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Quantification of Neisseria meningitidis Adherence to Human Epithelial Cells by Colony Counting
通过菌落计数定量脑膜炎奈瑟菌对人上皮细胞的粘附能力   

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PLOS Pathogens
Apr 2017

Abstract

To cause an infection, the human specific pathogen Neisseria meningitides must first colonize the nasopharynx. Upon tight interaction with the mucosal epithelium, N. meningitidis may cross the epithelial cellular barrier, reach the bloodstream and cause sepsis and/or meningitis. Since N. meningitidis niche is restricted to humans the availability of relevant animal models to study host-pathogen interactions are limiting. Therefore, most findings that involve N. meningitidis colonization derive from studies using cultured human cell lines. Human epithelial cells have been successfully used to examine and identify molecular effectors involved in initial adherence of the pathogen. Here, we describe a standard protocol to quantify the adherence of N. meningitidis to epithelial pharyngeal FaDu cells. Colony counts of cell lysates collected after infection are used to quantify adherence to the epithelial cells.

Keywords: Neisseria (奈瑟菌), Colonization (定植), Adherence (粘附), Adhesion assay (粘附测定), Type IV pili (IV型菌毛)

Background

Upon entry to a new host, adherence to specific host tissues serves as an important step in bacterial pathogenesis. Molecular interaction between bacterial adhesins and receptors on the host cell surface determines colonization sites (Soto and Hultgren, 1999). The epithelial layer in the nasopharynx forms the first cellular barrier that the human restricted pathogen N. meningitidis encounters and colonizes asymptomatically. Tight adherence and interaction with the host cells can lead to penetration of the epithelium and entry into the bloodstream, resulting in life-threatening sepsis and/or meningitis (Stephens, 2009). Long filaments extending from the bacterial membrane, called type IV pili (Tfp), containing PilC1 tip-located adhesin play a key role in initial adherence of N. meningitidis to the nasopharyngeal epithelium (Marceau et al., 1995; Rudel et al., 1995). Tfp does not only promote interaction with host cells but is also involved in the development of bacterial aggregates, that can contribute to a high level of adherence and resistance against shear stress (Helaine et al., 2005; Mikaty et al., 2009; Engman et al., 2016). Apart from the Tfp, other surface expressed molecules like the opacity proteins, LPS, NadA, NhhA, App and MspA have been shown to affect the level of adhesion to the epithelial surface (Hill et al., 2010).

Animal models to study N. meningitidis colonization are limiting due to human host specificity. Consequently, the majority of the studies over the years have relied on cultured human cell lines (Merz and So, 2000). Here, we provide a step-by-step protocol adapted from Sigurlásdóttir et al. (2017) to quantify adherence of N. meningitidis to human epithelial pharyngeal FaDu cells in culture. In the following protocol, human epithelial cells are infected with both wild-type and an adhesion-deficient ΔpilC1 strain at a multiplicity of infection (MOI) of 10 for an incubation time of 4 h. The procedure described herein for N. meningitidis adherence to cultured epithelial cells could be easily applicable to a range of different bacterial species and cell lines with adaptation of the growth media (de Klerk et al., 2017).

Materials and Reagents

  1. Lab coat and protective gloves
  2. Marker pen
  3. Cell culture flasks T75 (SARSTEDT, catalog number: 83.1813.001 )
  4. Cell culture plates 24-well (SARSTEDT, catalog number: 83.1836 )
  5. Serological pipettes
    2 ml pipette (SARSTEDT, catalog number: 86.1252.001 )
    5 ml pipette (SARSTEDT, catalog number: 86.1253.001 )
    10 ml pipette (SARSTEDT, catalog number: 86.1254.001 )
    25 ml pipette (SARSTEDT, catalog number: 86.1685.001 )
  6. Sterile plastic loops
    1 µl plastic loops (SARSTEDT, catalog number: 86.1567.050 )
    10 µl plastic loops (SARSTEDT, catalog number: 86.1562.050 )
  7. Falcon tubes
    15 ml tubes (SARSTEDT, catalog number: 62.554.502 )
    50 ml tubes (SARSTEDT, catalog number: 62.547.254 )
  8. Pipette tips
    20-200 µl capacity (SARSTEDT, catalog number: 70.760.502 )
    50-1,000 µl capacity (SARSTEDT, catalog number: 70.762.100 )
  9. 5 µm pore filter (VWR, catalog number: 514-4106 )
  10. Cell culture plates 96-well (SARSTEDT, catalog number: 83.3924 )
  11. Bacteriological Petri plates, 92 x 16 mm (SARSTEDT, catalog number: 82.1473 )
  12. 5 ml syringe (VWR, catalog number: 613-3940 )
  13. 250 ml vacuum filtration unit, 0.22 μm (SARSTEDT, catalog number: 83.1822.001 )
  14. Bacterial strain: Neisseria meningitidis serogroup C strain FAM20 wild-type and ΔpilC1 (Rahman et al., 1997). The bacterial strain FAM20 is a nalidixic acid-resistant mutant of FAM18 that is available at ATCC (ATCC, catalog number: 700532 )
    Note: The bacterial stocks are stored in 25% glycerol:75% GC liquid medium (see Recipes) at -80 °C.
  15. Cell line: pharyngeal epithelial cell line FaDu (ATCC, catalog number: HTB-43 )
    Note: The cell line is stored in 90% FBS:10% DMSO at -140 °C.
  16. 70% ethanol
  17. Glycerol (Sigma-Aldrich, catalog number: G5516 )
  18. DMEM high glucose, GlutaMAXTM Supplement, pyruvate (Thermo Fisher Scientific, catalog number: 31966047 )
  19. Fetal bovine serum (FBS), heat inactivated (Sigma-Aldrich, catalog number: F9665 )
  20. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D8418 )
  21. Phosphate-buffered saline (PBS), 10x concentrated (Statens Veterinärmedicinska Anstalt, catalog number: 992442 )
  22. GC agar medium base (NEOGEN, Acumedia, catalog number: 7104A )
  23. D-glucose (Sigma-Aldrich, catalog number: G8270 )
  24. L-glutamine (Sigma-Aldrich, catalog number: G8540 )
  25. Ferric nitrate (Sigma-Aldrich, catalog number: F3002 )
    Note: This product has been discontinued.
  26. Cocarboxylase (Sigma-Aldrich, catalog number: C8754 )
  27. Saponin (Sigma-Aldrich, catalog number: S7900 )
  28. Protease peptone (Oxoid, catalog number: LP0085 )
  29. Starch, soluble (Sigma-Aldrich, catalog number: S9765 )
  30. Potassium phosphate dibasic (Sigma-Aldrich, catalog number: 60353 )
  31. Potassium phosphate monobasic (Sigma-Aldrich, catalog number: 60218 )
  32. Sodium chloride (Sigma-Aldrich, catalog number: S3014 )
  33. Trypsin-EDTA (0.5%), no phenol red, 10x (Thermo Fisher Scientific, GibcoTM, catalog number: 15400054 )
  34. GC agar plates (see Recipes)
  35. Kellogg’s supplement (see Recipes)
  36. Cocarboxylase solution (see Recipes)
  37. 2x trypsin (see Recipes)
  38. 1% saponin (see Recipes)
  39. Phosphate-based GC liquid medium (see Recipes)

Equipment

  1. Class II biosafety cabinet (e.g., Esco Micro, model: Airstream® Max )
  2. Incubator at 37 °C and with a 5% CO2 environment (e.g., Thermo Fisher Scientific, Thermo ScientificTM, model: HeracellTM 150i )
  3. Cell culture hood (e.g., ESCO laminar flow cabinet, Esco Micro, model: Airstream® Gen 3 )
  4. Inverted microscope (e.g., Carl Zeiss, model: Axiovert 40 C )
  5. Spectrophotometer (e.g., Bio-Rad Laboratories, model: SmartSpec Plus )
  6. Hemocytometer (e.g., VWR, catalog number: 631-0923 )
  7. Pipette boy (e.g., Fisher Scientific, model: Fisherbrand Electric Pipet Controller )
  8. Pipettes
    10-100 µl capacity (e.g., Eppendorf, catalog number: 4924000053 )
    100-1,000 µl capacity (e.g., Eppendorf, catalog number: 4924000088 )
  9. Multichannel pipette, 10-100 µl capacity (e.g., Eppendorf, catalog number: 3125000036 )
  10. Water bath set to 37 °C (e.g., Grant Instruments, model: Sub Aqua Pro, catalog number: SAP12 )
  11. Centrifuge
  12. 1 L flask
  13. Autoclave

Procedure

Caution: N. meningitidis is designated a class II pathogen. Preparation of all bacterial cultures should be performed carefully in a Class II biosafety cabinet and according to national biosafety guidelines. Wear a lab coat and protective gloves during handling of the bacteria. Decontaminate work surface with 70% ethanol before and after use. Wash hands after removing gloves. All material that has been in contact with bacteria should be discarded as an Infectious/Biohazardous Waste.
Day 1

  1. Growth of N. meningitidis
    1. Wild-type and ΔpilC1 mutant bacteria are streaked from glycerol stocks on GC agar plates (see Recipes).
    2. Incubate plates overnight for 16-18 h at 37 °C in an incubator with a 5% CO2 environment.
  2. Preparation of cell cultures (work in a cell hood under sterile conditions)
    Note: The cell line, FaDu, is maintained in DMEM containing 10% FBS and split at ratio 1:3 or 1:6 every 3-5 days. The cells from a fully confluent cell flask of 75 cm2 (T75) are required to prepare one 24-well plate the day before the experiment. All volumes indicated below in Steps 2a-2d are given for one 75 cm2 flask as starting material. The cell line is categorized biosafety level 1.
    1. Prewarm DMEM supplemented with 10% FBS, sterile 1x PBS and 2x trypsin (see Recipes) for at least 30 min in a 37 °C water bath.
    2. Wash the cells once with 5 ml of 1x PBS.
    3. Add 1 ml of 2x trypsin and allow the cells to detach in a 37 °C incubator for 5 min. Examine the cells under an inverted microscope and make sure that no aggregates are visible.
    4. Resuspend the detached cells in 20 ml of pre-warmed DMEM supplemented with 10% FBS. Use a 10 ml serological pipette to mix the solution until homogenized.
    5. Count number of cells per ml with a hemocytometer. Seed cells at a density of 1.5 x 105 cells/well to a 24-well plate.
    6. Incubate the cell culture plate overnight and allow to adhere and grow to 100% confluence (2 x 105) at 37 °C in an incubator with a 5% CO2 environment. It is important to examine cell confluence and check for contamination before using cells in an adhesion assay.
      Note: Having the cultured epithelial cells at 100% confluence, eliminates the possibility of N. meningitidis being stuck in between the intracellular gap. The procedure ensures that the adherence observed is solely due to N. meningitidis adhered to the epithelial cells and not to any abiotic surface.

Day 2
  1. Adhesion assay (work in a Class II biosafety cabinet)
    Note: Before the adhesion assay, the OD600 nm that is equal to the viable count 108 cfu/ml has to be determined for the bacterial stock used. This can be determined by serial dilutions and plating to GC plates. For the bacterial stocks used in this experiment, OD600 nm: 0.36 is equal to 108 cfu/ml.
    1. Prewarm DMEM. Before the experiment wash the cells gently once and add 800 µl of fresh DMEM. While preparing bacterial cultures, keep the cell culture plate in an incubator at 37 °C with a 5% CO2 environment.
      Note: During the adhesion assay, DMEM is used without FBS supplementation.
    2. For each bacterial strain, pick bacteria (half fill a 10 μl loop) from the GC agar plate grown overnight and resuspend in a 15 ml Falcon tube containing 2 ml of DMEM. Use 1 ml pipette to pipette up and down 4 times to mix the bacterial solutions. Let the solution stand at room temperature for 3 min to let the largest bacterial aggregates sediment to the bottom of the tube.
      Note: The N. meningitidis bacterial cultures that are used in adhesion assays should never be vortexed as this might shear the Tfp and influence the results.
    3. Take 1 ml of the supernatant and transfer to a 50 ml Falcon tube containing 5 ml DMEM. Filter the bacterial solutions through 5 µm pore filter.
      Note: The level of N. meningitidis aggregation can influence the number of adhered bacteria. Therefore we chose to filter the bacterial suspension, to have only single bacterial cells in the solution before starting the adhesion assay. Filtering can thus prevent artefactual variations in the level of adhesion that would stem from differences in re-suspension of the bacteria. (Eriksson et al., 2012; Sigurlásdóttir et al., 2017).
    4. Measure the absorbance of the bacterial solution with a spectrophotometer. Adjust the absorbance to OD600 = 0.36 (equivalent to 108 cfu/ml) by diluting the bacterial solution with DMEM.
    5. After the bacterial solution has been adjusted to OD600 = 0.36, dilute the solution 10-fold (900 µl:100 µl, equivalent 107 cfu/ml). Pipette the solution up and down 4 times with a 1 ml pipette. Infect cells at MOI of 10 by adding 200 µl of the bacterial solution (2 x 106 cfu/ml) to relevant wells of the cell culture plate. The total volume in the wells should be 1 ml since 800 µl of fresh DMEM is added as indicated in Step 1a. To ensure equal distribution of the inoculum, add 200 µl of the bacterial solutions gently drop by drop apart from each other within the well. Each bacterial strain should be added preferably in triplicates, at least in duplicates.
      Note: The multiwell plate containing infected cells can be centrifugated (200 x g for 5 min) before infection starts. Centrifugation can enhance contact between host and bacteria. If adhesion assay is performed in parallel to time-lapse imaging, then centrifugation can be useful to accelerate the binding of bacteria to host cells (Eriksson et al., 2012; Sigurlásdóttir et al., 2017).
    6. Transfer the cell culture plate to a 37 °C incubator with a 5% CO2 environment. Incubate for 4 h.
    7. To ensure the viable count for every experiment performed, plate serial dilutions of the prepared bacterial cultures for each strain in duplicates immediately after the incubation of the adhesion assay starts. Plate to GC plates and incubate overnight in a 37 °C incubator with 5% CO2 overnight. The actual MOI for every experiment should be calculated from the viable count.
    8. During the 4 h incubation, leave 2 GC agar plates in the biosafety hood turned upside down to dry. Divide the GC agar plates into 12 squares with the help of a marker pen (Figure 1A). The division will help to spot 12 different dilutions on the same plate, thereby saving media (Figure 1B). As an alternative approach that will require 24 GC plates, spread 100 µl of each dilution to a GC agar plate by using a 10 µl loop. Prepare for serial dilutions, prepare five 1:10 dilution series in DMEM with a total volume of 200 µl (180 µl DMEM) in a 96-well plate (Figure 2).


      Figure 1. Template for plating 12 samples on a GC agar plate. A. Illustration of the GC agar plate divided into 12 squares. B. For each bacterial strain (WT–wild type and ΔpilC1 in triplicates), four (-2 to -5) dilutions per sample are divided into the 12 squares on the agar plate.

    9. After the 4 h incubation, take the cell culture plate out from the incubator. Work in a Class II biosafety cabinet. Gently wash unbound bacteria away three times by removing all cell culture media and adding 1 ml fresh DMEM to the wells. Carefully try not to detach the cells. No unbound bacteria should be visible under the microscope when washing is finished. Under the microscope, unbound bacteria would appear as free bacteria floating in the growth media.
    10. Lyse cells with 1 ml of 1% saponin (see Recipes) in DMEM for 10 min in a 37 °C incubator.
    11. Take the plate out of the incubator and check lysis of the cells through a microscope. If cells have not detached completely from the bottom of the plate, use the end of a sterile plate loop to scrape the cells from the bottom. Use a 1 ml pipette and mix the cell lysates up and down 4 times and transfer 200 µl to row A of the 96-well plate (Figure 2). Use a 100 µl multipipette to mix all the samples 4 x and then transfer 20 µl to row B to perform the -1 dilution. Use a 100 µl multipipette to mix all the samples 4 x and then transfer 20 µl to the next row for the -2 dilution and so on. It is very important to change pipette tips between every dilution performed. Repeat until -5 dilution has been performed (Figure 2).


      Figure 2. Dilution series performed in a 96-well plate. Add 180 µl of DMEM to -1 to -5 wells. Transfer 20 µl of cell lysates (from row A) to row B containing 180 µl of DMEM. Mix the solution and continue with serial dilutions until the -5 dilution has been completed.

    12. Spot 50 µl of dilutions -2 (10-2), -3 (10-3), -4 (10-4) and -5 (10-5) for each sample to each square marked on the GC agar plates to acquire countable colonies (Figure 1B). Use the pipette tip or a 1 µl loop to spread the drop, if it does not occur naturally, on the GC agar plates. Allow the drops to dry in the bacterial hood before shifting to an incubator.
    13. Incubate the GC agar plates in a 37 °C incubator with 5% CO2 overnight.

Day 3
  1. After overnight incubation, count the number of colonies from the dilutions that have between 20-100 colonies.

Data analysis

  1. To calculate the number of adhered bacteria, multiply the number of colonies with 20 to get the number of bacteria in 1 ml per dilution, since only 50 µl was plated. In order to calculate cfu/ml multiply with 10 for every dilution performed. For example, if 50 colonies were counted from the 5th dilution. 50 x 20 x 105 = 1 x 107. The data can be either presented as adhered bacteria (cfu/ml) or adhered bacteria/cell. Calculate the average of the duplicates/triplicates. Results can be plotted graphically in Microsoft Excel as a column graph.
  2. Standard deviation can be calculated as well as two-tailed, unpaired Student’s t-test for analysis of statistical significance from three independent adhesion assays.
  3. Example of data presentation can be observed in Sigurlásdóttir et al. (2017).

Recipes

  1. GC agar plates (1 L)
    To a 1 L flask, add 36 g medium base and fill up to 600 ml of distilled H2O
    Autoclave
    Add up to 1 L with sterile cold distilled H2O
    Add 10 ml of Kellogg’s supplement (Recipe 2) when the temperature is about 60 °C
    Pour the agar into Petri dishes and allow the plates to cool down
    Store the plates at 4 °C
  2. Kellogg’s supplement (200 ml)
    Dissolve 80 g of D-glucose in 100 ml of distilled H2O
    Add 1 g of L-glutamine, 0.1 g of ferric nitrate and 2 ml of 0.2% Cocarboxylase solution (Recipe 3)
    Add up to 200 ml with distilled H2O when everything is dissolved
    Sterile filter with 0.22 µm vacuum filtration unit and store at 4 °C
  3. Cocarboxylase solution
    0.02 g in 100 ml of distilled H2O, sterile filter with a 0.22 µm filter, store at 4 °C
  4. 2x trypsin
    Dilute to 2x trypsin by adding 5 ml to 20 ml of 1x PBS
  5. 1% saponin
    0.1 g saponin, dissolved in DMEM and filled up to 10 ml
    6. Phosphate-based GC liquid medium (500 ml)
    Dissolve 7.5 g of protease peptone, 0.5 g of soluble starch, 2 g of potassium phosphate dibasic, 0.5 g of potassium phosphate monobasic and 2.5 g of sodium chloride in 450 ml of distilled H2O. When dissolved, fill up to 500 ml with distilled H2O and autoclave

Acknowledgments

This work was adapted from a protocol we used for studies previously published (Sigurlásdóttir et al., 2017). This work was funded by the Swedish Research Council (Dnr 2006-4112, 2012-2415, 2013-2434), The Swedish Cancer Society and Torsten Söderbergs Stiftelse. The authors declare no conflicts on interest.

References

  1. de Klerk, N., Saroj, S. D., Wassing, G. M., Maudsdotter, L. and Jonsson, A. B. (2017). The host cell transcription factor EGR1 is induced by bacteria through the EGFR-ERK1/2 pathway. Front Cell Infect Microbiol 7: 16.
  2. Engman, J., Negrea, A., Sigurlasdottir, S., Georg, M., Eriksson, J., Eriksson, O. S., Kuwae, A., Sjolinder, H. and Jonsson, A. B. (2016). Neisseria meningitidis polynucleotide phosphorylase affects aggregation, adhesion, and virulence. Infect Immun 84(5): 1501-1513.
  3. Eriksson, J., Eriksson, O. S. and Jonsson, A. B. (2012). Loss of meningococcal PilU delays microcolony formation and attenuates virulence in vivo. Infect Immun 80(7): 2538-2547.
  4. Helaine, S., Carbonnelle, E., Prouvensier, L., Beretti, J. L., Nassif, X. and Pelicic, V. (2005). PilX, a pilus-associated protein essential for bacterial aggregation, is a key to pilus-facilitated attachment of Neisseria meningitidis to human cells. Mol Microbiol 55(1): 65-77.
  5. Hill, D. J., Griffiths, N. J., Borodina, E. and Virji, M. (2010). Cellular and molecular biology of Neisseria meningitidis colonization and invasive disease. Clin Sci (Lond) 118(9): 547-564.
  6. Marceau, M., Beretti, J. L. and Nassif, X. (1995). High adhesiveness of encapsulated Neisseria meningitidis to epithelial cells is associated with the formation of bundles of pili. Mol Microbiol 17(5): 855-863.
  7. Merz, A. J. and So, M. (2000). Interactions of pathogenic Neisseriae with epithelial cell membranes. Annu Rev Cell Dev Biol 16: 423-457.
  8. Mikaty, G., Soyer, M., Mairey, E., Henry, N., Dyer, D., Forest, K. T., Morand, P., Guadagnini, S., Prevost, M. C., Nassif, X. and Dumenil, G. (2009). Extracellular bacterial pathogen induces host cell surface reorganization to resist shear stress. PLoS Pathog 5(2): e1000314.
  9. Rahman, M., Kallstrom, H., Normark, S. and Jonsson, A. B. (1997). PilC of pathogenic Neisseria is associated with the bacterial cell surface. Mol Microbiol 25(1): 11-25.
  10. Rudel, T., Scheurerpflug, I. and Meyer, T. F. (1995). Neisseria PilC protein identified as type-4 pilus tip-located adhesin. Nature 373(6512): 357-359.
  11. Sigurlásdóttir, S., Engman, J., Eriksson, O. S., Saroj, S. D., Zguna, N., Lloris-Garcera, P., Ilag, L. L. and Jonsson, A. B. (2017). Host cell-derived lactate functions as an effector molecule in Neisseria meningitidis microcolony dispersal. PLoS Pathog 13(4): e1006251.
  12. Soto, G. E. and Hultgren, S. J. (1999). Bacterial adhesins: common themes and variations in architecture and assembly. J Bacteriol 181(4): 1059-1071.
  13. Stephens, D. S. (2009). Biology and pathogenesis of the evolutionarily successful, obligate human bacterium Neisseria meningitidis. Vaccine 27 Suppl 2: B71-77.

简介

为了引起感染,人类特定的病原体脑膜炎奈瑟氏脑必须首先在鼻咽中定居。 与粘膜上皮紧密相互作用时, 脑膜炎双球菌可能穿过上皮细胞屏障,到达血流并引起败血症和/或脑膜炎。 由于 N meningitidis niche限于人类。 因此,大多数涉及N 脑膜炎菌群定植来源于使用培养的人类细胞系的研究。 在最初的病原体坚持。 在这里,我们描述了一个标准协议来量化N的遵守情况。 脑膜炎双球菌对上皮细胞FaDu细胞的作用。 感染后收集的细胞裂解物的集落计数用于量化对上皮细胞的粘附。

【背景】作为细菌发病机制的重要一步。细菌粘附素与宿主细胞表面受体之间的分子相互作用决定了定植位点(Soto and Hultgren,1999)。鼻咽中的上皮层形成人类限制性病原体N的第一个细胞屏障。 meningitidis 遇到和无症状殖民。牢固粘附,并与宿主细胞相互作用可导致上皮细胞和进入血液的渗透,在危及生命的败血症和/或脑膜炎(斯蒂芬斯,2009)得到的。从细菌膜延伸出来的长细丝(称为IV型菌毛(Tfp))与PilC1尖端定位的粘附素在初始依从性中起关键作用。脑膜炎到鼻咽上皮细胞(Marceau等,1995; Rudel等,1995)。 Tfp不仅促进与宿主细胞的相互作用,而且参与细菌聚集体的发育,这可以有助于高水平的粘附和抵抗剪切应力(Helaine等,2005,Mikaty ,2009; Engman 等,2016)。除了Tfp之外,LPS,NadA,NhhA,App和MspA已经显示影响与上皮表面的粘附水平(Hill等,2010) )。

学习动物模型脑膜炎奈瑟菌定殖由于人类宿主的特异性而受到限制。因此,多年来的大多数研究都依赖于培养的人类细胞系(Merz and So,2000)。在这里,我们提供了一个由Sigurlásdóttir等人(2017)改编的逐步协议来量化N的依从性。脑膜炎双球菌对培养的人上皮细胞FaDu细胞。在以下方案中,用感染复数(MOI)为10的野生型和粘附缺陷型ΔpilC1菌株感染人上皮细胞4小时的孵育时间。在中描述的过程。脑膜炎奈瑟氏球菌粘附培养的上皮细胞可以很容易地适用于各种不同的细菌物种和细胞系与生长培养基的适应性(德克勒克等人,2017年)。

关键字:奈瑟菌, 定植, 粘附, 粘附测定, IV型菌毛

材料和试剂

  1. 实验室外套和防护手套
  2. 记号笔
  3. 细胞培养瓶T75(SARSTEDT,目录号:83.1813.001)
  4. 细胞培养板24孔(SARSTEDT,目录号:83.1836)
  5. 血清移液器
    2毫升吸管(SARSTEDT,目录号:86.1252.001)
    5毫升吸管(SARSTEDT,目录号:86.1253.001)
    10毫升吸管(SARSTEDT,目录号:86.1254.001)
    25毫升吸管(SARSTEDT,目录号:86.1685.001)
  6. 无菌塑料环
    1μl塑料环(SARSTEDT,目录号:86.1567.050)
    10μl塑料环(SARSTEDT,目录号:86.1562.050)
  7. 猎鹰管
    15毫升管(SARSTEDT,目录号:62.554.502)
    50毫升管(SARSTEDT,目录号:62.547.254)
  8. 移液器提示
    20-200μl容量(SARSTEDT,目录号:70.760.502)
    50-1,000μl容量(SARSTEDT,目录号:70.762.100)
  9. 5μm孔径过滤器(VWR,目录号:514-4106)
  10. 细胞培养板96孔(SARSTEDT,目录号:83.3924)
  11. 细菌培养皿,92×16毫米(SARSTEDT,目录号:82.1473)
  12. 5毫升注射器(VWR,目录号:613-3940)

  13. 250毫升真空过滤装置,0.22微米(SARSTEDT,目录号:83.1822.001)
  14. 细菌菌株:脑膜炎奈瑟球菌血清群C菌株FAM20野生型和ΔpilC1(Rahman等,1997)。细菌菌株FAM20是在ATCC(ATCC,目录号:700532)可获得的FAM18的萘啶酮酸抗性突变体。
    25%甘油:75%GC液体培养基(见食谱)-80℃。
  15. 细胞系:咽上皮细胞系FaDu(ATCC,目录号:HTB-43)
    注意:细胞系在-140℃下储存在90%FBS:10%DMSO中。
  16. 70%乙醇
  17. 甘油(Sigma-Aldrich,目录号:G5516)
  18. DMEM高葡萄糖,GlutaMAX TM补充物,丙酮酸(Thermo Fisher Scientific,目录号:31966047)。

  19. 胎牛血清(FBS),热灭活(西格玛 - 奥德里奇,目录号:F9665)。
  20. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:D8418)。
  21. 磷酸缓冲盐水(PBS),10倍浓缩(StatensVeterinärmedicinskaAnstalt,产品目录号:992442)
  22. GC琼脂培养基(NEOGEN,Acumedia,目录号:7104A)。
  23. D-葡萄糖(Sigma-Aldrich,目录号:G8270)。
  24. L-谷氨酰胺(Sigma-Aldrich,目录号:G8540)。
  25. 硝酸铁(Sigma-Aldrich,目录号:F3002)
    注:此产品已停产。
  26. 羧化酶(Sigma-Aldrich,目录号:C8754)。
  27. 皂苷(Sigma-Aldrich,目录号:S7900)
  28. 蛋白酶蛋白胨(Oxoid,目录号:LP0085)。
  29. 淀粉,可溶(Sigma-Aldrich,目录号:S9765)
  30. 磷酸氢二钾(Sigma-Aldrich,目录号:60353)
  31. 磷酸二氢钾(Sigma-Aldrich,目录号:60218)
  32. 氯化钠(Sigma-Aldrich,目录号:S3014)
  33. 胰蛋白酶-EDTA(0.5%),无酚红,10x(Thermo Fisher Scientific,Gibco TM,目录号:15400054)。
  34. GC琼脂平板(见食谱)
  35. 凯洛格的补充(见食谱)
  36. 羧化酶溶液(见食谱)
  37. 2x胰蛋白酶(见食谱)
  38. 1%皂素(见食谱)
  39. 磷酸盐基GC液体培养基(见食谱)

设备

  1. II级生物安全柜(,,Esco Micro,型号:Airstream Max)
  2. 在37℃和5%CO 2亚环境(Thermo Fisher Scientific,Thermo Scientific TM,型号:Heracell > TM 150i)
  3. 细胞培养罩(例如,ESCO层流柜,Esco Micro,型号:Airstream Gen 3)
  4. 倒置显微镜(,,卡尔蔡司,型号:Axiovert 40℃)
  5. 分光光度计(例如,Bio-Rad Laboratories,型号:SmartSpec Plus)
  6. 血细胞计数器(例如,VWR,目录号:631-0923)。
  7. 吸管男孩(例如,Fisher Scientific,型号Fisherbrand电动移液管控制器)
  8. 移液器
    (例如,Eppendorf,产品目录号:4924000053)。
    (例如,Eppendorf,目录号:4924000088)。
  9. 多通道移液器,10-100μl容量(Eppendorf,产品目录号:3125000036)
  10. 设置在37℃的水浴(例如Grant Instruments,型号:Sub Aqua Pro,目录号:SAP12)。
  11. 离心机
  12. 1升烧瓶
  13. 高压灭菌器

程序

小心:脑膜炎奈瑟氏菌被指定为II类病原体。所有细菌培养物的制备都应在II类生物安全柜内按照国家生物安全指南进行仔细的处理。在处理细菌的过程中,穿上实验服和防护手套。使用前后用70%乙醇去除工作表面。取下手套后洗手。所有与细菌接触的物质都应作为传染性/生物危害性废物丢弃。
第1天

  1. N的增长脑膜炎奈瑟氏球菌
    1. 野生型和ΔpilC1突变细菌在GC琼脂平板上从甘油储液中划线(参见食谱)。
    2. 在5%CO 2环境的培养箱中37°C孵育板过夜16-18小时。
  2. 制备细胞培养物(在无菌条件下在细胞中工作)
    注:细胞系FaDu保持在含有10%FBS的DMEM中,每3-5天以1:3或1:6的比例分开。在实验前一天需要75cm2(T75)来制备一个24孔板,下面在步骤2a-2d中给出的所有体积都是作为起始材料给一个75cm 2的烧瓶。细胞系被分类为生物安全级别1.
    1. 在37℃水浴中加入10%FBS,无菌1x PBS和2x胰蛋白酶(参见食谱)至少30分钟的DMEM培养基。
    2. 用5毫升的1x PBS洗细胞一次。
    3. 加入1毫升的2x胰蛋白酶,让细胞分离在37°C的孵化器5分钟。在倒置显微镜下检查细胞,确保没有聚集体可见。
    4. 重悬于20毫升预热的补充有10%FBS的DMEM中的分离的细胞。使用10毫升血清移液管混合,直到均化。
    5. 用血细胞计数器计数每毫升细胞的数量。将细胞以1.5×10 5个细胞/孔的密度接种到24孔板中。
    6. 孵育细胞培养板过夜,并允许在37℃下在具有5%CO 2环境的培养箱中生长至100%汇合(2×10 5) ,


      注意:使培养的上皮细胞达到100%汇合,消除了脑膜炎奈瑟氏菌滞留在细胞间隙之间的可能性。该程序确保了粘附于上皮细胞而不是任何非生物表面。脑膜炎奈瑟菌粘附于上皮细胞

第2天
  1. 粘附分析(在II类生物安全柜中工作)
    注:粘附试验之前,OD <子> 600nm的 确实等于活菌数10 已经使用了8μg/ ml的cfu / ml用于所使用的细菌储备物。这可以通过系列稀释和电镀到GC板来确定。对于在该实验中所用的细菌种群,OD <子> 600nm的 :0:36等于10 8 cfu / ml。
    1. 预热DMEM。在实验之前,轻轻地洗涤细胞并加入800μl新鲜的DMEM。在制备细菌培养物时,将细胞培养板保持在37℃,5%CO 2环境的培养箱中。
      注意:在粘附测定过程中,使用不含FBS补充物的DMEM。
    2. GC,琼脂平板过夜生长,并重新悬浮在含有2ml DMEM的15ml Falcon管中。使用1毫升移液器上下吸移4次以混合细菌溶液。
      3分钟到管底 注意:用于粘附分析的脑膜炎奈瑟球菌细菌培养物不应该被涡旋,因为这可能剪切物质并影响结果。
    3. 取1毫升的上清液,转移到含有5毫升DMEM的50毫升Falcon试管中。
      过滤细菌溶液通过5微米的孔过滤器 注:脑膜炎奈瑟球菌聚集的水平可以增加附着的细菌数量。因此,我们选择在开始附着测定之前使用溶液中的细菌悬浮液。因此过滤可以防止粘附水平的人为变化。 (Eriksson等,2012;Sigurlásdóttir等,2017)。
    4. 用分光光度计测量细菌溶液的吸光度。通过用DMEM稀释细菌溶液,将吸光度调节至OD <600> = 0.36(相当于10 8 cfu / ml)。
    5. 后,将细菌溶液已被调整至OD <子> 600 = 0:36,稀释溶液10倍(900微升100微升,10当量 7 CFU / ml)中。用1毫升移液管上下移取溶液4次。将200μl的细菌溶液(2×10 6 cfu / ml)加到细胞培养板的相关细胞中。因为如步骤1a所示,指示800μl新鲜DMEM,所以孔中的总体积应为1ml。为确保接种物平均分布,在孔内轻轻地逐滴加入200μl细菌溶液。
      每个细菌菌株应加入三份,至少一式两份 注:含感染细胞的多孔板可在感染开始前离心(200×g,5分钟)。离心可以增强宿主与细菌的接触。如果粘着测定在平行进行的时间推移成像,然后离心可能是有用的加速了细菌对宿主细胞(Eriksson等人,2012 ;.Sigurlásdóttir等人2017)的结合。
    6. 将细胞培养板转移到具有5%CO 2环境的37℃培养箱中。孵育4小时。
    7. 为了确保进行的每个实验的活细胞计数,开始粘附试验孵育之后立即将连续稀释的每种菌株制备的细菌培养物重复进行两次。板上的GC板和孵化过夜,在37°C孵化器与5%CO 2过夜。
      每个实验的实际MOI应该从可行数量计算
    8. 孵育4小时期间,将生物安全罩中的2个GC琼脂平板颠倒干燥。用标记笔将GC琼脂平板分成12个正方形(图1A)。该部门希望帮助在同一盘子上发现12种不同的稀释物,从而节省媒体(图1B)。作为需要24个GC板的替代方法,通过使用10μl环将100μl的每种稀释液涂布到GC琼脂平板上。准备系列稀释液,在96孔板(图2)中,在DMEM中制备5个1:10稀释系列,总体积为200μl(180μlDMEM)。

      “”
      图1.在GC琼脂平板上电镀12个样品的模板。 A. GC琼脂平板分成12个正方形的插图。 B.对于每种细菌菌株(WT-野生型和ΔpilC1三重复),每个样品将四个(-2至-5)稀释液分成琼脂板上的12个方块。

    9. 孵育4小时后,将细胞培养板从培养箱中取出。在第二类生物安全柜中工作。通过去除所有的细胞培养基,并添加1毫升新鲜的DMEM轻轻洗三次未结合的细菌。小心尽量不要分离细胞。清洗完毕后,显微镜下应看不到未结合的细菌。在显微镜下,未结合的细菌会以漂浮在生长介质中的游离细菌的形式出现。
    10. 在37℃培养箱中用1ml 1%皂苷溶解细胞(参见食谱)于DMEM中10分钟。
    11. 从培养箱中取出培养皿,通过显微镜检查细胞。如果细胞没有完全从平板的底部去除,使用无菌平板的末端从底部刮细胞。使用1毫升移液器,混合细胞裂解物上下4次,并转移200微升到96孔板A行(图2)。使用100μl多支管将所有样品混合4次,然后将20μl转移至B列进行-1稀释。使用一个100μl的多管吸管将所有样品混合4次,然后将20μl转移到下一行进行-2次稀释,依此类推。在每次稀释之间更换枪头是非常重要的。重复进行至-5稀释(图2)。


      图2.在96孔板中进行稀释系列。向-1至-5孔中加入180μlDMEM。将20μl细胞裂解物(从A行)转移到含有180μlDMEM的B行。混合溶液并继续进行系列稀释,直至完成稀释。

    12. 点样50μl的稀释液-2(10 -2 -2),-3(10 -3 -3),-4(10 -4 -4)和-5(10 -5 -5)在GC琼脂平板上标记的每个正方形以获得可数克隆(图1B)。使用移液器吸头或1μl回路将滴落物(如果不是自然发生的话)滴在GC琼脂平板上。
      在转移到培养箱之前让滴液在细菌罩内干燥
    13. 将GC琼脂平板在含5%CO 2的37℃培养箱中培养过夜。

第3天
  1. 过夜孵育后,计算20-100个菌落稀释的菌落数。

数据分析

  1. 为了计算粘附的细菌数量,将菌落数量乘以20,通过稀释获得1ml中的细菌数量,因为只涂布了50μl。为了计算每次稀释的cfu / ml乘以10。例如,如果从第5次稀释计数50个菌落。 50×20×10 5 = 1×10 7。数据可以表示为粘附的细菌(cfu / ml)或附着的细菌/细胞。计算重复/一式三份的平均值。结果可以在Microsoft Excel中以图表形式绘制。
  2. 可以计算标准偏差,也可以计算双尾不成对学生t检验分析三种独立的粘附分析。
  3. 数据呈现的例子可以在Sigurlásdóttiret al。(2017)观察到。

食谱

  1. GC琼脂平板(1 L)
    向1L烧瓶中加入36g培养基并加入600ml蒸馏过的H 2 O 高压灭菌器
    用无菌冷蒸馏H 2 O加入至多1L
    当温度约为60°C时,加入10毫升凯洛格的补充剂(配方2) 把琼脂倒入陪替氏培养皿中,让培养皿冷却下来
    存放在4°C的板块
  2. 凯洛格的补充(200毫升)
    将80克D-葡萄糖溶于100毫升蒸馏过的H 2 O中 加入1克L-谷氨酰胺,0.1克硝酸铁和2毫升0.2%羧化酶溶液(方案3)。
    当一切都溶解时,加入蒸馏水至200ml。
    无菌过滤器,带0.22μm真空过滤装置,4°C储存
  3. 羧化酶溶液
    在100ml蒸馏水中加入0.02g,带有0.22μm过滤器的无菌过滤器,在4℃下储存。
  4. 2x胰蛋白酶

    加入5 ml至20 ml的1x PBS,稀释至2x胰蛋白酶
  5. 1%皂素
    0.1克皂甙,溶于DMEM中并充满10毫升
    6.基于磷酸盐的GC液体培养基(500毫升)
    溶解7.5克蛋白酶蛋白胨,0.5g的可溶性淀粉,将2克磷酸氢二钾,0.5g的磷酸二氢钾和在450ml蒸馏水<子> 2 O. 2.5克氯化钠溶解后,用蒸馏水和高压灭菌器充满500毫升

确认

这项工作从我们用于先前公布的研究的协议是什么改编(Sigurlásdóttir的等的,2017年)。其由瑞典研究理事会(DNR 2006-4112,2012至2415年,2013年至2434年),瑞典癌症协会和托斯滕•索德伯格Stiftelse资助这项工作。作者声明没有利益冲突。

参考

  1. 德克勒克,N.,Saroj,S.D.,Wassing,G.M.,Maudsdotter,L和琼森,B. A.(2017)。 的宿主细胞转录因子EGR1是由细菌通过EGFR-ERK1 / 2途径诱导
  2. Engman,J.,Negrea,A.,Sigurlasdottir,S.,乔治,M.,埃里克森,J.,埃里克森,O. S.,桑江,A.,Sjolinder,H。和琼森,B.A。(2016)。 脑膜炎奈瑟氏球菌多核苷酸磷酸影响聚集,粘附,和毒力。
  3. Eriksson,J.,Eriksson,O.S.和Jonsson,A.B.(2012)。脑膜炎球菌的毗卢损失延迟小菌落的形成和毒力减弱的体内 < /感染免疫 80(7):2538-2547。
  4. 克拉斯基•] Helaine,S.,Carbonnelle,E.,Prouvensier,L.,Beretti,J. L.纳西夫,X。和Pelicic,V.(2005)。 PilX,菌毛相关蛋白对细菌聚集必需的,是的菌毛促进附着的关键脑膜炎奈瑟氏球菌人体细胞 分子微生物学 55(1):. 65-77
  5. 山,D.J.,格里菲思,N.J.,Borodina,E。和Virji,M。(2010)。的细胞和分子生物学的脑膜炎奈瑟氏球菌定殖和侵入性疾病。 临床科学(林斯顿) 118(9):547-564
  6. Marceau,M.,Beretti,J.L。和Nassif,X。(1995)。的封装的脑膜炎奈瑟氏球菌上皮细胞与形成相关高粘合性的菌毛代码束。分子微生物学 17(5):. 855-863
  7. Merz,A.J。和So,M。(2000)。的致病 neisseriae 上皮细胞膜相互作用。 Annu Rev Cell Dev Biol 16:423-457。
  8. Mikaty,G.,索耶尔,M.,Mairey,E.,亨利,N.,代尔,D.,森林,KT,莫朗,P.,瓜达尼尼,S.普雷沃,MC,纳西夫,X。和Dumenil, G.(2009)。 胞外细菌病原体诱导宿主细胞表面重组以抵抗剪切应力。 公共科学图书馆Pathog 5(2):e1000314。
  9. Rahman,M.,Kallstrom,H.,Normark,S.和Jonsson,A.B.(1997)。 的致病PILC 奈瑟氏球菌与细菌细胞表面相关联。
  10. Rudel,T.,Scheurerpflug,I.和Meyer,T.F。(1995)。 奈瑟PILC蛋白鉴定为4-型菌毛尖端位于粘附。 自然 373(6512):357-359。
  11. Sigurlásdóttir,S.,Engman,J.,埃里克森,O. S.,Saroj,S. D.,Zguna,N.,洛里斯-Garcera,P.,Ilag,L. L.和琼森,B.A。(2017)。 宿主细胞来源的乳酸用作在脑膜炎奈瑟氏球菌菌落效应分子。扩散码的公共科学图书馆Pathog 13(4):e1006251
  12. Soto,G.E.和Hultgren,S.J.(1999)。 细菌粘附:.在建筑和汇编代码的Ĵ细菌学共同的主题和变化 181(4):1059-1071。
  13. Stephens,D.S.(2009)。 生物学和发病机制的进化成功,预留人类细菌脑膜炎奈瑟氏球菌。 疫苗 27增刊2:B71-77。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Sigurlásdóttir, S., Saroj, S. D., Eriksson, O. S., Eriksson, J. and Jonsson, A. (2018). Quantification of Neisseria meningitidis Adherence to Human Epithelial Cells by Colony Counting. Bio-protocol 8(3): e2709. DOI: 10.21769/BioProtoc.2709.
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