紧密联系 (White et al ., 2003) 将使得它们无法分离。




A.成熟卷曲提取纯化


细菌菌株和培养条件
1.生长菌株S 。 Typhimurium 14028-3b Δ bcsA在 5 mL Luria 肉汤(1% 盐)中在 37°C 下以 200 rpm 摇动过夜。
2.将无菌拭子浸入这种过夜培养物中,然后均匀地铺在 120 个大(150 毫米× 15 毫米)T 琼脂板上,以确保均匀的细菌草坪覆盖整个平板。
3.将接种板在 28°C 下孵育 48 小时。


成熟的卷曲提取
1.使用显微镜载玻片(视频 1)或无菌细胞刮刀刮去琼脂表面,收集细菌细胞和细胞外物质。


 


视频 1. 刮板


2.将每组 10 个板中的细胞材料悬浮在 20 mL 的 10 mM Tris-HCl(pH 8)中,并辅以 RNase A 和 DNase I(最终浓度为 0.1 mg/mL)。
3.涡旋悬浮细胞材料,直到没有可见的团块。
4.将 1 mL 的细胞浆液转移到含有 5 mm 不锈钢珠 (Qiagen) 的 2 mL Safe-Lock 管中(每 10 个培养板约 20 个管)。
5.将管子放入搅拌机中,在室温下以 30 Hz 均质化 5 分钟,以分解细胞外基质。
6.将匀浆的细胞材料转移并合并到 30 mL Oak Ridge 离心管中。
7.× 30 s 脉冲以 30% 幅度通过超声裂解细菌细胞,脉冲之间在冰上冷却 2 分钟(图 1 )。


 


图 1. 用于细菌细胞裂解的超声设备的参考图像。
细胞悬浮在 30 mL Oak Ridge 离心管中,该离心管嵌入在 500 mL 烧杯中的冰中。超声仪探头放置在离心管内液体细胞浆液表面下方约 5 mm 处。


8.超声处理后,加入 2 M MgCl 2至终浓度为 1 mM,在 37°C 下孵育混合物 20 分钟,不要摇晃。
9.孵育后,加入溶菌酶至终浓度为 1 mg/mL 将混合物在 37°C 下孵育 40 分钟,不要摇晃。
10.将 SDS 和 0.1 mg/mL DNase I 添加至终浓度为 1%,并在 37°C 下将此混合物孵育过夜,不要摇晃。
11.第二天,将混合物在 30 mL Oak Ridge 离心管中以 25,000 × g和 4°C 离心 25 分钟。
12.对于每个试管,去除上清液并将颗粒重新悬浮在 10 mL 的 10 mM Tris-HCl (pH 8) 中,煮沸 10 分钟,然后以 25,000 × g的速度将混合物离心25 分钟。重复此步骤两次。
13.用 10 mL 的 10 mM Tris-HCl (pH 8) 重悬沉淀,并添加 RNase A(0.1 mg/mL 最终浓度)、DNase I(0.1 mg/mL 最终浓度)和溶菌酶(1 mg/mL 最终浓度.) 并将混合物在 37°C 下孵育过夜。
注意:如果混合物高度粘稠(即看起来像一团粘液),此步骤可以重复多次,而不会降低最终纯化材料的数量或质量。
14.在混合物达到较低粘度(即变成水样)后,将混合物以 12,100 × g离心15 分钟。
15.用 10 mL 的 10 mM Tris-HCl (pH 8) 在 12,100 × g下离心15 分钟,清洗颗粒两次。降低粘度对于此步骤有效工作是必要的,并且可能因批次而略有不同。
16.将沉淀重悬于 3 ml 2 × SDS-PAGE 样品缓冲液中并煮沸 15 分钟(粗蛋白样品)。


SDS-PAGE凝胶中的成熟卷曲纯化。
1.准备一个 4 mm 厚的 SDS-PAGE 凝胶,带有 5% 的堆叠凝胶和 12% 的分离凝胶(图 2A)。
2.将步骤 16(见上文)中的 3 mL 粗蛋白样品装入制备 SDS-PAGE 凝胶(图 2B)的孔中,并在 100V 下连续运行,直到样品中的所有染料都穿过底部(图 2C)。用冰在电泳装置的底部周围包装,以防止凝胶过热(图 2B)。


 


用于从细菌细胞中纯化 curli 的SDS-PAGE 凝胶应用程序。
(A) 凝胶浇注系统的参考图像。 (B) 在 SDS-PAGE 之前将三毫升粗蛋白样品装入井中。注意:SDS-PAGE 凝胶装置用冰包好或在 4 ° C的冷室中进行实验,以防止凝胶过热。 (C ) 进行电泳,直到上样染料到达底部。


3.使用带有 18 号针头的 5 mL 注射器收集保留在孔顶部的不溶性物质(图 3;箭头)。纯化的 curli 纤维不会进入 SDS-PAGE 凝胶,除非它们用 ˃90% FA (Collinson et al. 1991) 或 100% HFIP (Zhou et al. , 2013) 解聚。


 


图 3. 电泳后从凝胶顶部收集的 curli 聚集体。
红色箭头表示收集前井顶部的纯化卷曲原纤维。使用注射器和 18 号针头,研究人员可以从凝胶顶部收集白色物质。


4.用 10 mL 的无菌蒸馏水和沉淀物洗涤回收的不溶性物质,在16,000 × g和 4 °C下离心10 分钟。重复此洗涤步骤三遍。
5.用 5 ml 95% 乙醇溶解沉淀,并在 16,000 × g和 4°C 下离心 10 分钟。重复此步骤两次。
6.用 10 mL 无菌蒸馏水重悬沉淀,转移到合适的容器中(例如30 mL Oakridge 离心管;图 4A ),并在 -80°C 下冷冻混合物。
7.kimwipes包裹冷冻管,用橡皮筋固定,冻干 24 小时。白色的絮状材料应出现在每个管的底部(图 4B)。
注意:管可以用 1 层kimwipes或铝箔包裹。如果管子用铝箔包裹,则要打几个孔,以便对冷冻材料进行适当的冻干。这些孔允许冷冻材料从固态变为气态。


 


图 4. 纯化 curli 聚集体的冻干。
(A) 纯化的 curli 在水中的溶液在 -80°C 的 30 mL Oakridge 离心管中冷冻,用无绒组织包裹,并冻干 24 小时。 (B) 冻干后每个管底部存在的白色絮状物质 (红色箭头) 代表卷曲原纤维。


8.将冻干材料溶解在 2 mL 的 0.2 M 甘氨酸(pH 1.5)中,并将该混合物煮沸 10 分钟以溶解可能存在的任何 I 型菌毛(Müller等人, 1991)。
9.以 27,500 × g离心样品25 分钟。
10.用无菌蒸馏水洗涤颗粒五次,以 25,000 × g离心25 分钟。
11.用 5 mL 蒸馏水重新悬浮颗粒,并将混合物转移到预先称重的玻璃小瓶中。
12.将混合物冷冻在 -80 °C并冻干 24 小时。
13.冻干后,称量装有样品的玻璃小瓶以获得粗略的样品重量。最终纯化的材料(图 5 )应为白色至灰白色,并且比步骤 6 中描述的材料“更粉”。


 


图 5. 纯化卷曲的定量和长期储存。
在最终冻干步骤后收集的所有材料都被转移到预先称重的无菌玻璃瓶中,并计算粗略重量。这种纯化的材料可以储存在-20 °C 。


14.将冻干材料(Curli 标准品)储存在 -20°C。储备溶液的最佳制备方法是将所需数量的纯化卷曲称重到玻璃小瓶中,然后重悬于蒸馏水中以达到所需浓度,例如 1 mg/ mL。准备好储备溶液后,将混合物分装到 Eppendorf 管中。由于纯化的卷曲纤维不溶于水,因此请确保将原液混合均匀并使用大口径移液器转移等分试样。
15.要制备解聚卷曲,将 ˃90% FA 或 100% HFIP 添加到成熟卷曲(步骤 13 中纯化的卷曲)。
注意: HFIP 和 FA 是刺激性和腐蚀性化学品。始终在化学通风橱下使用它们,并在处理时佩戴适当的防护设备。将它们存放在通风良好、凉爽的地方。
16.对于 HFIP 治疗,称量 1 mg curli,将其溶解在 1 mL HFIP 中,然后将它们等分到 0.5 mL Eppendorf 管中。对于 FA 处理,在无菌水中制备 1 mg/mL 的 curli,将 10 μL 的库存等分到 0.5 mL Eppendorf 管中,并添加 92 μL 的 FA。
17.立即将混合物在 -80°C 下冷冻 1 小时,在盖子上打一个洞(即,使用无菌针头),然后冻干 24 小时。
注意:含有酸的样品可能会损坏某些冻干机。
18.用 30 µl 1 × SDS-PAGE 样品缓冲液溶解成熟(来自步骤 13)和 FA/HFIP 处理或解聚的卷曲(来自步骤 15 – 16),并将它们加载到每个 SDS-PAGE 凝胶泳道中。
19.通过免疫印迹分析检查成熟和解聚卷曲的纯度(图 6A 和 6C )。


 


图 6. 通过免疫印迹分析检测到的不同形式的 curli 的比较。
(A 和 B) 样品通过 SDS-PAGE 进行解析,SDS-PAGE 由 5% 丙烯酰胺浓缩胶和 12% 丙烯酰胺分离胶组成。使用iBlot系统将蛋白质转移到硝酸纤维素膜上。对于所有免疫印迹,使用兔抗卷曲多克隆血清检测CsgA的单体 (M)、二聚体 (D) 和更高分子量的寡聚体,然后使用IRDye 680RD 山羊抗兔 IgG 并使用 Odyssey CLx成像系统 (Li -Cor 生物科学)。 (A) 在 SDS-PAGE 之前,用 90% FA 对 20 μg 和 10 μg 的纯化卷曲进行解聚。 (B)如前所述 (Miller et al. , 2020) ,在 SDS-PAGE 之前,用 90% FA 连续处理 3 次来自感染鼠伤寒沙门氏菌的小鼠的 50 mg 均质组织样本。 (C) 将 10 µg 纯化的全长 curli(成熟)、curli 中间体( Interm )和 FA 或 HFIP 处理的 curli 上样并直接在预制的Novex TM 4 – 12% Tris-甘氨酸迷你凝胶上分离。白星表示 25 kDa蛋白质标准。


B.Curli中间体的纯化


curli 中间体的纯化依赖于产生 ∆ msbB突变菌株(Nicastro等人,2019) 。
对人类淀粉样蛋白的研究证明,在淀粉样蛋白聚合的多步过程中会形成不同类型的中间结构。其中,可溶性人淀粉样蛋白单体首先形成寡聚体,然后聚合成原纤维结构,然后将它们交叉组装成稳定、成熟的原纤维。尽管肠杆菌科中卷曲纤维组装的过程已经得到充分研究,但直到最近 Nicastro等人才发现或研究了卷曲的中间寡聚结构。 (2019)。作者使用∆ msbB菌株了解 curli 的原纤维化动力学,它们的中间体,以及成熟的 curli 原纤维聚集体在细菌细胞外基质组装中的作用。沙门氏菌中的∆ msbB基因编码一种酶,该酶催化完成脂质 A 生物合成的两个二级酰化反应之一。它合成了一个全长的含 O 抗原的 LPS 分子,该分子仅缺乏预期的二级酰基链,并且在体外和体内条件下都不太能够诱导细胞因子和可诱导的一氧化氮合酶反应。他们发现细菌淀粉样蛋白的中间原纤维结构( Curli中间体)更具细胞毒性,细菌 DNA 的添加加速它们形成成熟的纤维结构,限制了细胞毒性作用。有关卷曲纤维化动力学及其稳定性的更详细信息可以在其他地方找到(Nicastro等人,2019 年)。我们使用 lambda red 重组酶敲除程序( Datsenko和Wanner ,2000)来生成S。鼠伤寒 14028s ∆ msbB 。我们第一次尝试产生ΔmsbB菌株是不成功的,因为我们很难在 LB 琼脂上恢复抗生素抗性转化体。据报道,沙门氏菌msbB突变菌株在 LB 琼脂上生长不良,通过改用 MSB 琼脂(不含 NaCl 并添加 Mg 2+和 Ca 2+的 LB) ,生长可以恢复到接近野生型水平(Murray等人,2001)。一旦将程序更改为在 MSB 琼脂而不是 LB 琼脂上回收 CM R (耐氯霉素)转化体,λ red 程序就有效地工作了。
1.生长菌株S 。 Typhimurium 14028s Δ msbB在 5 mL 含 34 µg/mL 氯霉素的 MSB 肉汤中,在 37°C 下以 200 rpm 振摇过夜。
2.将 5 mL 过夜培养物添加到 500 mL 的 YESCA 肉汤中,并在 1-L 烧瓶中添加 4% DMSO,并在 26°C 下孵育 72 小时,并以 200 rpm 的速度摇动。
3.孵育后,以 10,000 × g离心10 分钟收集细菌沉淀。
4.将颗粒重悬于 30 mL 的 10 mM Tris-HCl (pH 8.0) 中,并用 RNase A(0.1 mg/mL 最终浓度)、DNase I(0.1 mg/mL 最终浓度)和 MgCl 2 (1 mM 最终浓度)在 37°C 下保持 30 分钟。
5.× 30 s 的脉冲以 30% 的幅度打开细菌,脉冲之间在冰上冷却 1 分钟。
6.向混合物中加入溶菌酶至终浓度为 1 mg/mL,并在 37°C 下孵育 40 分钟。
7.向混合物中添加 SDS 至终浓度为 1%,并在 37°C 下孵育 20 分钟,同时以 200 rpm 的速度摇动。
8.孵育后,离心沉淀 curli(10,000 × g ,4°C 10 分钟)。
9.将 curli 颗粒重新悬浮在 10 mL 的 10 mM Tris-HCl (pH 8.0) 中并煮沸 10 分钟。
10.2 (1 mM )之前,在冰上冷却样品 最终浓度)。将该混合物在 37°C 下孵育 2 小时。重复此步骤,直到达到所需的粘度(参见 A 部分的步骤 13 和 14)。
11.将混合物在 10,000 × g和 4°C 下离心 10 分钟。
12.用 10 mL 10 mM Tris-HCl (pH 8.0) 洗涤颗粒 3 次,并重新悬浮在 3 mL 的 2 × SDS-PAGE 样品缓冲液中并煮沸 10 分钟。
13.将 3 mL 的粗蛋白样品(来自上一步)加载到 4 mm 厚的 SDS-PAGE 凝胶中,该凝胶具有 5% 的浓缩凝胶和 12% 的分离凝胶,并在 100 V 下连续运行,直到样品中的所有染料都通过底部。
14.电泳后,使用 5 mL 注射器和 18 号针头从凝胶顶部收集 curli 聚集体,重悬于 5 – 10 mL 无菌水中,离心沉淀(10,000 x g × g 10 分钟)并洗涤用无菌水两次。
15.x g离心10分钟,然后用乙醇再洗涤两次。
16.将卷曲重新悬浮在无菌水中,并将混合物转移到预先称重的玻璃小瓶中。
17.在 -80°C 下冷冻混合物并冻干 24 小时。
18.冻干后,称量装有样品的玻璃小瓶以获得粗略的样品重量,并将冻干材料储存在-20°C。 curli 中间体的纯度可以通过免疫印迹分析来检查(图 6C ) 。


C.宿主组织/样本中的卷曲检测


小鼠组织分析程序改编自Miller等人。 (2020 年)。相同的程序可用于分析其他动物物种的组织或其他类型的样本。
1.将组织样本(即肝脏、盲肠、结肠和小肠)放入锡纸包中,在液氮中快速冷冻,并在 -80°C 下储存。
2.通过储存在 -20°C 下预冷研钵和研杵。在液氮下将冷冻组织样品研磨成细粉。可以使用金属刮刀将粉末样品从研钵和研杵上刮到称重纸上,然后转移到 2 mL 螺旋盖冷冻小瓶中,并在 -80°C 下储存。
3.称取 50 mg 粉末组织样品,转移到 Eppendorf 管中,重悬于 500 μL的 1 × SDS-PAGE 样品缓冲液中,煮沸 10 分钟。此步骤将溶解大部分可溶性细胞蛋白。
4.在冰上冷却溶液,并以 25,000 × g离心5 分钟使细胞碎片沉淀。
5.500 重悬沉淀 μL无菌蒸馏水并以 25,000 × g离心5 分钟。重复此步骤两次。这会洗掉 SDS-PAGE 样品缓冲液的任何剩余残留物。
6.用 500 μL的 90% FA 重悬沉淀,并在 -80°C 下冷冻样品至少 1 小时。
7.将混合物冻干 16-24小时。重复步骤 6 和 7 三次。对于纯化的 curli 标准品,FA 处理只需进行一次。
注意:含有酸的样品可能会损坏某些冻干机。
8.FA 处理后,将冻干样品重新悬浮在 50 μL的 1 × SDS-PAGE 样品缓冲液中,并在 25,000 × g下将样品离心2 分钟。
注意:不要煮沸样品,因为它会导致 curli 单体聚集。
9.将 20 μL等分的上清液装入每个 SDS-PAGE 凝胶泳道。我们使用了自行制备的 5% 丙烯酰胺浓缩胶和 12% 丙烯酰胺分离胶或市售的 4 – 12% 丙烯酰胺梯度胶。加载至少一个带有商业预染蛋白梯的凝胶泳道。
10.在 110 V 下进行电泳。小心取出凝胶并将其放在硝酸纤维素膜上,并在 Trans-Blot SD 半干转移细胞中以 25V 进行 40 分钟的蛋白质转移。或者,可以使用自动化iBlot系统将蛋白质转移到硝酸纤维素膜上。
11.使用任何标准免疫印迹程序检测组织样本中存在的 curli 蛋白。我们通常使用兔抗卷曲多克隆血清作为一抗,在 TBST 的 5% 脱脂牛奶中按 1:500 稀释,并在 4 °C下孵育过夜或在 37 °C下孵育1 小时。使用的二抗是 1:10,000 稀释的IRDye 680RD 山羊抗兔 IgG(用于荧光应用)或山羊抗兔 IgG 碱性磷酸酶偶联物(在 TBST 中 5% 脱脂牛奶中以 1:2,000 稀释)。在室温下在黑暗中孵育 1 小时 30 分钟用于荧光检测,或在 37°C 下在倾斜的平台摇床上孵育 1 小时用于比色检测。
12.抗体结合通过 Odyssey CLX 成像系统和 Image Studio 4.0 软件包使用荧光进行可视化(图 6B )。对于 curli 的比色检测,可溶性 BCIP/NBT 用作底物。碱性磷酸酶会产生稳定的蓝紫色产品,遇光不会褪色,无需特殊设备即可观察。


D.从卷曲中提取 DNA


Tükel实验室以前的工作表明,在成熟生物膜的发育过程中,细胞外 DNA (eDNA) 与卷曲原纤维结合,纯化的卷曲含有 eDNA (Gallo et al. , 2015)。由于 eDNA 的存在会影响免疫识别以及 TLR2/9 刺激( Tursi和Tükel ,2018 年),因此每次纯化批次时确定有多少 eDNA 与纯化的 curli 相关联是一种很好的做法。
1.μL的 TE 缓冲液、30 μL的 10% SDS 和 70 μL的蛋白酶 K(20 mg/mL)中重新悬浮 500 μg的纯化 curli 。
2.充分混合并在 37°C 下孵育 1 小时。
3.添加 100 μL的 5 M NaCl 并通过移液器混合。
4.添加 80 μL的 CTAB/NaCl 溶液,并通过使用宽孔吸头移液彻底混合。
5.在 37°C 下孵育 10 分钟。
6.加入 300 – 400 μL苯酚/氯仿/异戊醇,倒置试管进行混合
7.以 15,682 × g离心 和4°C 5分钟。
8.将上相转移到新的 Eppendorf 管中。
9.加入 700 μL氯仿并通过移液器混合。
10.以 15,682 × g离心 和4°C 5分钟。
11.将上相转移到新的 Eppendorf 管中,加入等体积的异丙醇并用手摇动管子。
12.-20°C 孵育 30 分钟。
13.在 13,362 × g和 4°C 下离心 5 分钟。
14.× g下离心 5 分钟。
15.将最终的 DNA 沉淀重悬于 30 μL TE缓冲液或水中。
16.使用DeNovix DS-11 FX分光光度计/荧光计测量 DNA 含量。




数据分析


为了从动物组织中检测细菌产生的卷曲,建议从每种组织类型中检测至少两个 50 mg 样本,并从感染组中筛选所有个体小鼠。在我们之前的工作中,我们没有在每只感染沙门氏菌的小鼠中检测到卷曲的存在(米勒等人,2020 年)。 我们不确定这是因为 curli 的随机表达(即,它不是在每只小鼠中产生)还是由于检测的技术困难。出于这个原因,作者建议对一组中的每只动物进行测试。




食谱


1.MSB 肉汤 (1,000 毫升)
胰蛋白胨 10 克
酵母提取物 2.5 克
1 mL 的 1 M CaCl 2
1 mL 1 M MgSO 4
在标准液体循环(15 psi 下 20 分钟)上对溶液进行高压灭菌。


2.YESCA 肉汤(1,000 毫升)
酪蛋白氨基酸 10 克
酵母提取物 1 克
在标准液体循环(15 psi 下 20 分钟)上对溶液进行高压灭菌,并添加 DMSO(最终浓度 4%)。


3.T 琼脂 (1,000 毫升)
胰蛋白胨 10 克
琼脂 15 克
在标准液体循环(15 psi 下 20 分钟)上对溶液进行高压灭菌。


4.2 × SDS-PAGE 样品缓冲液 (100 mL)
Tris (1 M, pH 6.8) 8 毫升
SDS (20 %) 10 毫升
甘油 10 毫升
溴酚蓝 (0.1 %) 600 微升
β-巯基乙醇(新鲜添加)4 mL
蒸馏水 67.4 毫升


5.10 × SDS-PAGE 运行缓冲液 (1,000 mL)
Tris碱 30.3 克
甘氨酸 144.4 克
SDS 10 克


6.10 × Towbin缓冲液或传输缓冲液 (1,000 mL)
Tris碱 30.3 克
甘氨酸 144.4 克
通过添加 100 mL 的 10 ×转移缓冲液和 200 mL 的甲醇并加入 700 mL 的蒸馏水来制备 1 ×转移缓冲液。


7.10 × TBS (1,000 毫升)
Tris基地 24 克
氯化钠 88 克
将组件溶解在 900 mL 的蒸馏水中。用 HCl 将 pH 值调节至 7.6,并用蒸馏水调至最终体积。通过添加 100 mL 的 10 × TBS 和 900 mL 的蒸馏水来制备 1 × TBS。


8.1 × TBST
将 0.5 mL 的 Tween 20 添加到 1,000 mL 的 1 × TBS。


9.12% 分离胶(2 块胶)
蒸馏水 82.56 毫升
1.5 M Tris-HCl pH 8.8 48 mL
丙烯酰胺/双 (40%) 57.6 毫升
10% SDS 1.92 毫升
10% APS 1.92 毫升
96微升_


10.5% 浓缩胶(2 块凝胶)
蒸馏水 23.84 毫升
0.5 M Tris-HCl pH 6.8 9.6 mL
丙烯酰胺/双 (40%) 4.5 毫升
10% SDS 400 μL 
10% APS 480微升
40微升


11.AP 缓冲液(500 毫升)
100 mM Tris 6.06 克
100 毫米氯化钠 2.92 克
5 mM MgCl 2 · 6H 2 O 0.51 g
将组件溶解在 450 mL 的蒸馏水中。使用 3 M HCl 将 pH 值调节至 9.5,并使用蒸馏水和高压灭菌器将其调至 500 mL。


12.NBT/BCIP开发方案
AP 缓冲液 10 毫升
NBT 66微升 
BCIP 33微升




致谢


这项研究得到了自然科学和工程研究委员会 (NSERC) 发现补助金(Grant #2017-05737)、 Jarislowsky生物技术主席以及萨斯喀彻温大学授予 APW CT 的医学院研究奖的支持,并得到了 NIH 的支持授予 AI153325、AI151893 和 AI148770。 EGH,得到了 NSERC 本科生研究奖的支持。我们感谢 VIDO 的 Neil Rawlyk提供一般技术援助。资助者在研究设计、数据收集和分析、发表决定或手稿准备方面没有任何作用。我们最近的出版物(Miller et al ., 2020)中使用了成熟的 curli 纯化方案和来自动物组织的 curli 免疫检测。用于纯化 curli 中间体和 DNA 提取的方案发表在 Nicastro等人。 (2019)。作为 VIDO 手稿系列第 968 号出版。




利益争夺


作者没有可竞争的利益报告。




伦理


根据加拿大动物护理委员会的指导方针和萨斯喀彻温大学动物护理和供应委员会的规定,按照萨斯喀彻温大学批准的动物使用协议 #20110057 或 20170080 照顾和使用小鼠动物研究伦理委员会。




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引用:Sivaranjani, M., Hansen, E. G., Perera, S. R., Flores, P. A., Tukel, C. and White, A. P. (2022). Purification of the Bacterial Amyloid “Curli” from Salmonella enterica Serovar Typhimurium and Detection of Curli from Infected Host Tissues. Bio-protocol 12(10): e4419. DOI: 10.21769/BioProtoc.4419.
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