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May 2018
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Tracking the Subcellular Localization of Surface Proteins in Staphylococcus aureus by Immunofluorescence Microscopy
用免疫荧光显微镜追踪金黄色葡萄球菌表面蛋白的亚细胞定位   

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Abstract

Surface proteins of Staphylococcus aureus and other Gram-positive bacteria play essential roles in bacterial colonization and host-microbe interactions. Surface protein precursors containing a YSIRK/GXXS signal peptide are translocated across the septal membrane at mid-cell, anchored to the cell wall peptidoglycan at the cross-wall compartment, and presented on the new hemispheres of the daughter cells following cell division. After several generations of cell division, these surface proteins will eventually cover the entire cell surface. To understand how these proteins travel from the bacterial cytoplasm to the cell surface, we describe a series of immunofluorescence microscopy protocols designed to detect the stepwise subcellular localization of the surface protein precursors: surface display (protocol A), cross-wall localization (protocol B), and cytoplasmic/septal membrane localization (protocol C). Staphylococcal protein A (SpA) is the model protein used in this work. The protocols described here are readily adapted to study the localization of other surface proteins as well as other cytoplasmic or membrane proteins in S. aureus in general. Furthermore, the protocols can be modified and adapted for use in other Gram-positive bacteria.


Graphic abstract:



Tracking the subcellular localization of surface proteins in S. aureus


Keywords: Immunofluorescence microscopy (免疫荧光显微技术), Staphylococcus aureus (金黄色酿脓葡萄球菌), Surface proteins (表面蛋白), YSIRK/GXXS signal peptide (YSIRK / GXXS信号肽), Protein A (SpA) (蛋白A(SpA)), SecA (SecA), Surface display (表面显示), Cross-wall localization (穿壁定位), Septal localization (间隔定位)

Background

Staphylococcus aureus is a Gram-positive bacterium and an opportunistic pathogen. It frequently colonizes human nares and skin and is a leading cause of both hospital- and community-acquired infections (von Eiff et al., 2001; Tong et al., 2015). The cell envelope of S. aureus consists of a cytoplasmic membrane and a thick cell wall peptidoglycan layer. To replicate, S. aureus undergoes binary fission by forming a division septum at the mid-cell. The cell wall biosynthesis machinery is recruited to the septum during cell division (Pinho and Errington, 2003). New cell wall peptidoglycan is synthesized to form a cross-wall ring and eventually a cross-wall disc coupled with the invagination of the septal membrane (Zhou et al., 2015). Once the cross-wall disc is fully synthesized, specific cell wall hydrolases cleave at the outer edges of the cross-wall to split the two daughter cells (Oshida et al., 1995; Sugai et al., 1995; Yamada et al., 1996; Kajimura et al., 2005). Due to high internal turgor pressure, the two daughter cells separate from each other and the newly synthesized cross-wall discs become the new hemispheres of the daughter cells (Monteiro et al., 2015; Zhou et al., 2015).


Cell wall peptidoglycan-anchored surface proteins are key components of the Gram-positive bacterial cell envelope. Many of them perform virulence functions in S. aureus, such as adhesion, biofilm formation, nutrient acquisition, antibiotic resistance, and immune evasion (Foster et al., 2014; Schneewind and Missiakas, 2019). Many surface protein precursors contain a specific N-terminal signal peptide with a highly conserved YSIRK/GXXS motif (Rosenstein and Götz, 2000; Tettelin et al., 2001). The secretion, cell wall anchoring, and surface display of YSIRK/GXXS proteins are tightly coupled with the bacterial cell cycle (Carlsson et al., 2006; Raz et al., 2012; Yu et al., 2018). In the early stages, the YSIRK/GXXS signal peptide promotes localized protein secretion at the division septum (Carlsson et al., 2006; DeDent et al., 2008). Subsequently, septal secreted surface proteins are covalently anchored to the cross-wall peptidoglycan by sortase A (Mazmanian et al., 1999). Upon cell division and separation, cross-wall-anchored surface proteins are displayed on the surface of the new hemisphere of the daughter cells (Cole and Hahn, 1962; Swanson et al., 1969; Raz et al., 2012; Yu et al., 2018). Eventually, surface proteins are displayed on the entire cell surface after several generations of cell division (DeDent et al., 2008; Raz et al., 2012; Yu et al., 2018).


Proper imaging methods are essential in revealing the subcellular localization of proteins. Here, we describe a series of protocols to track the subcellular localization of surface proteins. While it is straightforward to localize proteins on bacterial cell surface (protocol A, Figure 1), a “pulse-chase” type of method is used to reveal the localization of newly anchored surface proteins. In their classical paper, Cole and Hahn (1962) described an immunofluorescence staining method in which streptococcal cells were incubated with fluorescently labeled surface protein M antibody and subsequently with non-fluorescent antibody. In another classical study, streptococci were trypsin-treated to digest the existing M protein on the bacterial surface; new surface-deposited M protein was observed after re-incubating the bacteria in fresh medium without trypsin (Swanson et al., 1969). The method of trypsinization followed by regeneration has subsequently been used to localize newly anchored surface proteins on the cell surface of both streptococci and staphylococci (Carlsson et al., 2006; DeDent et al., 2008; Raz et al., 2012; Yu et al., 2018). Here, we provide a detailed description of the protocol that is specifically tailored to S. aureus (protocol B, Figure 1). The model protein we use is protein A (SpA), one of the major staphylococcal surface proteins that binds to host immunoglobulin and disrupts host immune responses (Forsgren and Sjöquist, 1966).


We have previously shown that SpA engages the SecA-mediated secretion pathway for translocation across the cytoplasmic membrane (Yu et al., 2018). To reveal where SpA precursors accumulate in the cytoplasm upon secA depletion, we developed a protocol to detect the localization of intracellular proteins (protocol C, Figure 1) based on methods described earlier by Harry et al., (1995) and Pinho and Errington (2003). In this protocol, staphylococcal cells are fixed with paraformaldehyde and glutaraldehyde, which adhere to the poly-L-lysine-coated glass slide. Cells are digested on the slide with a robust staphylococcal cell wall hydrolase, lysostaphin, to generate protoplasts (Schindler and Schuhardt, 1964). The protoplasts are fixed and permeabilized with methanol and acetone, respectively, and subsequently subjected to immunofluorescence staining. Depending on the genetic background of different strains, protocol C can be used to localize membrane-bound or cytoplasmic-localized surface protein precursors. Furthermore, protocol C is not restricted to surface proteins; it can be used to localize cytoplasmic or membrane proteins in S. aureus in general. The protocols described here can also be adapted for use in other Gram-positive bacteria.



Figure 1. Schematic overview of the protocols described in this work

Materials and Reagents

  1. 8-well glass slides (MP Biomedicals/Thermo Fisher Scientific, catalog number: 096040805E)

  2. Disposable borosilicate glass tubes, 16 mm diameter, 125 mm length (Thermo Fisher Scientific, catalog number: 1496130)

  3. 1,250 µl XL graduated tips (USA Scientific, catalog number: 1112-1720)

  4. 200 µl graduated tips (USA Scientific, catalog number: 1110-1700)

  5. 10 µl graduated tips (USA Scientific, catalog number: 1111-3700)

  6. 10 ml serological pipets (Thermo Fisher Scientific, catalog number: 1367811E)

  7. 25 ml serological pipets (Thermo Fisher Scientific, catalog number: 13-678-11)

  8. 1.5 ml microcentrifuge tubes (Thermo Fisher Scientific, catalog number: 05-408-129)

  9. 2.0 ml microcentrifuge tubes (Thermo Fisher Scientific, catalog number: 05-408-138)

  10. Scienceware microcentrifuge tube rack (Thermo Fisher Scientific, catalog number: 10029259)

  11. 1.5 ml microcentrifuge tubes (Thermo Fisher Scientific, catalog number: 05-408-129)

  12. 2.0 ml microcentrifuge tubes (Thermo Fisher Scientific, catalog number: 05-408-138)

  13. Nunc 15 ml conical sterile tubes (Thermo Fisher Scientific, catalog number: 12565269)

  14. Nunc 50 ml conical sterile tubes (Thermo Fisher Scientific, catalog number: 12565271)

  15. Corning PES syringe filters (Thermo Fisher Scientific, catalog number: 09-754-29)

  16. 20 ml filter syringes (Thermo Fisher Scientific, catalog number: 14-955-460)

  17. Round Petri dishes (100 × 15 mm) (Thermo Fisher Scientific, catalog number: FB0875712)

  18. Transfer pipettes (Thermo Fisher Scientific, catalog number: 13-711-7M)

  19. Kimberly-Clark ProfessionalTM KimwipesTM Delicate Task Wipers (Thermo Fisher Scientific, catalog number: 06-666A)

  20. BD BactoTM Tryptic Soy Broth (TSB) (Thermo Fisher Scientific, catalog number: DF0370-07-5)

  21. BD Tryptic Soy Agar (TSA) (Thermo Fisher Scientific, catalog number: DF0369-07-8)

  22. Sodium chloride (NaCl) (Thermo Fisher Scientific, catalog number: S271-1)

  23. Hydrochloric acid (HCl) (Thermo Fisher Chemical, catalog number: 187066)

  24. Potassium chloride (KCl) (Thermo Fisher Scientific, catalog number: AM9640G)

  25. Potassium phosphate dibasic (K2HPO4) (Thermo Fisher Scientific, catalog number: BP363-500)

  26. Sodium phosphate dibasic, anhydrous (Na2HPO4) (Thermo Fisher Scientific, catalog number: BP332-1)

  27. Ethylenediamine tetraacetic acid, EDTA (Thermo Fisher Scientific, catalog number: BP118-500)

  28. Tris base (Thermo Fisher Scientific, catalog number: BP152-5)

  29. D-(+)-glucose (Sigma-Aldrich, catalog number: G8270-1KG)

  30. Acetone (Thermo Fisher Scientific, catalog number: A929-4)

  31. Methanol (Thermo Fisher Scientific, catalog number: A454-4)

  32. Ethanol (Thermo Fisher Scientific, catalog number: A405P-4)

  33. Bovine Serum Albumin (BSA) (Thermo Fisher Scientific Bioreagents, catalog number: BP1600-100)

  34. 0.1% poly-L-lysine solution (Sigma-Aldrich, catalog number: P8920-100ML)

  35. Paraformaldehyde (PFA) 4% in PBS (Thermo Fisher Scientific, catalog number: AAJ19943K2)

  36. Glutaraldehyde 50% in H2O (Sigma-Aldrich, catalog number: 340855-25ML)

  37. Primary antibody: SpAKKAA antiserum (Kim et al., 2010). Store at 4°C

  38. Secondary antibodies:

    1. Goat anti-rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Thermo Fisher Scientific, catalog number: A-11034). Store at 4°C

    2. Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 (Thermo Fisher Scientific, catalog number: A-21244). Store at 4°C

  39. Molecular ProbesTM SlowFadeTM Diamond Antifade Mountant (Invitrogen, catalog number: S36963)

  40. Nile Red (Sigma-Aldrich, catalog number: 19123-10MG)

  41. Hoechst 33342 DNA dye, 10 mg/ml (Thermo Fisher Scientific, catalog number: H3570), store at 4°C.

  42. BODIPYTM Vancomycin-FL (Thermo Fisher Scientific, catalog number: V34850)

  43. CorningTM Rectangular Cover Glasses No.1 (22 × 50 mm) (Thermo Fisher Scientific, catalog number: 12-553-461) (see Note 1)

  44. Clear nail polish (cheapest one in any grocery store)

  45. Trypsin, from bovine pancreas (Sigma-Aldrich, catalog number: T1426)

  46. Trypsin inhibitor, from glycine max soybean (Sigma-Aldrich, catalog number: T9128)

  47. Lysostaphin (AMBI, catalog number: LSPN-50)

  48. Phosphate-buffered saline (PBS) (see Recipes)

  49. Fixation solution (see Recipes)

  50. GTE solution (see Recipes)

  51. Trypsin stock solution (see Recipes)

  52. Trypsin inhibitor stock solution (see Recipes)

  53. BSA blocking solution (see Recipes)

  54. Lysostaphin stock solution (see Recipes)

  55. Nile Red stock solution (see Recipes)

  56. BODIPYTM Vancomycin-FL stock solution (see Recipes)

Equipment

  1. Eppendorf pipettes 100–1,000 µl, 20–200 µl, 2–20 µl, 0.1–2.5 µl (Eppendorf, catalog number: 2231000714)

  2. Eppendorf Easypet®3 (Eppendorf, catalog number: 4430000018)

  3. FisherbrandTM TraceableTM Multi-Colored Timer (Thermo Fisher Scientific, catalog number: 02-261-840)

  4. Forceps (MilliporeSigmaTM Filter Forceps/Thermo Fisher Scientific, catalog number: XX6200006P)

  5. In-house vacuum

  6. Shaker (Eppendorf New BrunswickTM Innova® 42 shaker, catalog number: EPM1335-0010)

  7. Table centrifuge (Eppendorf, model: Centrifuge 5425, catalog number EP5405000441)

  8. Spectrophotometer (Thermo Scientific Genesys GENESYSTM 30 Visible Light Spectrophotometer, catalog number: 14-380-442)

  9. Mini-tube rotator (FisherbrandTM Mini Tube Rotator, catalog number: 88-861-051)

  10. Incubator microbiological (Fisher Scientific, catalog number: 51030513)

  11. -20°C freezer

  12. 4°C refrigerator

  13. LP Vortex Mixer (Thermo Fisher Scientific, catalog number: 88880017)

  14. Leica SP5 2-photon Laser Scanning Confocal microscope (Leica Microsystems, product name: Leica TCS SP5 Confocal)

Software

  1. Image J (Rasband W.S./U. S. NIH, Bethesda, Maryland, USA, https://imagej.nih.gov/ij/)

  2. Leica microscope software LAS_AF Leica (Leica microscopes)

  3. Prism GraphPad Software for statistical analysis (https://www.graphpad.com/scientific-software/prism/)

  4. Adobe Illustrator to assemble figures

Procedure

  1. Slide preparation

    1. Add 50 µl 0.1% poly-L-lysine to each well of an 8-well glass slide and allow to sit for 5 min at room temperature (see Note 2).

    2. Briefly rinse the wells with ddH2O, remove the excess liquid using a vacuum, and allow the slide to air dry completely (see Notes 3 and 4).


  2. Bacterial cultures

    1. Prepare an overnight culture: inoculate one single colony from a streaked agar plate to 3 ml TSB in a glass test tube; add the appropriate antibiotics, if needed.

    2. Grow the overnight culture at 37°C with rigorous shaking at 220 rpm.

    3. The next morning, inoculate 30 µl overnight culture to 3 ml fresh TSB (1:100 dilution).

    4. Grow the cultures at 37°C with rigorous shaking at 220 rpm.

    5. Measure the optical density OD600 of the culture every hour in a spectrophotometer.

    6. Place the cultures on ice when OD600 reaches 0.8–1.0 (see Note 5).

    7. Continue the sample preparation in Part C below according to the different protocols (A, B, or C).


  3. Sample preparation

    Protocol A – Surface display:

    1. Transfer 2 ml bacterial culture into a 2 ml microcentrifugation tube.

    2. Spin at 18,000 x g for 3 min in a tabletop centrifuge to pellet the bacterial cells.

    3. Remove the supernatant without disturbing the pellet.

    4. Resuspend the pellet in 1 ml PBS and vortex well.

    5. Spin at 18,000 × g for 3 min and remove the supernatant (steps 4–5 are “wash with PBS” steps).

    6. Resuspend the pellet in 1 ml PBS, vortex thoroughly; mix 250 µl bacterial suspension with 250 µl fixation solution (see Recipes) in a clean 1.5 ml microcentrifuge tube, briefly vortex to mix, and incubate for 20 min at room temperature (fixation step).

    7. Wash twice with PBS, as described in steps 4–5 (see Note 6).

    8. Resuspend the pellet in 150 µl PBS, vortex thoroughly (see Note 7).

    9. Add 50 µl bacterial suspension to poly-L-lysine-coated glass slides and allow to sit for 5 min.

    10. Remove the excess liquid (non-adherent cells) using a vacuum.

    11. Add one drop of PBS to each well using a plastic disposable transfer pipette and remove using a vacuum (this is the “drop and remove on-slide wash” step).

    12. Repeat the drop and remove on-slide wash step.

    13. Continue with immunofluorescence in Part D.


    Protocol B – Cross-wall localization:
    1. Transfer 2 ml bacterial culture into a 2 ml microcentrifugation tube.

    2. Spin at 18,000 × g for 3 min in a tabletop centrifuge to pellet the bacterial cells.

    3. Remove the supernatant without disturbing the pellet.

    4. Wash once with PBS.

    5. Resuspend the pellet in 900 µl PBS, vortex thoroughly; add 100 µl 5 mg/ml trypsin stock solution (see Recipes) and briefly vortex (trypsin final concentration: 0.5 mg/ml).

    6. Incubate the tubes in a Mini-tube rotator at 37°C for 1 h at a rotation speed of 16 (medium speed).

    7. Wash twice with PBS.

    8. Resuspend the pellet in 900 µl fresh TSB, vortex thoroughly; add 100 µl 10 mg/ml soybean trypsin inhibitor stock solution (see Recipes) and briefly vortex to mix (final concentration of soybean trypsin inhibitor: 1 mg/ml).

    9. Incubate the tubes in a Mini-tube rotator at 37°C for exactly 20 min at a rotation speed of 16 (see Note 8).

    10. Add 250 µl fixation solution to a clean 1.5 ml microcentrifuge tube during the 20 min incubation.

    11. At the 20-min timepoint, quickly transfer 250 µl bacterial sample to the microcentrifuge tubes prepared in the previous step.

    12. Vortex to mix and allow the sample to sit at room temperature for 20 min.

    13. Wash twice with PBS.

    14. Resuspend the pellet in 150 µl PBS and vortex thoroughly (adjust the volume depending on the pellet size).

    15. Add 50 µl bacterial suspension to a glass slide coated with poly-L-lysine and allow to sit for 5 min.

    16. Remove the liquid (non-adherent cells) using a vacuum.

    17. Perform the drop and remove on-slide wash twice for each well as described above.

    18. Continue with immunofluorescence in Part D.


    Protocol C – Cytoplasmic/septal membrane localization:

    1. Place two 50 ml tubes containing approximately 25 ml methanol and 25 ml acetone, respectively, into a -20°C freezer.

    2. Normalize all the bacterial cultures to OD600 = 1.

    3. Transfer 2 ml normalized bacterial culture to a 2 ml microcentrifuge tube (this step is to have same cell numbers for the following enzymatic digestion step).

    4. Spin at 18,000 × g for 3 min in a tabletop centrifuge to pellet the bacterial cells.

    5. Remove the supernatant without disturbing the pellet.

    6. Wash once with PBS.

    7. Resuspend the pellet in 900 µl PBS and vortex thoroughly; add 100 µl 5 mg/ml trypsin stock solution and briefly vortex (see Note 9).

    8. Incubate in a Mini-tube rotator at 37°C for 1 h at a rotation speed of 16.

    9. Wash twice with PBS.

    10. Resuspend the pellet in 500 µl PBS, vortex thoroughly; add 500 µl fixation solution and vortex to mix.

    11. Incubate the sample for 15 min at room temperature and then on ice for 15 min to fix.

    12. Wash three times with PBS.

    13. Resuspend the pellet in 1 ml freshly made GTE buffer (see Recipes) and vortex thoroughly (see Note 10).

    14. Add 50 µl cell suspension to poly-L-lysine-coated glass slides (this is the control without lysostaphin digestion) (see Note 11).

    15. Prepare a timer, add 10 µl lysostaphin working solution (see Recipes) to the rest of the cell suspension; quickly vortex and immediately add 50 µl to the glass slides (see Note 12).

    16. Incubate for 2 min on the slide (see Note 13).

    17. Remove the liquid using a vacuum until completely dry.

    18. Immediately place the slide into prechilled methanol at -20°C for 5 min.

    19. Take out the slide using forceps and place into prechilled acetone at -20°C for 30 s (see Note 14).

    20. Take out the slide using forceps and allow to air-dry completely.

    21. Once the slide is dry, apply 50 µl PBS to the sample well to rehydrate.

    22. Perform the drop and remove on-slide wash twice for each well.

    23. Continue with immunofluorescence in Part D.


  4. Immunofluorescence

    1. Remove PBS, add BSA blocking solution (see Recipes), and incubate for 30 min at room temperature.

    2. Remove the blocking solution, add 50 µl primary antibody solution (rabbit serum SpAKKAA 1:4,000 dilution in BSA blocking solution), and incubate overnight at 4°C or at room temperature for 1 h (see Note 15).

    3. Remove the unbound primary antibody solution and wash 8 times with PBS with the last wash step for 5 min.

    4. Remove the washing solution, add 50 µl secondary antibody diluted in BSA blocking solution (e.g., Alexa Fluor 647-IgG or Alexa Fluor 488-IgG, 1:500 dilution), and incubate in the dark for 1 hour at room temperature (see Note 16).

    5. Perform the on-slide wash 10 times with PBS.

    6. Take a clean 1.5 ml microcentrifuge tube, add 1 ml PBS, 5 µl Hoechest stock solution (1:200 dilution), 2 µl Van-FL stock solution (1:500 dilution), or 5 µl Nile Red stock solution (1:200 dilution) (see Recipes) and mix well; add 50 µl staining solution to each well.

    7. Incubate in the dark for 10 min at room temperature.

    8. Perform the on-slide wash three times with PBS.

    9. After the last wash, remove all the excess liquid from the well.

    10. Add a 5-μl drop of Slow Fade Diamond Antifade Mountant at 3 different places between the sample wells (see Note 17).

    11. Brush a thin layer of nail polish around the edges of the slide and seal with a cover slip; gently press the cover slip and use a Kim wipes to remove the excess antifade solution around the edges (see Note 18).

    12. Image the samples using a microscope with the appropriate fluorescent channels (Part E).

    13. The prepared slides can be stored at 4°C for a few days and at -20°C for a longer period; however, immediate imaging is recommended.


  5. Imaging

    1. The samples prepared above are suitable to be imaged by different imaging systems, including epi-fluorescence microscopy, confocal microscopy, or deconvolution microscopy. However, to reveal bacterial cellular features and define protein localization in tiny bacterial cells, a microscope with high resolution is recommended. A 60× or 100× objective lens with a higher numerical aperture is needed. We used a Leica DM 2000 coupled with a sensitive CCD camera, a Leica SP5 2-photon Laser Scanning Confocal microscope, and a Leica SP8 3X STED Laser Confocal Microscope. All showed good imaging results.

    2. Representative images of protocols A, B, and C are displayed in Figure 2. The images were captured using a Leica SP5 2-photon Laser Scanning Confocal microscope.



      Figure 2. Representative images from (A) protocol A, showing surface display of SpA; (B) protocol B, showing cross-wall localization of SpA; and (C) protocol C, showing septal localization of SpA in the presence of SecA and cytoplasmic localization of SpA precursors in the absence of SecA. Van-FL: BODIPYTM Vancomycin-FL cell wall staining; BF: brightfield images; Nile Red: membrane staining; scale bar: 2 µm in panels A and B and 1 µm in panel C. Images are adapted from Yu et al. (2018).

Data analysis

  1. Take at least three images with random views for each sample in each experiment. One has to take more random images, especially when there are only a few cells on the slides or when there are different phenotypes on one slide.

  2. Perform the experiment independently at least three times.

  3. To quantitate the percentage of SpA cross-wall localization, open images from protocol B in ImageJ, split the channels, and enlarge the images to allow better visualization.

  4. Open the “cell counter” tool in ImageJ.

    Select cell type 1, manually count pairs of diplococci in Van-FL-stained images, and record the number. Count at least 50 pairs of diplococci per image. Diplococci are defined as two connecting daughter cells that have just been split but not yet separated (see sample images in Figure 3) (see Note 19).

  5. Select cell type 2, manually count cross-wall localized SpA signals in the merged images, and record the number. Cross-wall localized SpA signals are defined as clear lines at the cross-wall. To be rigorous, dots are not counted.

  6. Calculate the average of three images per experiment.

  7. Input the average values of three independent experiments to GraphPad Prism.

  8. In GraphPad Prism, use a t-test to statistically analyze significant differences between two groups; use one-way ANOVA for multiple group comparisons; and use Tukey’s multiple comparison test to analyze differences among multiple groups.



    Figure 3. Sample images with the cell counting window, demonstrating how to quantitate the percentage of SpA cross-wall localization . Images are adapted from Yu et al. (2018).

Notes

  1. The choice of thickness of the cover slip depends on the imaging system.

  2. A 1:10 dilution of poly-L-lysine to 0.01% also works.

  3. It takes about 15 min to air-dry the poly-L-lysine-coated slides. One can also use a vacuum to dry the slides.

  4. To assemble the vacuum system, connect an in-house vacuum to a tube, cut the extremity off a 200 µl pipette tip, and insert the tip into the tube. It is important not to touch the samples on the glass slides during drying.

  5. It usually takes about 2–3 h for S. aureus to reach an OD600 of 0.8–1.0 under standard lab culture conditions.

  6. Cells tend to clump after fixation; a longer vortex may be needed.

  7. The volume can be adjusted according to the pellet size; if unsure, one can add less PBS and make dilutions. The key point here is to have a proper number of cells on the slide so that most of the cells are well separated. Too many cells will lead to bacterial clumping and cause artifacts in immunostaining; too few cells will not provide reliable results.

  8. The time of re-generation was determined experimentally in our protocol. It should be tested and optimized depending on different growth conditions and antigens.

  9. This step is not critical for protocol C, as lysostaphin digestion will remove most of the cell wall as well as the existing SpA. We include this step in our protocol to minimize any potential background caused by existing SpA.

  10. GTE buffer is an osmotic stabilizing buffer. Lysostaphin is a zinc-dependent endopeptidase (Sabala et al., 2014). Although EDTA in the GTE buffer can chelate zinc, it does not have any obvious negative effects in our experiments. We have tried other osmotic stabilizing buffers without EDTA, such as TSM [50 mM Tris-HCl (pH 7.5), 0.5 M sucrose, 10 mM MgCl2], which also works.

  11. It is important to have this control. Staphylococcal cells after lysostaphin digestion will become more translucent in brightfield images, whereas undigested cells have a dark cell contour. Moreover, the two closely attached daughter cells will separate after lysostaphin digestion (see Figure 2C).

  12. Other cell wall hydrolases can substitute lysostaphin if this protocol is to be adapted for another bacterium. Lysozyme, for example, has been used in Bacillus subtilis (Harry et al., 1995). Most S. aureus strains are lysozyme-resistant, which limits its use in S. aureus. The digestion time and buffer will have to be adjusted experimentally for a different enzyme or bacterium.

  13. It is critical to perform on-slide digestion to stabilize the protoplasts.

  14. This step stabilizes the protoplast after lysostaphin digestion and permeabilizes the cytoplasmic membrane. Depending on the bacterial strain and abundance of antigens, Triton X-100 can be used to further permeabilize the cytoplasmic membrane.

  15. For any new antigen, serial dilution of primary antibody is necessary to determine the optimal concentration. A negative control that does not express the antigen is an essential control. If there is no mutant available, one should include at least a control without primary antibody. Minimal background signals should be seen in the negative control.

  16. Depending on the microscope system, one can choose different fluorescent-labeled secondary antibodies. We consistently use the secondary antibody at a 1:500 dilution.

  17. Different kinds of antifade solution are commercially available. One should choose the antifade solution compatible with the imaging system.

  18. Make sure that the antifade solution covers every well as a very thin layer without bubbles, and that the cover slip is leveled on the slide.

  19. The reason for counting diplococci is because cross-wall localized SpA signals can only be detected at the cross-wall of these diplococci under our experimental conditions; however, as defining diplococci may be subjective, it can introduce bias. Thus, one can count total cell numbers instead of diplococci.

Recipes

  1. Phosphate-buffered saline (PBS)

    137 mM NaCl

    2.7 mM KCl

    10 mM Na2HPO4

    1.8 mM K2HPO4

    pH 7.4

    1. Dissolve 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, and 0.24 g K2HPO4 in 1 L ddH2O

    2. Adjust the pH to 7.4 with HCl and autoclave at 121°C for 20 min

  2. Fixation solution

    2.4% paraformaldehyde and 0.01% glutaraldehyde in PBS

    Mix 30 ml 4% PFA and 10 µl 50% glutaraldehyde and add PBS to a 50-ml total volume. Store at 4°C (stable for at least two weeks).

  3. GTE solution

    50 mM glucose

    10 mM EDTA

    20 mM Tris-HCl pH 7.5

    Note: Make fresh and filter-sterilize before use.

    1. Make stock solutions of 0.5 M EDTA (pH 8) and 1 M Tris-HCl (pH 7.5)

    2. Add 0.9 g D-glucose, 2 ml 0.5 M EDTA, and 2 ml 1 M Tris-HCl to a final volume of 80 ml ddH2O

    3. Adjust the pH to 7.5 with HCl, add ddH2O to 100 ml, filter-sterilize, and store at 4°C

  4. Trypsin stock solution

    5 mg/ml trypsin in PBS, filter-sterilize, and store at -20°C

  5. Trypsin inhibitor stock solution

    10 mg/ml trypsin inhibitor in ddH2O, filter-sterilize, store at -20°C

  6. BSA blocking solution: 3% BSA in PBS

    Dissolve 0.3 g BSA powder in 10 ml PBS; make fresh and filter-sterilize before use, store at 4°C

  7. Lysostaphin stock solution

    1. Make a stock solution of 10 mg/ml in 20 mM sodium acetate (pH 4.5), store at -20°C

    2. Dilute with 200 mM Tris-HCl (pH 8) to 2 mg/ml as a working solution, store at 4°C

  8. Nile Red stock solution

    1. Dissolve in 100% ethanol to make a 0.5 mg/ml stock solution, store at -20°C

    2. Add 5 µl Nile Red stock solution to 1 ml PBS (1:200 dilution) to stain the samples

  9. BODIPYTM Vancomycin-FL stock solution

    1. Dissolve 100 µg in 100 µl DMSO to make a 1 µg/µl stock solution, store at -20°C

    2. Add 2 µl Van-FL stock solution to 1 ml PBS (1:500 dilution) to stain the samples

Acknowledgments

This work was supported by the start-up funds to W.Y. from the University of South Florida. We thank Olaf Schneewind and Dominique Missiakas for their mentorship during the initial development of the protocols. We thank Vytas Bindokas, Robert Hill, and Byeong Cha for their assistance with microscope facilities. We thank lab members for suggestions regarding the manuscript. This work reports the fluorescence microscopy methods used in our previous paper (Yu et al., 2018). Images from Yu et al. (2018) have been adapted in this report to demonstrate the methods.

Competing interests

The authors declare that there are no conflicts of interest or competing interests.

References

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简介

[摘要]金黄色葡萄球菌和其他革兰氏阳性细菌的表面蛋白在细菌定植和宿主-微生物相互作用中起着至关重要的作用。含有YSIRK / GXXS信号肽表面蛋白前体在细胞中间横跨隔膜易位,在横壁隔室锚定到细胞壁肽聚糖,并呈现在子细胞的新半球以下细胞分裂。经过几代细胞分裂,这些表面蛋白最终将覆盖整个细胞表面。为了了解这些蛋白质如何从细菌细胞质传播到细胞表面,我们描述了一系列的免疫荧光显微镜检查 设计用于检测表面蛋白前体的逐步亚细胞定位的协议:表面展示(协议A),跨壁定位(协议B)和细胞质/隔膜膜定位(协议C)。葡萄球菌蛋白A(SpA )是这项工作中使用的模型蛋白。此处所述的协议易于研究一般在金黄色葡萄球菌中其他表面蛋白以及其他细胞质或膜蛋白的定位。此外,可以修改方案并使其适用于其他革兰氏阳性细菌。


图形摘要:


追踪金黄色葡萄球菌表面蛋白的亚细胞定位



[背景]金黄色葡萄球菌是革兰氏阳性细菌和机会病原体。它经常定居在人的鼻孔和皮肤上,并且是医院和社区获得性感染的主要原因(von Eiff等,2001; Tong等,2015)。金黄色葡萄球菌的细胞包膜由细胞质膜和厚的细胞壁肽聚糖层组成。为了复制,金黄色葡萄球菌通过在中间细胞处形成分裂隔膜而经历二元裂变。细胞壁生物合成机制在细胞分裂过程中被募集到隔膜(Pinho和Errington ,2003)。合成新的细胞壁肽聚糖以形成跨壁环,并最终形成跨壁椎间盘并与隔膜的内陷结合(Zhou等人,2015)。一旦横壁盘被完全合成,特定细胞壁在交叉壁的外边缘水解酶切割来分割两个子细胞(押田等人,1995;菅等人,1995;山田等人,1996; Kajimura等人,2005)。由于较高的内部膨胀压力,两个子细胞彼此分离,新合成的跨壁圆盘成为子细胞的新半球(Monteiro等,2015; Zhou等,2015)。

细胞壁肽聚糖锚定的表面蛋白是革兰氏阳性细菌l细胞包膜的关键成分。许多的这些执行在毒力功能的金黄色葡萄球菌,如粘合性,生物膜形成,营养采集,抗生素抗性,和免疫逃避(福斯特等人,2014; Schneewind和Missiakas ,2019) 。许多表面蛋白前体包含具有高度保守的YSIRK / GXXS基序的特定N端信号肽(Rosenstein和Götz ,2000; Tettelin等,2001)。分泌,细胞壁锚定,和表面YSIRK / GXXS蛋白的显示紧密结合的细菌细胞周期(Carlsson的等人,2006;拉兹等人,2012;余等人,2018) 。在早期阶段,YSIRK / GXXS信号肽促进分裂间隔处的局部蛋白分泌(Carlsson等,2006; DeDent等,2008)。随后,通过分选酶A将间隔分泌的表面蛋白共价锚定到跨壁肽聚糖上(Mazmanian等,1999)。当细胞分裂和分离,横壁-锚定的表面蛋白被显示在子细胞的新半球的表面上(科尔和哈恩,1962; Swanson的。等人,1969;拉兹。等人,2012;于等。,2018)。最终,在几代细胞分裂后,表面蛋白显示在整个细胞表面上(DeDent等,2008; Raz等,2012; Yu等,2018)。

适当的成像方法是必不可少的揭示荷兰国际集团蛋白的亚细胞定位。在这里,我们描述了一系列协议来跟踪表面蛋白的亚细胞定位。虽然这是直截了当来本地化细菌细胞表面蛋白(协议A,图1)中,“脉冲追踪”类型的方法被用于揭示LOCA莉莎新锚定的表面蛋白的灰。在他们的论文古典,山口È和哈恩(1962)DES cribed一个immunofluorescen CE ,其中链球菌细胞与荧光染色方法LY标记表面M蛋白的抗体,随后与非-荧光抗体。在另一项经典研究中,对链球菌进行了胰蛋白酶处理,以消化细菌表面上现有的M蛋白。在没有胰蛋白酶的新鲜培养基中重新培养细菌后,观察到了新的表面沉积的M蛋白(Swanson等,1969)。胰蛋白酶消化,随后再生该方法后来被用于定位在细胞表面上新锚定表面蛋白的两个链球菌和葡萄球菌(Carlsson的等人,2006; DEDENT语言等人,2008;拉兹等人,2012;于等等人,2018)。在这里,我们提供了专门针对金黄色葡萄球菌量身定制的协议的详细说明(协议B,图1)。我们使用的模型蛋白是蛋白质A(SpA公司)之一的主要葡萄球菌表面蛋白结合到宿主免疫球蛋白和破坏宿主的免疫反应(Forsgren和Sjöquist ,1966年)。

我们以前曾表明,SpA的接合的SecA的介导的分泌途径通过细胞质膜的易位(于等人,2018) 。为了揭示其中的SpA前体在细胞学累积血浆在SECA枯竭,我们开发的协议来检测的细胞内的定位的蛋白质根据方法(协议C,图1)由哈里前面描述等。,(1995)以及Pinho和Errington (2003)。在该亲母育酚,葡萄球菌细胞固定机智ħ多聚甲醛和戊二醛,其附着于聚L-赖氨酸-包被的玻璃载玻片上。用健壮的葡萄球菌细胞壁水解酶溶葡萄球菌素在玻片上消化细胞以产生原生质体(Schindler and Schuhardt ,1964)。将原生质体固定并分别用甲醇和丙酮渗透,然后进行免疫荧光染色。根据不同的菌株的遗传背景,协议C可以被用于定位膜结合或胞质-局部表面蛋白前体。此外,方案C不仅限于表面蛋白;反之亦然。通常,它可用于定位金黄色葡萄球菌的细胞质或膜蛋白。这里描述的协议也可以适用于其他革兰氏阳性细菌。

图1.本工作中描述的协议的示意图

关键字:免疫荧光显微技术, 金黄色酿脓葡萄球菌, 表面蛋白, YSIRK / GXXS信号肽, 蛋白A(SpA), SecA, 表面显示, 穿壁定位, 间隔定位


材料和试剂
8孔载玻片小号(MP Biomedicals公司/赛默飞世尔科技,产品目录号:096040805E)
一次性硼硅酸盐玻璃管,直径16毫米,长度125毫米(Thermo Fisher Scientific,目录号:1496130)
1,250 µl XL g定量针头(美国科学公司,目录号:1112-1720)
将200μl克raduated提示(美国科学,目录号:1110年至1700年)
10个微升克raduated提示(美国科学,目录号:1111-3700)
10 ml的移液吸管(Thermo Fisher Scientific,目录号:1367811E)
25毫升小号erological吸管(赛默飞世尔科技,产品目录号:13-678-11)
1.5毫升米icrocentrifuge管(赛默飞世尔科技,产品目录号:05-408-129)
2.0毫升米icrocentrifuge管(赛默飞世尔科技,产品目录号:05-408-138)
Scienceware米icrocentrifuge吨UBE ř ACK(赛默飞世尔科技,产品目录号:10029259)
1.5毫升米icrocentrifuge管(赛默飞世尔科技,产品目录号:05-408-129)
2.0毫升米icrocentrifuge管(赛默飞世尔科技,产品目录号:05-408-138)
Nunc公司15ml锥形无菌管小号(赛默飞世尔科技,产品目录号:12565269)
Nunc公司50ml锥形无菌管小号(赛默飞世尔科技,产品目录号:12565271)
康宁PES注射器式过滤器(Thermo Fisher Scientific,目录号:09-754-29)
20毫升˚F ILTER注射器小号(赛默飞世尔科技,产品目录号:14-955-460)
圆P ETRI菜ES (100 × 15mm)中(赛默飞世尔科技,产品目录号:FB0875712)
移液管(Thermo Fisher Scientific,目录号:13-711-7M)
Kimberly-Clark Professional TM Kimwipes TM精致任务刮水器(Thermo Fisher Scientific,目录号:06-666A)
BD Bacto TM胰蛋白So大豆汤(TSB)(Thermo Fisher Scientific,目录号:DF0370-07-5)
BD胰蛋白酶大豆琼脂(TSA)(Thermo Fisher Scientific,目录号:DF0369-07-8)
氯化钠(NaCl)(Thermo Fisher Scientific,目录号:S271-1)
盐酸(HCl)(Thermo Fisher Chemical,目录号:187066)             
氯化钾(KCl )(Thermo Fisher Scientific,目录号:AM9640G)
磷酸氢二钾(K 2 HPO 4 )(Thermo Fisher Scientific,目录号:BP363-500)              
无水磷酸氢二钠(Na 2 HPO 4 )(Thermo Fisher Scientific,目录号:BP332-1)
乙二胺四乙酸EDTA(Thermo Fisher Scientific,目录号BP118-500)
Tris基座(Thermo Fisher Scientific,目录号:BP152-5)
D-(+)- g葡萄糖(Sigma-Aldrich,目录号:G8270-1KG)
丙酮(Thermo Fisher Scientific,目录号:A929-4)
甲醇(Thermo Fisher Scientific,目录号:A454-4)
乙醇(Thermo Fisher Scientific,目录号:A405P-4)
牛血清白蛋白(BSA)(Thermo Fisher Scientific Bioreagents,目录号BP1600-100)
0.1%p OLY-L-升ysine溶液(Sigma-Aldrich公司,目录号:P8920-100ML)
PBS中4%的低聚甲醛(PFA)(Thermo Fisher Scientific,目录号:AAJ19943K2)
戊二醛在H 2 O中为50%(Sigma-Aldrich,目录号:340855-25ML)
一抗:SpA KKAA抗血清(Kim等,2010)。储存在4°C
二级antibod IES :
山羊抗- [R abbit的IgG(H + L)高交吸附第二抗体的Alexa Fluor488的(赛默飞世尔科技,产品目录号:A-11034)。储存在4°C
山羊抗兔IgG(H + L)交叉吸附二级抗体,Alexa Fluor 647(Thermo Fisher Scientific,目录号:A-21244 )。储存在4°C
分子探针TM的SlowFade TM钻石抗荧光淬灭封固剂(Invitrogen公司,目录号:S36963)
尼罗河红(Sigma-Aldrich,目录号:19123-10MG)
Hoechst 33342 DNA染料,10 mg / ml(Thermo Fisher Scientific,目录号:H3570),在4°C下储存。
BODIPY TM万古霉素-FL(Thermo Fisher Scientific,目录号:V34850)
康宁TM 1号矩形盖玻片(22 × 50 mm)(Thermo Fisher Scientific,目录号:12-553-46 1)(参见注释1)
透明指甲油(在任何杂货店中最便宜的一种)
胰蛋白酶,来自牛胰腺(Sigma-Aldrich,目录号:T1426)
胰蛋白酶抑制剂,来自最大大豆甘氨酸(Sigma-Aldrich,目录号:T9128)
溶葡萄球菌素(AMBI,目录号:LSPN-50)
磷酸盐缓冲盐水(PBS)(请参阅食谱)
固定解决方案(请参阅食谱)
GTE解决方案(请参阅食谱)
胰蛋白酶原液(请参阅食谱)
胰蛋白酶抑制剂原液(请参阅食谱)
BSA阻止解决方案(请参阅食谱)
溶葡萄球菌素原液(请参阅食谱)
尼罗河红原液(请参阅食谱)
BODIPY TM万古霉素-FL储备溶液(请参阅食谱)


设备




的Eppendorf移液器100 - 1 ,000微升,20 - 200微升,2 - 20微升,0.1 - 2.5微升仪(Eppendorf,Ç atalog编号:2231000714)
的Eppendorf Easypet ® 3仪(Eppendorf,Ç atalog编号:4430000018)
Fisherbrand TM Traceable TM多色计时器(Thermo Fisher Scientific,目录号:02-261-840)
镊子(MilliporeSigma TM过滤镊子/ Thermo Fisher Scientific,目录号:XX6200006P)
内部真空
摇床(的Eppendorf氖瓦特不伦瑞克TM伊诺® 42摇床,目录号:EPM1335-0010)
台式离心机(Eppendorf ,型号:Centrifuge 5425,目录号EP5405000441)
分光光度计(Thermo Scientific Genesys GENESYS TM 30可见光分光光度计,目录号:14-380-442)
迷你管旋转器(Fisherbrand TM M ini Tube Rotator ,目录号:88-861-051 )
微生物培养箱(Fisher Scientific,目录号:51030513)
-20°C冷冻室
4℃ - [R efrigerator
LP涡旋混合器(Thermo Fisher Scientific,目录号:88880017)
Leica SP5 2光子激光扫描共聚焦显微镜(Leica Microsystems,产品名称:Leica TCS SP5共聚焦)


软件




图片J(Rasband WS / US NIH,美国马里兰州贝塞斯达,https://imagej.nih.gov/ij/)
徕卡显微镜软件LAS_AF徕卡(徕卡显微镜)
用于统计分析的Prism GraphPad软件(https://www.graphpad.com/scientific-software/prism/)
Adobe Illustrator组装图形


程序


幻灯片准备
向8孔载玻片的每个孔中加入50 µl 0.1%p -L-赖氨酸,并在室温下静置5分钟(请参见注2)。
简言之冲洗用的DDH的孔2 O,除去所述过量的液体使用真空,并允许滑动空气干燥完全(见注3和4)。


细菌培养
准备过夜培养:在玻璃试管中,从条纹琼脂板上接种一个单菌落到3 ml TSB中;添加了合适的抗生素,如果需要的话。
生长的过夜培养物在37℃与振摇严谨在220转。
n个分机早晨,接种30微升过夜培养至3ml新鲜TSB(1:100稀释)。
生长培养物在37℃下严谨振摇在220rpm下。
每小时在分光光度计中测量培养物的光密度OD 600 。
P花边在冰上培养时OD 600达到0.8 - 1.0 (见注5)。
根据不同的方案(A,B或C),在下面的P C中继续进行样品制备。


样品p赔偿
协议A –表面显示:


将2 ml细菌培养物转移到2 ml微量离心管中。
旋在18000 ×g离心3分钟以表格顶部离心机以沉淀细菌细胞。
除去上清液,不要干扰沉淀。
将沉淀重悬于1 ml PBS中并充分涡旋。
以18,000 × g的转速旋转3分钟,然后除去上清液(步骤4 – 5是“用PBS洗涤”的步骤)。
将沉淀重悬于1 ml PBS中,充分涡旋;在干净的1.5 ml微量离心管中将250 µl细菌l悬浮液与250 µl固定液混合(请参见食谱),短暂涡旋混合,并在室温下孵育20分钟(固定步骤)。
按照步骤4至5所述,用PBS洗涤两次(请参阅注6)。
将沉淀重悬于150 µl PBS中,充分涡旋振荡(请参见注释7)。
加入50μl的细菌悬浮液与聚-L-赖氨酸-包被的玻璃载玻片,并允许静置5分钟。
使用真空除去多余的液体(非粘附细胞)。
PBS一滴添加到每个孔中使用一次性塑料移液管,并删除利用一个真空(这是在“滴,拆下上滑动洗涤”步骤)。
重复下降操作,并删除滑梯上的清洗步骤。
用免疫荧光继续P艺术D.


协议B –跨墙本地化:


将2 ml细菌培养物转移到2 ml微量离心管中。
旋在18000 ×克在表3分钟顶离心机以沉淀细菌细胞。
除去上清液,不要干扰沉淀。
用PBS洗涤一次。
将沉淀重悬于900 µl PBS中,充分涡旋;添加100微升5毫克/毫升胰蛋白酶原液(见配方)和简要LY涡流(胰蛋白酶终浓度:0.5毫克/毫升)。
孵育在Mini-试管旋转器中的管子在37℃下1个小时在旋转速度的16(中速)。
用PBS洗涤两次。
将沉淀重悬于900 µl新鲜TSB中,充分涡旋;加入100微升10毫克/毫升大豆胰蛋白酶抑制剂储液(见配方)和简要LY涡旋混合(胰蛋白酶抑制剂大豆终浓度:1毫克/毫升)。
在微型离心管旋转器中,以16的旋转速度将离心管在37°C下温育20分钟(请参见注释8)。
在20分钟的孵育过程中,将250 µl固定液添加到干净的1.5 ml微量离心管中。
在20分钟的时间点,将250 µl细菌样品快速转移到上一步中制备的微量离心管中。
涡旋混合,使样品在室温下静置20分钟。
用PBS洗涤两次。
将沉淀重悬于150 µl PBS中并充分涡旋(根据沉淀大小调整体积)。
将50 µl细菌悬液添加到涂有聚L-赖氨酸的载玻片上,静置5分钟。
用真空除去液体(非粘附细胞)。
执行d ROP,拆下上滑动洗涤两次对每个孔上面所描述的。
与免疫继续在荧光P艺术D.


方案C –细胞质/隔膜的定位:


将两个50ml试管含有大约25毫升甲醇和25毫升丙酮,分别,成-20℃冷冻机中。
将所有细菌培养物标准化为OD 600 = 1。
转移2毫升归一化细菌培养于2ml的microcentrifug ë管(这一步是具有相同的细胞数为以下酶消化步骤)。
旋在18000 ×克在表3分钟顶离心机以沉淀细菌细胞。
除去上清液,不要干扰沉淀。
用PBS洗涤一次。
将沉淀重悬浮在900μlPBS中并充分涡旋; 添加100微升5毫克/毫升胰蛋白酶原液和简要LY涡流(见注9)。
孵育在37℃的迷你试管旋转器1个小时在旋转速度的16。
用PBS洗涤两次。
将沉淀重悬于500 µl PBS中,充分涡旋;加入500 µl固定液并涡旋混合。
孵育所述样品,在室温下15分钟,然后在冰上进行15分钟到修复。
用PBS洗涤3次。
将沉淀重悬于1 ml新鲜制成的GTE缓冲液中(请参阅食谱),并彻底涡旋(请参见注释10)。
加入50μl的细胞悬浮液,以聚-L-赖氨酸-包被的玻璃载玻片(这是不溶葡球菌酶消化的控制)(见注11)。
准备一个计时器,加10 μ升溶葡球菌酶工作溶液(见配方)向细胞悬浮液的剩余部分; 快速涡旋并立即向载玻片中加入50 µl(请参见注释12)。
在载玻片上孵育2分钟(请参阅注释13)。
除去液体使用真空,直到完全干燥。
立即将载玻片放入-20°C的预冷甲醇中5分钟。
取出来的滑动用在-20℃下进行30秒钳子并放入预冷的丙酮(见注14)。
就拿出来幻灯片使用镊子和允许完全风干。
玻片干燥后,将50 µl PBS加到样品孔中以重新水化。
进行滴落,并为每个孔去除两次滑上清洗液。
用免疫荧光继续P艺术D.


免疫荧光
除去PBS,添加BSA封闭溶液(请参阅“食谱”),并在室温下孵育30分钟。
除去封闭溶液,添加50 µl一抗溶液(兔血清SpA KKAA 1:4 ,在BSA封闭溶液中稀释000),并在4℃或室温下孵育过夜1小时(请参见注释15)。
除去未结合的一抗溶液,并用PBS洗涤8次,最后一个洗涤步骤为5分钟。
除去洗涤液,加入50 µl在BSA封闭液中稀释的二抗(例如,Alexa Fluor 647-IgG或Alexa Fluor 488-IgG,按1:500稀释),并在室温下于黑暗中孵育1小时(请参阅“注意”)。 16)。
用PBS进行O型玻片洗涤10次。
取一个干净的1.5 ml微量离心管,加入1 ml PBS,5 µl Hoechest储备液(1:200稀释),2 µl Van-FL储备液(1:500稀释)或5 µl Nile Red储备液(1 :200稀释)(请参见食谱)并充分混合;向每个孔中添加50 µl染色液。
在室温下于黑暗中孵育10分钟。
用PBS进行O型滑梯清洗3次。
经过最后一次洗涤,去除所有的多余的液体从井。
添加一个5 -微升的一滴慢慢退出钻石抗淬灭封固在3个样品池之间不同的地方(见注17)。
在滑轨的边缘刷一层薄的指甲油,并用盖玻片密封; 轻轻按下盖玻片,然后使用Kim擦拭纸去除边缘周围多余的防褪色溶液(请参见注释18)。
图像使用具有显微镜样品的适当的荧光信道(E部分)。
将制备的玻片可以储存在4 ° C ^为几天,并在-20 ℃下为一个较长的时间; ħ H但是,推荐即时成像。


影像学
以上制备的样品适合通过不同的成像系统成像,包括落射荧光显微镜,共聚焦显微镜或解卷积显微镜。但是,要揭示细菌的细胞特征并定义微小细菌细胞中的蛋白质定位,建议使用高分辨率的显微镜。甲60 ×或100 ×与物镜一个需要更高的数值孔径。我们使用一个徕卡DM 2000加上一个敏感的CCD照相机,一个徕卡SP5双光子激光扫描共聚焦显微镜,和一个徕卡SP8 3X STED激光共聚焦显微镜。所有节目编着不错的成像效果。
协议的代表性图像小号A,B,和C被显示在图2中捕获图像使用莱卡SP5双光子激光扫描共聚焦显微镜。




图2. (A)协议A的代表性图像,显示了SpA的表面显示;(B)协议B,显示SpA的跨壁定位;和(C)协议C,示出的间隔定位的SpA在SecA的和细胞质定位的存在SpA的在不存在SecA的的前体。范-FL:BODIPY TM万古霉素FL细胞壁染色; BF:明场图像;尼罗河红:膜染色;比例尺:2微米面板小号A和B和1μm的面板C.图像被改编自于等。,(2018)。




数据分析




每个实验中的每个样品至少要拍摄三张具有随机视图的图像。一个人必须拍摄更多的随机图像,尤其是当幻灯片上只有几个单元格或一张幻灯片上有不同的表型时。
至少独立进行3次实验。
到孔定量泰特的百分比SpA的横壁定位,从在ImageJ的协议B打开的图像,分割信道,并放大图像,以允许更好的可视化。
打开ImageJ中的“单元格计数器”工具。
选择单元格类型1,手动计算Van-FL染色图像中的双球菌数,并记录数量。每张图像至少计数50对双球菌。Diplococci被定义为两个刚刚分裂但尚未分离的连接子细胞(请参见图3中的示例图像)(请参见注释19)。


选择单元格类型2,在合并的图像中手动计算跨壁局部SpA信号,并记录编号。跨壁局部SpA信号定义为跨壁处的清晰线条。严格地说,不计点。
计算每个实验中三张图像的平均值。
将三个独立实验的平均值输入到GraphPad Prism。
在GraphPad Prism中,U SE一个牛逼测试至统计学分析显著差异小号两组之间; 使用单因素ANOVA对多个组比较小号; 并使用Tukey的多重比较测试来分析多个组之间的差异。




图3.样本图像与所述细胞计数窗口,演示如何孔定量泰特所述的百分比SpA的横壁定位。图像改编自Yu等人。,(2018)。




笔记




盖玻片厚度的选择取决于成像系统。
一个1:10稀释聚L-赖氨酸0.01%也有效。
大约需要15分钟,空气干燥的聚-L-赖氨酸-包被的载玻片。人们也可以使用一个真空来干燥载片。
为了组装真空系统,连接一个内部真空到一个管中,切割末端关闭200μl的移液管尖端,并且插入尖端在到管上。重要的是在干燥过程中不要触摸载玻片上的样品。
它通常需要约2 - 3小时金黄色葡萄球菌以达到一个OD 600 0.8 - 1.0标准实验室文化性下ë条件。
固定后细胞倾向于结块; 一个可能需要更长的涡流。
体积可根据颗粒大小调节;如果未确认,可以少加PBS并进行稀释。这里的关键是要有一个载玻片上细胞的适当数量,使大部分细胞得到良好的分离。过多的细胞会导致细菌升聚集而引起的免疫染色的文物; 牛逼○○少数细胞不会提供可靠的结果。
再生时间是在我们的实验方案中通过实验确定的。应该根据不同的生长条件和抗原对其进行测试和优化。
该步骤对于方案C并不关键,因为溶葡萄球菌素消化将去除大部分细胞壁以及现有的SpA 。我们将这一步骤包括在我们的协议中,以最大程度地减少现有SpA所引起的任何潜在背景。
GTE缓冲液是一种渗透稳定缓冲液。溶葡萄球菌素是锌依赖性内肽酶(Sabala等,2014)。尽管GTE缓冲液中的EDTA可以螯合锌,但在我们的实验中它没有任何明显的负面影响。我们尝试了其他没有EDTA的渗透稳定缓冲液,例如TSM [50 mM Tris-HCl(pH 7.5),0.5 M蔗糖,10 mM MgCl 2 ],也可以使用。
拥有此控制很重要。溶葡萄球菌素消化后的葡萄球菌细胞在明视场图像中将变得更加半透明,而未消化的细胞则具有暗细胞轮廓。此外,溶葡萄球菌素消化后,两个紧密连接的子细胞将分离(参见图2C )。
如果此协议适用于其他细菌,则其他细胞壁水解酶可以替代溶葡萄球菌素。例如,溶菌酶已被用于枯草芽孢杆菌中(Harry等,1995)。大多数金黄色葡萄球菌菌株是耐溶菌酶的,这限制了其在金黄色葡萄球菌中的使用。对于不同的酶或细菌,必须通过实验调整消化时间和缓冲液。
进行载玻片消化以稳定原生质体至关重要。
此步骤可在溶葡萄球菌素消化后稳定原生质体并透化细胞质膜。d epending上的抗原的细菌菌株和丰度,曲通X-100可被用于进一步透化细胞质膜。
对于任何新的抗原,斯里人初级抗体的稀释度是必要的,以确定最佳的浓度。不表达抗原的阴性对照是必需的对照。我˚F没有突变体可用,一个至少应包括无第一抗体的对照。在阴性对照中应观察到最小的背景信号。
根据显微镜系统的不同,可以选择不同的荧光标记的二抗。我们始终以1:500的稀释度使用二抗。
不同种类的防褪色溶液是可商购的。人们应该选择与成像系统兼容的防褪色解决方案。
中号肯定AKE该抗褪色溶液覆盖每以及非常薄的层无气泡,并且该盖片平整的幻灯片。
其原因为计数ING双球菌是因为横壁局部SpA的信号只能在我们的实验条件下,这些双球菌的横壁被检测; ħ H但是,作为定义双球菌可能是主观的,它可以引入偏倚。因此,可以计数总细胞数而不是双球菌。


菜谱




磷酸盐缓冲盐水(PBS)
137毫米氯化钠


2.7毫米氯化钾


10毫米Na 2 HPO 4


1.8毫米K 2 HPO 4


pH值7.4


将8 g NaCl ,0.2 g KCl ,1.44 g Na 2 HPO 4和0.24 g K 2 HPO 4溶解在1 L ddH 2 O中
用HCl将pH调节至7.4,并在121°C高压灭菌20分钟
固定解决方案
PBS中的2.4%低聚甲醛和0.01%戊二醛


拌30毫升4%PFA和10微升50%的戊二醛并添加PBS到一个50 -毫升总体积。储存在4°C(稳定至少两周)。


GTE解决方案
50 mM葡萄糖


10毫米EDTA


20 mM Tris-HCl pH 7.5


注:中号阿克清新过滤灭菌后使用。


制备0.5 M EDTA(pH 8)和1 M Tris-HCl(pH 7.5)的储备溶液
添加0.9克d葡萄糖溶液,2ml的0.5M EDTA ,和2毫升的1M的Tris-HCl至终体积80毫升的DDH 2 ö
调整的pH值至7.5,用HCl,添加的DDH 2 O操作100毫升,过滤器-灭菌,并在4℃下存储
胰蛋白酶原液
PBS中5 mg / ml胰蛋白酶,过滤-灭菌,并在-20°C储存


胰蛋白酶抑制剂原液
ddH 2 O中的10 mg / ml胰蛋白酶抑制剂,过滤-灭菌,在-20°C储存


BSA封闭液:PBS中含3%BSA
将0.3 g BSA粉末溶于10 ml PBS中; 使新鲜并过滤-使用前灭菌,在4°C下储存


Ly sostaphin储备液
使一个的10毫克/毫升在20mM乙酸钠(pH 4.5)中,储存在-20℃下储备溶液
稀用200mM的Tris-HCl(pH 8)中至2mg / ml的作为一个工作溶液,储存于4℃下
尼罗河红原液
溶解在100%乙醇以使一个0.5毫克/毫升的储备溶液,贮存在-20℃下
将5 µl尼罗河红原液加到1 ml PBS(1:200稀释液)中以对样品染色
BODIPY TM万古霉素-FL储备液
溶解100微克在100μlDMSO中以制备一个1微克/微升原液,储存在-20℃下
将2 µl Van-FL储备液加到1 ml PBS (1:500稀释液)中以对样品染色


致谢




这项工作WA ■通过从启动资金,以支持WY的南佛罗里达大学。我们感谢奥拉夫Schneewind和Dominique Missiakas为他们辅导的初期发展过程中的协议。感谢Vytas Bindokas ,Robert Hill和Byeong Cha在显微镜设施方面的协助。我们感谢建议实验室成员有关的手稿。这项工作报告中所使用的荧光显微镜方法我们以前的论文(俞等人。,2018 )。来自Yu等人的图片。(2018 )已在本报告中进行了修改以演示方法。




利益争夺




作者宣称没有利益冲突或利益冲突。




参考




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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Scaffidi, S. J., Shebes, M. A. and Yu, W. (2021). Tracking the Subcellular Localization of Surface Proteins in Staphylococcus aureus by Immunofluorescence Microscopy. Bio-protocol 11(10): e4038. DOI: 10.21769/BioProtoc.4038.
  2. Yu, W., Missiakas, D. and Schneewind, O. (2018). Septal secretion of protein A in Staphylococcus aureus requires SecA and lipoteichoic acid synthesis. Elife 7.
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