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Live-cell Imaging of Neisseria meningitidis Microcolony Dispersal Induced by Lactate or Other Molecules
乳酸盐或其他分子诱导的脑膜炎奈瑟氏菌小菌落扩散的活细胞成像   

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参见作者原研究论文

本实验方案简略版
PLOS Pathogens
Apr 2017

Abstract

To efficiently colonize the nasopharyngeal epithelium, the human restricted pathogen Neisseria meningitidis follows a multistep adhesion cascade. First, the bacteria adhere to host cells and aggregate into spherical shaped structures called microcolonies. Several hours later, single bacteria start dispersing from the microcolonies and form a monolayer on top of the host cells. Once in proximity to host cells meningococci can adhere tightly to the epithelial surface or become internalized. This can eventually result in invasion of the mucosal surfaces and gain access to the bloodstream, causing a life-threatening disease. Lactate, a metabolite derived from human epithelial cells, has been previously shown to induce rapid dispersal of N. meningitidis from microcolonies. Here, we describe a host-cell free method based on live-cell imaging to examine the effect of host derived lactate on the timing of N. meningitides microcolony dispersal. Although in this protocol we use lactate, it can be easily modified to test the effects of other molecules.

Keywords: Neisseria (奈瑟氏菌), Aggregation (聚集), Microcolonies (小菌落), Dispersal (扩散), Colonization (定植), Live-cell imaging (活细胞成像)

Background

N. meningitidis is an obligate human pathogen that is responsible for septicemia and/or meningitis. Initial attachment to the nasopharyngeal epithelium and subsequent formation of microcolonies are the first steps in the establishment of infection. In order to cause a disease N. meningitidis must cross the epithelial barrier in the nasopharyngeal mucosa, its natural reservoir, and enter the bloodstream (Stephens, 2009; Trivedi et al., 2011). Dispersal of bacteria from microcolonies plays an important role in progression to an invasive disease as it allows the bacteria to come in close contact with the host epithelium (Pujol et al., 1997 and 1999). Despite its importance, not much is known about the underlying mechanism that governs neisserial detachment from the microcolonies. Recently, we reported that lactate, a common metabolite produced and released from human host cells, is involved in inducing microcolony dispersal (Sigurlásdóttir et al., 2017). Here, we provide a step by step method adapted from Sigurlásdóttir et al. (2017) that can be used to examine the effect of small molecules on N. meningitidis microcolony dispersal in a host-cell free manner by time-lapse imaging. In the described method the focus is to examine microcolony dispersal after addition of lactate, however the effect of other molecules derived from the host or the microbiota can also be tested.

Materials and Reagents

  1. Glass bottom 24-well cell culture plates (MatTek, catalog number: P24G-1.5-13-F )
  2. Sterile plastic loops
    1 µl plastic loops (SARSTEDT, catalog number: 86.1567.050 )
    10 µl plastic loops (SARSTEDT, catalog number: 86.1562.050 )
  3. Falcon tubes
    15 ml Falcon tubes (SARSTEDT, catalog number: 62.554.502 )
    50 ml Falcon tubes (SARSTEDT, catalog number: 62.547.254 )
  4. Pipette tips
    0.1-10 µl capacity (SARSTEDT, catalog number: 70.1130.100 )
    20-200 µl capacity (SARSTEDT, catalog number: 70.760.502 )
    50-1,000 µl capacity (SARSTEDT, catalog number: 70.762.100 )
  5. Bacteriological Petri plates (SARSTEDT, catalog number: 82.1473 )
  6. 5-µm pore filter (VWR, catalog number: 514-4106 )
  7. 5 ml syringe (VWR, catalog number: 613-3940 )
  8. Serological pipettes
    5 ml pipette (SARSTEDT, catalog number: 86.1253.001 )
    25 ml pipette (SARSTEDT, catalog number: 86.1685.001 )
  9. 250 ml vacuum filtration unit, 0.22 μm (SARSTEDT, catalog number: 83.1822.001 )
  10. Bacterial strain: Neisseria meningitidis serogroup C strain FAM20 (Rahman et al., 1997). FAM20 is a nalidixic acid-resistant mutant of FAM18 (ATCC, catalog number: 700532 )
    Note: The bacterial stock is stored in 25% glycerol at -80 °C.
  11. Glycerol (Sigma-Aldrich, catalog number: G5516 )
  12. GC agar (NEOGEN, catalog number: 7104A )
  13. D-glucose (Sigma-Aldrich, catalog number: G8270 )
  14. L-glutamine (Sigma-Aldrich, catalog number: G8540 )
  15. Ferric(III) nitrate nonahydrate (FeNO3·9H2O) (Sigma-Aldrich, catalog number: F3002 )
    Note: This product has been discontinued.
  16. Cocarboxylase (Sigma-Aldrich, catalog number: C8754 )
  17. DMEM, no glucose, no glutamine, no phenol red (Thermo Fisher Scientific, GibcoTM, catalog number: A1443001 )
  18. GlutaMAXTM Supplement (Thermo Fisher Scientific, GibcoTM, catalog number: 35050038 )
  19. Sodium pyruvate (Thermo Fisher Scientific, catalog number: 11360039 )
  20. Fetal bovine serum (FBS), heat inactivated (Sigma-Aldrich, catalog number: F9665 )
  21. Sodium L-lactate (Sigma-Aldrich, catalog number: L7022 )
  22. Sodium D-lactate (Sigma-Aldrich, catalog number: 71716 )
  23. GC agar plates (see Recipes)
  24. 0.2% Cocarboxylase solution (see Recipes)
  25. Kellogg’s supplement (see Recipes)
  26. 2.5 mM glucose (see Recipes)
  27. DMEM (see Recipes)
  28. DMEM/1%FBS (see Recipes)
  29. Lactate solutions (see Recipes)

Equipment

  1. Class II biosafety cabinet (e.g., Esco Micro, model: Airstream® Class II Biological Safety Cabinet )
  2. Humidified 5% CO2 incubator at 37 °C (e.g., Thermo Fisher Scientific, Thermo ScientificTM, model: HeracellTM 150i )
  3. Water bath set to 37 °C (e.g., Grant Instruments, model: SAP12 )
  4. Spectrophotometer (e.g., Bio-Rad Laboratories, model: SmartSpec Plus )
  5. Inverted live-cell observer, connected to an incubator with a controlled temperature (temperature module) and CO2 module (e.g., Carl Zeiss, model: Axio Observer Z1 )
  6. Pipette boy (e.g., Fisher Scientific, model: FisherbrandTM Electric Pipet Controller )
  7. Pipettes
    0.5-10 µl capacity (e.g., Eppendorf, catalog number: 4924000029 )
    10-100 µl capacity (e.g., Eppendorf, catalog number: 4924000053 )
    100-1,000 µl capacity (e.g., Eppendorf, catalog number: 4924000088 )
  8. 1 L bottle
  9. 500 ml flask

Software

  1. AxioVision (Release: 4.8.2 SP3)
  2. Fiji (http://fiji.sc/; version: ImageJ 2.0.0-rc-44/1.50e)
  3. Microsoft Excel

Procedure

Caution: N. meningitidis is classified as a class II pathogen. Therefore, all work with bacterial solutions must be done in a Class II biosafety cabinet and according to a national regulation of biosafety. In addition, all bacterial waste should be discarded as Infectious/Biohazardous.

Note: Before performing the experiment the OD600 value corresponding to viable count 108 cfu/ml has to be determined. For the bacterial stock used in this experiment, the OD600: 0.36 is 108 cfu/ml. This can be done by performing serial dilutions of cultures containing bacteria that have been grown on a GC plate for 16-18 h. The dilutions should then be plated on GC plates and incubated overnight at 37 °C with 5% CO2 atmosphere.

Day 1
Preparation of overnight bacterial cultures. Work in a Class II biosafety cabinet.

  1. N. meningitidis is streaked from a glycerol stock on a GC agar plate (see Recipes) supplemented with 10% Kellogg’s supplement (see Recipes) and incubated overnight for 16-18 h at 37 °C in an incubator with a 5% CO2 environment.

Day 2
  1. Induction assay: Preparation and incubation of bacterial liquid cultures. Work in a Class II biosafety cabinet.
    1. The Axio Observer microscope should be turned on and pre-warmed to 37 °C at least 30 min prior to your experiment. The CO2 should be set to 5% before inserting the multiwell plate to the microscope.
    2. Prepare 30 ml pre-warmed DMEM (see Recipes) containing 1% FBS (DMEM/1% FBS, see Recipes). Fill 900 µl of the medium to the wells of 24-well glass bottom plate (MaTek) that will be used in the experiment (Figure 1). The multiwell plate should be warmed in a 37 °C incubator until needed in Step 1f.
      Note: Cultivation of N. meningitidis should always be performed with pre-warmed medium. In addition, the bacterial cultures should never be vortexed prior to experiments as this might induce shedding of bacterial pili, involved in microcolony formation.
    3. Half fill a 10 µl loop with bacteria from the overnight culture and resuspend in 2 ml of DMEM/1% FBS. Mix the bacterial solution 4 times with a 1 ml pipette. Wait for 3 min and let the largest bacterial aggregates settle down to the bottom of the tube.
    4. Transfer 1 ml of the uppermost bacterial solution, leaving the large aggregates behind, to a 50 ml Falcon tube containing 5 ml DMEM/1% FBS. Use a syringe to take up the bacterial solution and filter through a 5 µm-pore filter to a 15 ml Falcon tube.
      Note: In order to prevent variations in microcolony size that can occur due to differences in bacterial re-suspension, we filter the bacterial solution through 5 µm filter to break all pre-existing aggregates (Eriksson et al., 2012; Engman et al., 2016; Sigurlásdóttir et al., 2017).
    5. Use a spectrophotometer to measure the absorbance of the bacteria solution. Adjust the absorbance to OD600 = 0.36 (equivalent to 108 cfu/ml) by diluting with DMEM/1%FBS.
    6. Take the pre-warmed glass bottom plate from the incubator. Add 100 µl of bacterial solution to the wells containing 900 µl of DMEM/1%FBS (Figure 1). Mix 4 times with a 1 ml pipette. Start timing your 3 h incubation exactly when bacterial cultures are added to the multi-well plate. Write down the exact time when the cultures are added to the wells. Keep the 15 ml Falcon tube containing the bacterial solution for viable count (see Step 2a).
    7. Immediately transfer the multi-well plate to the pre-warmed microscope. Incubate for 3 h at 37 °C and 5% CO2 to allow bacteria to form microcolonies.


      Figure 1. Schematic drawing of experimental setup in the multiwell plate. Initially, bacterial culture is added to all relevant wells and incubated for 3 h under a microscope. After the incubation, lactate solutions are added to wells according to the scheme and the time-lapse started.

  2. Preparation during the 3 h incubation
    1. Perform serial dilutions and plating of the bacterial culture.
      Note: Immediately after the 3 h incubation starts, the viable count (108 cfu/ml) of the bacterial culture has to be confirmed by performing serial dilutions and plating.
      1. Dilute the bacterial solution (OD600 = 0.36) by 105 (five 10x dilutions, 50 µl:450 µl) in duplicates and plate 100 µl (10x dilution) of the last dilution to GC plates.
      2. Incubate the GC plates in a 5% CO2 incubator at 37 °C overnight.
      3. Count colonies and calculate the viable count (cfu/ml).
    2. Prepare the microscope.
      1. In AxioVision software, open multidimensional acquisition from the acquisition on the main tab. In the multidimensional window, click the experiment tab and choose time-lapse and position-list (Figure 2A). Choose a name for your experiment. Settings for experiment should be set to brightfield (BF). The Colibri, which is used for fluorescent microscopy only, should always be turned off and the halogen lamp (HAL), which is used for BF, should be turned off after the experiment.
      2. In the C tab (channel) choose brightfield in the Dye section and make sure that BF is turned on during acquisition and HAL off after acquisition. Set focus to current (Figure 2B).


        Figure 2. Settings in the AxioVision multidimensional acquisition experiment (A) and channel (B) tabs

      3. In the T tab (time) choose time-lapse and interval (Figure 3A). To follow the effect on microcolony dispersal, set the multidimensional acquisition to capture image every 5 or 10 min for 5 h total (8 h total incubation).
      4. In the XY tab, choose both position-list and apply settings before/after timepoint per position (Figure 3B). Click Mark Find and a new window will open. Choose a 24-well plate from the slide/dish collection. Choose the first well containing bacterial solution, well B2 according to the scheme in Figure 1. Click on the live button on the main tab or through acquisition on the main tab. By using bright field illumination and a 40x lens, adjust the focus and set three random positions per well. Place the positions not too close to each other and not too close to the edge of the well, see graphic selected well in Figure 4. Save your positions.
    3. Start preparing and pre-warming lactate solutions (see Recipes) at different concentrations in DMEM 1 h before addition to microcolonies. The prepared lactate solutions can be kept either in 37 °C incubator or a heating block until addition to microcolonies. For a control, pre-warm DMEM as well.


      Figure 3. Settings in the AxioVision multidimensional acquisition time (A) and xy (B) tabs


      Figure 4. Mark Find window in AxioVision. The image shows how three random positions within a well should be located.

  3. Induction assay: addition of lactate to microcolonies and time-lapse imaging
    1. After 3 h incubation, open the incubator unit and add 1 ml of each lactate solutions and DMEM control to relevant wells (see scheme in Figure 1). Note that the solutions have to be added quickly but also carefully, so the microcolonies will not be flushed away from the position chosen. Close the incubator unit.
    2. After all the solutions have been added to relevant wells, quickly examine the positions chosen in the microscope and make sure that the microcolonies chosen have not moved out of position upon addition of lactate solutions. If they do not contain microcolonies, quickly relocate the position. The exposure time needs to be adjusted before starting time lapse. All the adjustments have to be done within 10 min so the time-lapse can be started exactly at 10 min after addition of lactate.
    3. Start image acquisition, at every 5 or 10 min for 5 h. To avoid differences in timing, capture images in the same order as lactate was added to the wells. Analyze the time-lapse with either the AxioVision software or the Fiji software (see Data analysis).

Data analysis

Acquired images are either analyzed with the software AxioVision or with the license free alternative Fiji (Image J). In Fiji, the files should be opened in XYCZT hyperstack. Evaluate and write down the timing of dispersal phase for every position within the samples, which is the first frame when bacteria start detaching from microcolonies and the last frame when microcolonies are present. If the time-lapse starts exactly 10 min after addition of lactate then the first frame (T1) will be 190 min (3 h and 10 min) and T2 will be 195 min or 200 min depending on if images have been acquired every 5 min or 10 min (Figure 5).


Figure 5. Example of image analysis. In the following time-lapse, 10 mM L-lactate has been added to the well after a 3 h incubation of bacteria. The time-lapse was started 10 min after the addition of lactate (190 min). In frame T3, detachment of bacteria from microcolonies is visible. In frame T8, no aggregates are present anymore. Note that the frames have been cropped in order to create the figure. Scale bar = 20 μm.

  1. In Excel, make a table with the timing of aggregation phase and dispersal phase for every position within the samples. Aggregation phase is the time from the start of the incubation (0 h) until the last frame before dispersal from microcolonies starts. To get the exact length in time of dispersal phase, subtract the timing when dispersal ends with the timing when dispersal starts. Calculate the average of the three positions. Convert the minutes to hours by dividing the minutes by 60. For length of time of planktonic phase, subtract 8 h (total hours) by length of time in aggregation phase and dispersal phase. Use stacked column chart in Excel to make a graph. According to the example in Figure 5, the dispersal phase occurs between 200 and 220 min, aggregation phase will therefore be from 0-200 min. Planktonic phase will last from 220 min until the end of the timelapse or 480 min (8 h). However, the planktonic phase should be calculated either from the average of the three positions for a single experiment or the average of three independently performed experiments. Data presentation can be seen in Sigurlásdóttir et al. (2017).
  2. For lactate induced dispersal, three independently performed experiments are sufficient for statistical analysis. However, the number of replicates required to obtain statistically significant data might increase depending on the molecule tested and/or other variabilities in the experimental settings and procedure.

Recipes

  1. GC agar plates (1 L)
    1. Add 36 g GC agar medium base to a 1 L bottle
    2. Add 600 ml of distilled H2O and autoclave at 120 °C for 20 min
    3. Add up to 1 L of sterile cold distilled H2O
    4. When the temperature is approximately 60 °C, add 10 ml of Kellogg’s supplement (Recipe below)
    5. Pour into Petri dishes and allow the plates to cool down and store at 4 °C
  2. 0.2% Cocarboxylase solution
    0.02 g in 100 ml of distilled water
    Sterile filter with 0.22 µm filter
    Store at 4 °C
  3. Kellogg’s supplement (200 ml)
    1. Add 80 g of D-glucose slowly to 100 ml of distilled H2O and allow it to dissolve
    2. Add 1 g of L-glutamine, 0.1 g of ferric nitrate nonahydrate (FeNO3·9H2O) and 2 ml of 0.2% Cocarboxylase solution (Recipe 2)
    3. When everything is dissolved, adjust the volume to 200 ml with distilled H2O
    4. Sterile filter with 0.22 µm filter and store at 4 °C
  4. 2.5 mM glucose
    45.04 g glucose in 100 ml of distilled water
    Sterile filter with 0.22 µm filter
    Store at 4 °C
  5. DMEM
    For a 500 ml flask of DMEM add the following:
    5 ml of GlutaMAX
    5 ml of 2.5 mM glucose (Recipe 4)
    5 ml of sodium pyruvate
  6. DMEM/1%FBS
    For every 30 ml of DMEM add 0.3 ml of FBS
  7. Lactate solutions
    Notes:
    1. L-lactate and D-lactate solutions are prepared in the same way.
    2. The lactate solutions are added to the bacterial culture at 1:1 ratio. Therefore the concentration of solutions prepared has to be twice the final concentration.
    Lactate stock solution (1 M): 1.1206 g lactate in 10 ml of distilled water
    4 mM (final concentration 2 mM): add 4 µl of 1 M lactate solution to DMEM at a final volume of 1 ml
    20 mM (final concentration 10 mM): add 20 µl of 1 M lactate solution to DMEM at a final volume of 1 ml
    100 mM (final concentration 50 mM): add 100 µl of 1 M lactate solution to DMEM at a final volume of 1 ml

Acknowledgments

The work was adapted from a protocol previously published in Sigurlásdóttir et al. (2017). We thank Dr. Sunil D. Saroj for comments on the manuscript. 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 of interest.

References

  1. Engman, J., Negrea, A., Sigurlásdóttir, 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.
  2. 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.
  3. Pujol, C., Eugene, E., de Saint Martin, L. and Nassif, X. (1997). Interaction of Neisseria meningitidis with a polarized monolayer of epithelial cells. Infect Immun 65(11): 4836-4842.
  4. Pujol, C., Eugene, E., Marceau, M. and Nassif, X. (1999). The meningococcal PilT protein is required for induction of intimate attachment to epithelial cells following pilus-mediated adhesion. Proc Natl Acad Sci U S A 96(7): 4017-4022.
  5. 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.
  6. 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.
  7. Stephens, D. S. (2009). Biology and pathogenesis of the evolutionarily successful, obligate human bacterium Neisseria meningitidis. Vaccine 27 Suppl 2: B71-77.
  8. Trivedi, K., Tang, C. M. and Exley, R. M. (2011). Mechanisms of meningococcal colonisation. Trends Microbiol 19(9): 456-463.

简介

为了有效地定居鼻咽上皮,人类限制性病原体脑膜炎奈瑟氏球菌遵循多步粘附级联。首先,细菌粘附到宿主细胞并聚集成称为微菌落的球形结构。几个小时后,单细菌开始从微菌落分散并在宿主细胞上形成单层。一旦接近宿主细胞,脑膜炎球菌可紧密地粘附在上皮表面或内化。这最终可能导致粘膜表面的侵入并进入血液,导致危及生命的疾病。乳酸是一种来源于人类上皮细胞的代谢物,之前已被证明能诱导N的快速分散。 meningitidis 来自微菌落。在这里,我们描述基于活细胞成像的宿主细胞自由方法来检查宿主来源的乳酸对N的时间的影响。 meningitides microcolony扩散。虽然在这个协议中我们使用乳酸盐,它可以很容易地修改,以测试其他分子的影响。

【背景】ñ。脑膜炎是引起败血症和/或脑膜炎的专性人类病原体。鼻咽上皮的初始附着和随后形成的小菌落是建立感染的第一步。为了导致疾病N。脑膜炎奈瑟球必须穿过鼻咽粘膜上皮屏障,其天然储库,并进入血液(Stephens,2009; Trivedi等人,2011)。微菌落中的细菌分散在侵入性疾病发展中起重要作用,因为它使细菌与宿主上皮细胞紧密接触(Pujol等人,1997和1999)。尽管它的重要性,但是从微菌落控制neisserial分离的基本机制知之甚少。最近,我们报道了从人类宿主细胞产生和释放的一种常见代谢物乳酸盐参与诱导小菌落扩散(Sigurlásdóttir et al。,2017)。在这里,我们提供了一个由Sigurlásdóttir等人(2017)改编的一步一步的方法,可以用来检查小分子对N的影响。通过延时成像以无宿主细胞的方式传播脑膜炎微生物菌落。在所描述的方法中,重点是检查添加乳酸后的微菌落扩散,然而也可以测试来自宿主或微生物群的其他分子的作用。

关键字:奈瑟氏菌, 聚集, 小菌落, 扩散, 定植, 活细胞成像

材料和试剂

  1. 玻璃底部24孔细胞培养板(MatTek,目录号:P24G-1.5-13-F)
  2. 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)
  3. 猎鹰管

    15毫升Falcon管(SARSTEDT,目录号:62.554.502)
    50毫升Falcon管(SARSTEDT,目录号:62.547.254)
  4. 移液器提示
    0.1-10μl容量(SARSTEDT,目录号:70.1130.100)
    20-200μl容量(SARSTEDT,目录号:70.760.502)
    50-1,000μl容量(SARSTEDT,目录号:70.762.100)
  5. 细菌培养皿(SARSTEDT,目录号:82.1473)
  6. 5-μm孔径过滤器(VWR,目录号:514-4106)
  7. 5毫升注射器(VWR,目录号:613-3940)
  8. 血清移液器
    5毫升吸管(SARSTEDT,目录号:86.1253.001)
    25毫升吸管(SARSTEDT,目录号:86.1685.001)

  9. 250 ml真空过滤装置,0.22μm(SARSTEDT,目录号:83.1822.001)
  10. 细菌菌株:脑膜炎奈瑟氏球菌血清组C菌株FAM20(Rahman等人,1997)。 FAM20是FAM18(ATCC,目录号:700532)的萘啶酮酸抗性突变体。 注意:细菌储存在-80°C的25%甘油中。
  11. 甘油(Sigma-Aldrich,目录号:G5516)
  12. GC琼脂(NEOGEN,目录号:7104A)
  13. D-葡萄糖(Sigma-Aldrich,目录号:G8270)
  14. L-谷氨酰胺(Sigma-Aldrich,目录号:G8540)
  15. 三价硝酸铁(III)九水合物(FeNO 3•9H 2 O)(Sigma-Aldrich,目录号:F3002)
    注:此产品已停产。
  16. 羧化酶(Sigma-Aldrich,目录号:C8754)
  17. DMEM,不含葡萄糖,不含谷氨酰胺,不含酚红(Thermo Fisher Scientific,Gibco TM,目录号:A1443001)
  18. GlutaMAX TM补充物(Thermo Fisher Scientific,Gibco TM,目录号:35050038)
  19. 丙酮酸钠(Thermo Fisher Scientific,目录号:11360039)
  20. 胎牛血清(FBS),加热灭活(Sigma-Aldrich,目录号:F9665)
  21. L-乳酸钠(Sigma-Aldrich,目录号:L7022)
  22. D-乳酸钠(Sigma-Aldrich,目录号:71716)
  23. GC琼脂平板(见食谱)
  24. 0.2%羧化酶溶液(见食谱)
  25. 凯洛格的补充(见食谱)
  26. 2.5毫米葡萄糖(见食谱)
  27. DMEM(见食谱)
  28. DMEM / 1%FBS(见食谱)
  29. 乳酸盐溶液(见食谱)

设备

  1. II级生物安全柜(,例如,Esco Micro,型号:Airstream®II级生物安全柜)
  2. 在37℃加湿的5%CO 2培养箱(例如,Thermo Fisher Scientific,Thermo Scientific TM,型号:Heracell TM TM) / sup> 150i)

  3. 水浴温度设定为37°C(例如,Grant Instruments,型号:SAP12)
  4. 分光光度计(例如,Bio-Rad Laboratories,型号:SmartSpec Plus)
  5. 倒置的活细胞观察器,连接到具有受控温度(温度模块)和CO 2模块(例如,Carl Zeiss,型号:Axio Observer Z1)的培养箱。 />
  6. 移液器男孩(例如,Fisher Scientific,Fisher Scientific,Fisher Scientific TM电动移液管控制器)
  7. 移液器
    (例如,Eppendorf,目录号:4924000029)
    (例如,Eppendorf,产品目录号:4924000053)。
    (例如,Eppendorf,产品目录号:4924000088)。
  8. 1升瓶
  9. 500毫升的烧瓶

软件

  1. AxioVision(版本:4.8.2 SP3)
  2. 斐济( http://fiji.sc/ ;版本:ImageJ 2.0.0-rc-44 / 1.50e)< br />
  3. Microsoft Excel

程序

小心:脑膜炎奈瑟球菌属于II类病原体。因此,所有使用细菌解决方案的工作都必须在二级生物安全柜内进行,并根据国家生物安全法规进行。此外,所有的细菌废物应作为感染/生物危害物丢弃。

注意:在进行实验之前,必须确定对应于活细胞计数10 8 cfu / ml的OD 600值。对于本实验中使用的细菌原液,OD 600:0.36是10 8 cfu / ml。这可以通过连续稀释含有在GC板上生长的细菌16-18小时的培养物来完成。然后将这些稀释物接种在GC平板上,并在37℃,5%CO 2气氛下温育过夜。

第1天
制备过夜细菌培养物。在第二类生物安全柜中工作。

  1. ñ。从补充有10%凯洛格补充剂的GC琼脂平板(参见食谱)的甘油储备液中划线培养脑膜炎奈瑟球菌(参见配方),并在37℃下在具有5%CO的培养箱中温育过夜16-18小时

第2天
  1. 诱导分析:制备和培养细菌液体培养物。在第二类生物安全柜中工作。
    1. 应该打开Axio Observer显微镜,并在实验前至少30分钟预热至37°C。在将多孔板插入显微镜之前,应将CO 2设定为5%。
    2. 准备30毫升预热DMEM(见食谱)含有1%FBS(DMEM / 1%FBS,见食谱)。向实验中使用的24孔玻璃底板(MaTek)的孔中加入900μl培养基(图1)。
      多孔板应在37°C培养箱中加热,直到步骤1f需要 注:脑膜炎奈瑟球菌的培养应始终使用预热的培养基进行。此外,细菌培养物在实验之前不应该被涡旋,因为这可能会导致细菌菌毛脱落,参与小菌落形成。
    3. 用过夜培养物中的细菌一半填充10μl环,并重悬于2ml DMEM / 1%FBS中。用1毫升移液管混合细菌溶液4次。等待3分钟,让最大的细菌聚集体沉降到管底。
    4. 转移1毫升最上面的细菌溶液,留下大的聚集体,到50毫升含有5毫升DMEM / 1%FBS的Falcon试管中。使用注射器吸收细菌溶液,并通过5微米孔过滤器过滤到15毫升猎鹰管。
      注意:为了防止由于细菌再悬浮的不同而可能发生的小菌落大小的变化,我们通过5μm过滤器过滤细菌溶液以破坏所有预先存在的聚集体(Eriksson等人,2012; Engman et al。,2016;Sigurlásdóttir等,2017)。
    5. 用分光光度计测量细菌溶液的吸光度。通过用DMEM / 1%FBS稀释,将吸光度调节至OD 600 = 0.36(相当于10 8 cfu / ml)。
    6. 从孵化器预热的玻璃底板。向含有900μlDMEM / 1%FBS的孔中加入100μl细菌溶液(图1)。用1毫升移液管混合4次。开始计时您的3小时孵化准确时,细菌培养物添加到多孔板。写下培养物添加到孔中的确切时间。保持15毫升含有细菌溶液的Falcon管进行活菌计数(见步骤2a)。
    7. 立即将多孔板转移到预热显微镜。在37℃和5%CO 2下孵育3小时以使细菌形成微菌落。


      图1.多孔板中实验装置的示意图。最初,将细菌培养物加入到所有相关的孔中,并在显微镜下温育3小时。孵化后,根据计划将乳酸盐溶液加入孔中,并开始时间推移。

  2. 在3小时的孵育期间进行准备
    1. 执行系列稀释和电镀细菌培养。
      注意:培养3小时后立即将细菌培养物的活菌计数(10 8 cfu / ml)通过连续稀释和电镀进行确认。
      1. 用10 5(5次10倍稀释,50μl:450μl)重复两次,并将100μl(10倍稀释)的细菌溶液(OD 600 = 0.36)稀释最后稀释到GC板。
      2. 在5%CO 2孵育器中在37℃孵育GC板过夜。
      3. 计数殖民地和计算可行的计数(cfu / ml)。
    2. 准备显微镜。
      1. 在AxioVision软件中,在主选项卡上从采集中打开多维采集。在多维窗口中,点击实验选项卡,然后选择延时和位置列表(图2A)。为您的实验选择一个名称。实验设置应设置为明场(BF)。用于荧光显微镜的Colibri应该始终关闭,用于BF的卤素灯(HAL)应该在实验后关闭。
      2. 在C选项卡(通道)中选择Dye区域中的明场,确保采集期间BF开启,采集后关闭HAL。将焦点设置为当前(图2B)。


        图2. AxioVision多维采集实验(A)和通道(B)选项卡中的设置

      3. 在T标签(时间)中选择延时和时间间隔(图3A)。为了遵循对小菌落扩散的影响,将多维采集设置为每5或10分钟捕获图像,总共5小时(总共孵育8小时)。
      4. 在XY选项卡中,选择位置列表并在每个位置的时间点之前/之后应用设置(图3B)。点击标记查找,将打开一个新窗口。从幻灯片/碟集合中选择一个24孔板。根据图1中的方案选择含有细菌溶液的第一口井B2。点击主选项卡上的实时按钮或通过主选项卡上的采集。通过使用明场照明和40倍镜头,调整焦点并设置每个井的三个随机位置。放置位置不要太靠近彼此,不要太靠近井的边缘,见图4中选择的图形。保存你的位置。
    3. 开始准备和预加热乳酸溶液(见食谱)不同浓度的DMEM 1小时,然后加入微菌落。制备的乳酸盐溶液可以保存在37℃培养箱或加热块中,直到加入微菌落。对于一个控制,预热DMEM。


      图3. AxioVision多维采集时间(A)和xy(B)选项卡中的设置


      图4. AxioVision中的标记查找窗口。 图片显示了一个井内三个随机位置应该如何定位。

  3. 诱导测定:将乳酸添加到微菌落和延时成像
    1. 培养3小时后,打开培养箱,向相关孔中加入1ml乳酸盐溶液和DMEM对照(见图1中的方案)。请注意,解决方案必须快速,但也要小心,所以小菌落不会被冲走所选位置。关闭孵化器。
    2. 将所有溶液添加到相关孔中后,快速检查显微镜中选择的位置,确保添加乳酸盐溶液后所选的微菌落没有移出位置。如果他们不包含微菌落,迅速重新安置这个位置。曝光时间需要在开始时间之前进行调整。所有的调整必须在10分钟内完成,所以时间推移可以在加入乳酸后10分钟开始。
    3. 开始图像采集,每5或10分钟5小时。为了避免时间上的差异,将与乳酸盐相同顺序的图像添加到孔中。
      使用AxioVision软件或斐济软件分析时间推移(请参阅数据分析)。

数据分析

获取的图像或者用软件AxioVision或者免费的替代斐济(Image J)进行分析。在斐济,文件应该在XYCZT hyperstack中打开。评估和记录样本内每个位置的扩散阶段的时间,这是细菌开始从微菌落中分离出来的第一帧,当微菌落存在时的最后一帧。如果时间推移正好从添加乳酸后10分钟开始,则第一帧(T1)将是190分钟(3小时和10分钟),T2将是195分钟或200分钟,这取决于是否每隔5分钟获取图像或10分钟(图5)。


图5.图像分析示例在下面的时间推移中,细菌孵育3小时后,10mM L-乳酸已被添加到孔中。乳酸盐加入10分钟后开始延时(190分钟)。在框架T3中,细菌从微菌落中分离是可见的。在T8帧中,不再有聚合体出现。请注意,框架已被裁剪,以创建图形。比例尺=20μm。

  1. 在Excel中,为样本内的每个位置制作聚合阶段和分散阶段的时间表。聚集阶段是从孵化开始(0h)直到从微菌落开始分散之前的最后一帧开始的时间。为了得到分散阶段的准确长度,减去分散结束的时间与分散开始的时间。计算三个位置的平均值。通过将分钟除以60来将分钟转换为小时。对于浮游阶段的时间长度,在聚集阶段和分散阶段中按时间长度减去8小时(总小时数)。在Excel中使用堆积的柱形图来绘制图形。根据图5中的例子,分散阶段发生在200到220分钟之间,因此聚集阶段将从0到200分钟。浮游阶段将持续220分钟,直到时间推移结束或480分钟(8小时)。然而,浮游阶段应该从三个位置的平均值进行单个实验或三个独立进行实验的平均值。数据呈现可以在Sigurlásdóttir等人看到。 (2017)。
  2. 对于乳酸诱导的分散,三个独立进行的实验足以进行统计分析。然而,获得统计学显着数据所需的重复次数可能会增加,取决于所测试的分子和/或实验设置和程序中的其他变化。

食谱

  1. GC琼脂平板(1L)
    1. 将36克GC琼脂培养基加入1升的瓶子中
    2. 加入600毫升蒸馏过的H 2 O和高压灭菌器在120℃下20分钟。
    3. 加1L无菌冷蒸馏H 2 O
    4. 当温度大约60°C时,加10毫升凯洛格的补充剂(下面的配方)
    5. 倒入培养皿,让盘子冷却下来,并在4°C存储
  2. 0.2%羧化酶溶液
    在100毫升蒸馏水中0.02克
    无菌过滤器与0.22微米过滤器
    在4°C储存
  3. 凯洛格的补充(200毫升)
    1. 将80克D-葡萄糖缓慢加入到100毫升蒸馏过的H 2 O中并使其溶解
    2. 加入1g L-谷氨酰胺,0.1g硝酸铁九水合物(FeNO 3•9H 2 O)和2ml 0.2%羧化酶溶液(配方2) />
    3. 当一切溶解后,用蒸馏水调节体积至200ml
    4. 无菌过滤器,0.22μm过滤器,4°C储存
  4. 2.5 mM葡萄糖
    45.04克葡萄糖在100毫升蒸馏水中
    无菌过滤器与0.22微米过滤器
    在4°C储存
  5. DMEM
    对于一个500毫升的DMEM烧瓶添加以下内容:
    5毫升的GlutaMAX
    5毫升的2.5毫米葡萄糖(方案4)
    5毫升的丙酮酸钠
  6. DMEM / 1%FBS
    每30毫升的DMEM添加0.3毫升的FBS
  7. 乳酸盐溶液
    注意:
    1. 以相同的方式制备L-乳酸盐和D-乳酸盐溶液。
    2. 将乳酸盐溶液以1:1的比例加入到细菌培养物中。因此,制备的溶液浓度必须是最终浓度的两倍。
    乳酸盐储备液(1M):1.1206克乳酸盐在10毫升蒸馏水中
    4 mM(终浓度2 mM):将4μl1M乳酸盐溶液加入到DMEM中,终体积为1 ml。
    20 mM(终浓度10 mM):将20μl1M乳酸盐溶液加入到DMEM中,终体积为1 ml。
    100 mM(终浓度50 mM):将100μl1M乳酸盐溶液加入DMEM中,终体积为1 ml。

致谢

该工作是根据之前在Sigurlásdóttir等人发表的一项议定书(2017年)进行的。我们感谢Sunil D. Saroj博士对手稿的评论。这项工作由瑞典研究委员会(Dnr 2006-4112,2012-2415,2013-2434),瑞典癌症协会和TorstenSöderbergsStiftelse资助。作者宣称没有利益冲突。

参考

  1. Engman,J.,Negrea,A.,Sigurlásdóttir,S.,Georg,M.,Eriksson,J.,Eriksson,O.S。,Kuwae,A.,Sjolinder,H.and Jonsson,A.B。(2016)。 脑膜炎奈瑟氏球菌多核苷酸磷酸化酶影响聚集,粘附和毒力。感染免疫84(5):1501-1513。
  2. Eriksson,J.,Eriksson,O.S.和Jonsson,A.B.(2012)。 脑膜炎球菌PilU的缺失延缓了小菌落的形成并减弱了体内的毒力。 /感染免疫 80(7):2538-2547。
  3. Pujol,C.,Eugene,E.,de Saint Martin,L.和Nassif,X。(1997)。 脑膜炎奈瑟氏球菌与上皮细胞的极化单层的相互作用感染免疫 65(11):4836-4842。
  4. Pujol,C.,Eugene,E.,Marceau,M.和Nassif,X。(1999)。 脑膜炎球菌PilT蛋白是诱导与菌毛介导的粘附后上皮细胞的紧密附着所必需的。美国国家科学院院刊96(7):4017-4022。
  5. Rahman,M.,Kallstrom,H.,Normark,S.和Jonsson,A.B。(1997)。 致病性奈瑟氏菌PilC 与细菌细胞表面相关。分子微生物学25(1):11-25。
  6. Sigurlásdóttir,S.,Engman,J.,Eriksson,O.S。,Saroj,S.D.,Zguna,N.,Lloris-Garcera,P.,Ilag,L.L。和Jonsson,A.B.(2017)。 宿主细胞衍生的乳酸在脑膜炎奈瑟氏球菌中的效应分子功能 microcolony (PLoS Pathog) 13(4):e1006251。
  7. Stephens,D. S.(2009)。 进化上成功的专性人类细菌脑膜炎奈瑟球菌的生物学和发病机制。 疫苗 27增刊2:B71-77。
  8. Trivedi,K.,Tang,C.M。和Exley,R.M。(2011)。 脑膜炎球菌定殖机制 趋势Microbiol 19(9 ):456-463。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Sigurlásdóttir, S., Eriksson, O. S., Eriksson, J. and Jonsson, A. (2018). Live-cell Imaging of Neisseria meningitidis Microcolony Dispersal Induced by Lactate or Other Molecules. Bio-protocol 8(2): e2695. DOI: 10.21769/BioProtoc.2695.
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