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Bacterial Survival in Dictyostelium
盘基网柄菌中细菌存活能力的检测   

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Abstract

We performed an assay to test the ability of different E. coli strains to survive inside amoebal cells after ingestion. In the assay we incubated bacteria together with cells of Dictyostelium discoideum for six hours. After co-incubation most of the uningested bacteria were removed by centrifugation and the remaining uningested bacteria were killed by gentamicin. Gentamicin is used because it does not penetrate into eukaryotic cells allowing the ingested bacteria to survive the antibiotic treatment, whereas bacteria outside the amoebal cells are killed.

Keywords: Bacteria(细菌), Amoebae(变形虫), Protozoa(原生动物), Protists(原生生物), Grazing(放牧), Digestion resistance(消化阻力)

Background

Bacteria have evolved several different strategies to avoid or resist protozoan predation (Rønn et al., 2012). Mechanisms that reduce chance of ingestion such as increased cell size, aggregation, biofilm formation and increased swimming speed are well documented (Matz and Kjelleberg, 2005; Rønn et al., 2012) whereas mechanisms that allow bacteria to resist digestion in protozoan food vacuoles after ingestion are less studied (Gong et al., 2016). Here we describe a method to investigate the ability of bacterial strains to survive after ingestion by the social amoeba Dictyostelium discoideum. We used this assay to investigate if copper resistant E. coli have higher chance of survival after ingestion than bacteria without copper resistance (Hao et al., 2016). We studied copper resistance because it is known that macrophages, which are phagocytotic cells with an important role in the immune system of vertebrates, use copper to kill bacteria in the phagosome (Hodgkinson and Petris, 2012). We hypothesized that this killing mechanism originally evolved in free-living protozoa long before multicellular life arose and hence copper resistance could be a factor that protects bacteria against protozoan predation.

The assay is a modification of the Gentamicin protection assay used for evaluation of the ability of macrophages to kill bacteria (see e.g., Kaneko et al., 2016) and it utilizes that gentamicin is not able to penetrate into eukaryotic cells. In the original assay the number of internalized bacterial cells in the macrophages is determined at two time points. First, macrophages are incubated with bacteria for a given time period and after this co-incubation the uningested bacteria are removed and the number of internalized bacteria is estimated. Second measurement is taken after macrophages are incubated without extracellular bacteria to allow them to digest the internalized bacteria. The ratio between these two estimates is a measure of the bacterial survival rate.

Rapid digestion of some of our strains led to reduced reliability of estimates of the disappearance rate and made the original procedure unsuitable for use with Dictyostelium. We therefore modified the procedure so that we only estimated the number of surviving internalized bacteria at one time point after co-incubation of bacteria and amoebae for 6 h. This estimate reflects an equilibrium between ingestion and digestion rate of bacteria. It should be noted that this will only allow comparison of the digestion rate of different bacterial strains if it can be assumed that the bacteria are consumed with similar rates. In our case we had evidence that the different E. coli strains were taken up by the amoebae to the same extent and therefore we used the assay as an indicator of digestion rate. The assay will still be useful even if it cannot be assumed that bacteria are ingested at the same rate but of course it should be interpreted accordingly. It must also be noted that the procedure can only be used with bacteria that are sensitive to gentamicin.

Materials and Reagents

  1. Pipettes and sterile tips
  2. Sterile centrifuge tubes (e.g., Falcon® 50 ml conical centrifuge tubes)
  3. Sterile cell culture flasks (e.g., 25 cm2 Nunc® EasYFlasksTM)*
  4. 24 well, cell culture plates, flat bottom (Corning, Costar®–or similar)
  5. Sterile 1.5 ml Eppendorf tubes
  6. 50 ml Falcon tubes (Corning®–or similar)
  7. Sterile filters (0.2 µm) for sterile filtration (Corning)
  8. Dictyostelium discoideum AX4 (or other axenic strain; can be obtained at the Dicty Stock Center, Northwestern University, Chicago, IL, USA)
  9. Bacterial cultures*
  10. Gentamicin (Sigma-Aldrich, catalog number: G1264 )
  11. Dihydrostreptomycin sulfate (Sigma-Aldrich, catalog number: PHR1517 )
  12. Triton X-100 (Sigma-Aldrich, catalog number: 93443 )
  13. OxoidTM tryptone (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: LP0042 )
  14. OxoidTM yeast extract (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: LP0021 )
  15. Sodium phosphate dibasic heptahydrate (Na2HPO4·7H2O) (Sigma-Aldrich, catalog number: S9390 )
  16. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P5655 )
  17. Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: 435570 )
    Note: This product has been discontinued.
  18. Glucose (Sigma-Aldrich, catalog number: G8270 )
  19. Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: C5670 )
  20. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: 310166 )
    Note: This product has been discontinued.
  21. Agar
  22. Sterile HL5 medium (see Recipes)
  23. SorC buffer (see Recipes)
  24. Sterile Luria Broth (LB) (see Recipes)
  25. Agar plates with Luria agar (see Recipes)

    *Note: This protocol describes the procedure we used to compare survival rate of different E. coli strains, but other bacteria may be used. It is a requirement that the bacteria are sensitive to gentamicin.

Equipment

  1. Pipette
  2. Laminar flow hood (Heraeus, model: Air HLB 2472 )
  3. 125 ml Erlenmeyer flasks
  4. Rotary shaker for incubation at 22 °C
  5. 37 °C constant incubator (Thermo Fisher Scientific)*
  6. Rotary shaker for incubation at 37 °C*
  7. Benchtop centrifuge for Eppendorf tubes (cooled at 4 °C) (Eppendorf, model: 5424 R )
  8. Centrifuge for 50 ml Falcon tubes (HERMLE LABORTECHNIK, model: Z 326 K )
  9. Spectrophotometer and respective cuvettes (600 nm)
  10. Autoclave (STIK, model: MJ-Series )
  11. Vortex mixer (Scientific Industries, model: Vortex-Genie 2 , catalog number: SI-0236)
  12. pH meter and calibration buffers
  13. Inverted microscope (Olympus, model: CKX31 )**
  14. Microscope (Nikon Instruments, model: ECILPSE 80i)
  15. Neubauer hemocytometer blood count with double counting chamber (Gizmo Supply, Germany)

    *Note: Incubators set at 37 °C are necessary for work with E. coli but if other bacteria are used, growth temperature conditions should of course be modified accordingly.
    **Note: An inverted microscope is very useful for inspection of culture flasks and cell culture plates during the experiment.

Procedure

Notes:

  1. All work is carried out under sterile conditions using a laminar flow hood.
  2. The experiments are carried out with liquid axenic cultures of Dictyostelium discoideum. Strains of D. discoideum are usually provided on plates (e.g., from the Dicty stock center). Before start of the experiments, the amoebae should be brought into liquid culture. Detailed and useful information about culturing Dictyostelium can be found at the homepage of the Dicty stock center, http://dictybase.org/ (Fey et al., 2007).
  3. Preculturing of Dictyostelium in cell culture flasks has the advantage that it allows direct inspection of the cultures, but it is also possible to grow the cultures in shaking liquid cultures e.g., in 125 ml Erlenmeyer flasks. This can make it easier to obtain high cell densities in the cultures.
  4. Growth of Dictyostelium can be improved if it is supplied with washed heat-killed bacteria. We have previously used overnight cultures of E. coli which were washed by centrifugation, resuspended in SorC and heat-killed at 65 °C for 10 min.
  5. In this protocol we used different strains of E. coli. The procedure could be used for other bacteria; in this case bacteria-specific growth conditions, optical densities (OD) for estimation of bacterial cell density etc. should be modified accordingly.
  1. Day 1. Start D. discoideum culture
    1. Add 500 µl of a 2-3 day old D. discoideum culture to 10 ml fresh sterile HL5 in sterile culture flasks.
    2. Incubate flasks at 22 °C (without shaking).

  2. Day 2(-3). Start bacterial cultures
    1. Start bacterial cultures by inoculating 25 ml sterile LB medium with 1% inoculum of E. coli culture (overnight growth) in 125 ml Erlenmeyer flasks.
    2. Incubate overnight on a rotary shaker at 37 °C, 200 rpm.

  3. Day 3(-4). Survival assay
    1. Harvest bacteria
      1. Harvest 1 ml of bacteria by centrifugation at 12,000 x g for 2 min.
      2. Remove supernatant by carefully pipetting the liquid without disturbing the pellet.
      3. Resuspend bacterial pellet in 1 ml of 1:4 HL5:SorC solution.
      4. Centrifuge at 12,000 x g for 2 min.
      5. Remove the supernatant and resuspend bacterial pellet in 1 ml of 1:4 HL5:SorC solution.
      6. Adjust bacterial density to OD600 of 0.1 (around 108 CFU ml-1).
    2. Harvest D. discoideum cells
      1. Add amoebal culture to 50 ml Falcon tubes and centrifuge at 500 x g for 4 min.
      2. Remove supernatant by carefully pipetting the liquid without disturbing the pellet.
      3. Resuspend amoebae in 25 ml of 1:4 HL5:SorC solution.
      4. Centrifuge at 500 x g for 4 min.
      5. Resuspend amoebae in 1:4 HL5:SorC solution.
      6. Estimate amoebal density in the suspension by counting in a hemocytometer.
      7. Adjust amoebal density to 1 x 106 cells ml-1.
    3. Start survival assay
      1. Add 900 µl amoebal suspension (from step C2g) to a number of wells in 24-well culture plates.
        Note: We use at least 6 replicates for each bacterial strain.
      2. Add 100 µl bacterial suspension adjusted to approximately 4.5 x 107 cells ml-1 in order to obtain a multiplicity of infection (MOI) of 5.
      3. To obtain other levels of MOI, the bacterial cell densities can be adjusted accordingly.
      4. Prepare a set of replicates with 900 µl amoebal suspension and 100 µl sterile 1:4 HL5:SorC.
        Note: This treatment serves as a negative control to ensure that the initial amoebal cultures are free from bacteria.
      5. For each of the bacterial strains, prepare a set of replicates with 100 µl bacterial suspension and 900 µl 1:4 HL5:SorC solution (without amoebae).
        Note: This treatment serves as a control for the efficiency of gentamicin to kill extracellular bacteria.
      6. Incubate at 22 °C for six hours (without shaking).
    4. Add gentamicin
      1. Transfer content of each well in the 24-well cell culture plates to a 1.5 ml Eppendorf tube.
      2. Centrifuge at 500 x g for 4 min.
      3. Remove supernatant by carefully pipetting the liquid without disturbing the pellet.
      4. Add 1 ml SorC buffer.
      5. Centrifuge at 500 x g for 4 min.
      6. Remove supernatant by carefully pipetting the liquid without disturbing the pellet.
      7. Repeat steps C4d-C4f.
      8. Add 1 ml SorC containing 400 µg ml-1 gentamicin.
      9. Incubate for 1 h at room temperature.
    5. Lyse amoebal cells and count internalized bacteria
      1. Centrifuge at 500 x g for 4 min.
      2. Remove supernatant by carefully pipetting the liquid without disturbing the pellet.
      3. Add 1 ml SorC buffer.
      4. Centrifuge at 500 x g for 4 min.
      5. Remove supernatant by carefully pipetting the liquid without disturbing the pellet.
      6. Repeat steps C5c-C5e.
      7. Add 1 ml of a solution with 0.4% Triton X-100 diluted in SorC buffer.
        Note: This will lyse the amoebal cells.
      8. Mix and prepare 10-fold dilution series of the lysate. The dilutions are performed in SorC buffer using a vortex mixer.
        Note: Preparation of dilution series and plating should be performed immediately after addition of Triton-X since longer exposure to the solvent may kill bacteria. The most important thing is that the different replicates are exposed for the solvent for approximately the same length of time.
      9. Plate 100 µl of relevant dilutions on LB agar plates.
        Note: We recommend using at least two replicate agar plates per dilution level in order to reduce variability at this step. In our experiment we had relatively low bacterial density so for our lowest dilution we simply plated 100 µl of sample directly from the experimental wells.
      10. Incubate for 16 h at 37 °C.
      11. Count colonies on agar plates.

Data analysis

Use agar plates with ~20-200 colonies to calculate the number of colony-forming units (CFU) per ml for each of the replicate wells. The number of CFU’s in different treatments is analyzed with one-way analysis of variance, followed by a test to compare means. We used Duncan’s test (Hao et al., 2016) but other tests would be equally suitable. In the example above we only compared two strains and here we used a t-test (Figure 1).
Note: The two control treatments (from steps C3d and C3e) should be free from bacteria. Presence of bacteria in the negative control without bacteria (step C3d) indicate that the amoebal cultures are contaminated with bacteria and experiments will have to be repeated with newly established axenic amoebal cultures without contaminant bacteria. Presence of bacteria in control treatments without amoebae (step C3e) indicates that the bacteria were not efficiently killed by gentamicin and data cannot be used to evaluate bacterial survival.


Figure 1. Number of intracellular bacteria in Dictyostelium cells co-incubated with a wild type strain of E. coli (W3110) and with the mutant, E345, with triple gene deletion. The assay was performed at MOI 1. Data were analyzed with a t-test and the two strains were significantly different at the 1% level (P = 0.0045, n = 6 for each strain). Further details and examples can be found in Hao et al., 2016.

Recipes

  1. Sterile modified HL5
    14.0 g tryptone (Oxoid)
    7.0 g yeast extract (Oxoid)
    0.35 g Na2HPO4·7H2O
    1.2 g KH2PO4
    Dissolve components in 937.5 ml distilled water, adjust pH with HCl to pH 6.4-6.7, and autoclave for 20 min to sterilize
    Prepare a sterile solution of glucose (224 g L-1) by filtering through 0.2 µm sterile membrane filters. Add 62.5 ml of this solution to 937.5 ml of the autoclaved solution above
    (0.05 g L-1 dihydrostreptomycin-sulfate*)
    *Note: Addition of dihydrostreptomycin-sulfate can be useful when cultures are initiated or have been contaminated with bacteria. However, for the precultures for the actual experiments we avoided the use of antibiotics, since antibiotics could interact with our assay of bacterial survival.
  2. SorC buffer
    2.0 g L-1 KH2PO4
    0.29 g L-1 Na2HPO4
    Dissolve in distilled water, check that pH is 6.0 ± 0.1, if not adjust with HCl
    Add 1 ml 50 mM CaCl2 to 1 L SorC solution, and autoclave
  3. Sterile Luria Broth (LB)
    10.0 g L-1 tryptone
    5.0 g L-1 yeast extract
    10.0 g L-1 NaCl
    Dissolve components in distilled water, adjust pH with HCl to pH 6.8-7.2, and autoclave for 20 min
  4. Agar plates with Luria agar
    Add 15.0 g L-1 agar to Luria Broth
    Autoclave and pour plates
    Note: Make sure to calculate the number of agar plates you need for the experiment. Remember that for each bacterial strain you need to plate samples both from the replicates with bacteria added (steps C3a-C3b) and for the control plates without amoebae (step C3e). Furthermore, in addition to the treatments with bacterial strains, you should also prepare plates for the control treatment with amoebae but without added bacteria (step C3d).

Acknowledgments

Research in the laboratory of CR was funded by startup funds from Fujian Agriculture and Forestry University and the 100 Talents program from Fujian province. Authors would like to thank the International Postdoctoral Exchange Fellowship Program (No. 20150079), and Twasol Research Excellence Program, ‘TRE Program’, King Saud University, Saudi Arabia for support. DLH thanks the Fulbright Kommission for a Fulbright Scholar grant at the University of Copenhagen. This protocol was adapted from the publication by Hao et al., 2016.

References

  1. Fey, P., Kowal, A. S., Gaudet, P., Pilcher, K. E. and Chisholm, R. L. (2007). Protocols for growth and development of Dictyostelium discoideum. Nat Protoc 2(6): 1307-1316.
  2. Gong, J., Qing, Y., Zou, S., Fu, R., Su, L., Zhang, X. and Zhang, Q. (2016). Protist-bacteria associations: Gammaproteobacteria and Alphaproteobacteria are prevalent as digestion-resistant bacteria in ciliated protozoa. Front Microbiol 7: 498.
  3. Hao, X., Luthje, F., Ronn, R., German, N. A., Li, X., Huang, F., Kisaka, J., Huffman, D., Alwathnani, H. A., Zhu, Y. G. and Rensing, C. (2016). A role for copper in protozoan grazing - two billion years selecting for bacterial copper resistance. Mol Microbiol 102(4): 628-641.
  4. Hodgkinson, V. and Petris, M. J. (2012). Copper homeostasis at the host-pathogen interface. J Biol Chem 287(17): 13549-13555.
  5. Kaneko, M., Emoto, Y. and Emoto, M. (2016). A simple, reproducible, inexpensive, yet old-fashioned method for determining phagocytic and bactericidal activities of macrophages. Yonsei Med J 57(2): 283-290.
  6. Matz, C. and Kjelleberg, S. (2005). Off the hook--how bacteria survive protozoan grazing. Trends Microbiol 13(7): 302-307.
  7. Rønn, R., Vestergård, M. and Ekelund, F. (2012). Interactions between bacteria, protozoa and nematodes in soil. Acta Protozool 51: 223-235.

简介

我们进行了一个测试来测试不同E的能力。 大肠杆菌菌株在摄入后在变形细胞内存活。 在测定中,我们将细菌与迪氏底盘藻细胞一起培养6小时。 共培养后,通过离心除去大部分未染色的细菌,剩余的未染色细菌被庆大霉素杀死。 使用庆大霉素,因为它不会渗透到真核细胞中,允许摄取的细菌在抗生素治疗中存活,而在变形细胞外的细菌被杀死。
【背景】细菌已经发展了几种不同的策略来避免或抵抗原生动物的捕食(Rønn等人,2012)。减少摄入机会,如增加细胞大小,聚集,生物膜形成和增加游泳速度的机制有充分的记录(Matz和Kjelleberg,2005;Rønn等人,2012),而机制允许细菌摄入后原生动物食物空泡中抵抗消化较少研究(龚等人,2016)。在这里,我们描述了一种方法来研究细菌菌株在通过社会变形虫的盘根盘菌摄入后存活的能力。我们使用这种测定法来研究铜是否耐药。大肠杆菌摄入后的生存机会比没有铜抗性的细菌更高(郝等,2016)。我们研究了铜电阻,因为已知巨噬细胞是在脊椎动物的免疫系统中具有重要作用的吞噬细胞,使用铜来杀死吞噬细胞中的细菌(Hodgkinson和Petris,2012)。我们假设这种杀伤机制最初是在多细胞生命发生之前自由生存的原生动物中发生的,因此铜抗性可能是保护细菌免受原生动物捕食的一个因素。
该测定法是用于评估巨噬细胞杀死细菌的能力的庆大霉素保护测定的修饰(参见例如,,Kaneko等人,2016),并且它利用庆大霉素不能渗入真核细胞。在原始测定中,在两个时间点确定巨噬细胞中的内化细菌细胞的数量。首先,将巨噬细胞与细菌一起培养给定的时间段,并且在这种共培养之后,去除未经检测的细菌并估计内化细菌的数量。巨噬细胞在没有细胞外细菌的情况下进行第二次测量,以使其消化内源性细菌。这两个估计之间的比率是细菌存活率的量度。
快速消化我们的一些菌株导致降低对失踪率估计的可靠性,并使原始程序不适合与使用。因此,我们修改了该程序,以便在细菌和变态反应蛋白共培养6小时后,我们只估计一个时间点存活的内部细菌的数量。这个估计反映了摄入和细菌消化率之间的平衡。应该注意的是,如果可以假设细菌以相似的速率消耗,这将仅允许比较不同细菌菌株的消化率。在我们的例子中,我们证明了不同的E。大肠杆菌菌株以相同的程度被变形虫吸收,因此我们使用测定法作为消化率的指标。即使不能假定细菌以相同的速度摄取,但是该测定仍然是有用的,但是当然应该相应地被解释。还必须注意的是,该程序只能用于对庆大霉素敏感的细菌。

关键字:细菌, 变形虫, 原生动物, 原生生物, 放牧, 消化阻力

材料和试剂

  1. 移液器和无菌提示
  2. 无菌离心管(例如,Falcon,50ml)圆锥形离心管)
  3. 无菌细胞培养瓶(例如,例如,25cm 2,Nunc< E& E> EASYFlasks<>)*
  4. 24孔,细胞培养板,平底(Corning,Costar ®/或类似)
  5. 无菌1.5 ml Eppendorf管
  6. 50ml Falcon管(康宁或类似)
  7. 无菌过滤器(Corning)的无菌过滤器(0.2μm)
  8. <盘>盘根杆菌AX4(或其他无性菌株;可在美国伊利诺斯州芝加哥西北大学Dicty Stock Center的Dicty Stock Center获得)
  9. 细菌培养*
  10. 庆大霉素(Sigma-Aldrich,目录号:G1264)
  11. 硫酸二氢链霉素(Sigma-Aldrich,目录号:PHR1517)
  12. Triton X-100(Sigma-Aldrich,目录号:93443)
  13. Oxoid TM 胰蛋白胨(Thermo Fisher Scientific,Thermo Scientific TM,目录号:LP0042)
  14. Oxoid TM 酵母提取物(Thermo Fisher Scientific,Thermo Scientific TM,目录号:LP0021)
  15. 磷酸氢二钠七水合物(Na 2 HPO 4·7H 2 O)(Sigma-Aldrich,目录号:S9390)
  16. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:P5655)
  17. 盐酸(HCl)(Sigma-Aldrich,目录号:435570)
    注意:本产品已停产。
  18. 葡萄糖(Sigma-Aldrich,目录号:G8270)
  19. 氯化钙(CaCl 2)(Sigma-Aldrich,目录号:C5670)
  20. 氯化钠(NaCl)(Sigma-Aldrich,目录号:310166)
    注意:本产品已停产。
  21. 琼脂
  22. 无菌HL5培养基(见食谱)
  23. SorC缓冲液(参见食谱)
  24. 无菌Luria肉汤(LB)(见食谱)
  25. 琼脂平板与Luria琼脂(见食谱)

    注意:本方案描述了我们用于比较不同大肠杆菌菌株的存活率的方法,但可以使用其他细菌。这是细菌对庆大霉素敏感的要求

设备

  1. 移液器
  2. 层流罩(Heraeus,型号:Air HLB 2472)
  3. 125ml锥形瓶
  4. 旋转振荡器,在22°C温育
  5. 37℃恒温箱(Thermo Fisher Scientific)*
  6. 旋转振荡器,在37℃下孵育*
  7. 用于eppendorf管的台式离心机(4℃冷却)(Eppendorf,型号:5424R)
  8. 离心机用于50ml Falcon管(HERMLE LABORTECHNIK,型号:Z 326 K)
  9. 分光光度计和各自的比色皿(600nm)
  10. 高压灭菌器(STIK,型号:MJ系列)
  11. 涡旋混合器(Scientific Industries,型号:Vortex-Genie 2,目录号:SI-0236)
  12. pH计和校准缓冲液
  13. 倒置显微镜(Olympus,型号:CKX31)**
  14. 显微镜(Nikon Instruments,型号:ECILPSE 80i)
  15. Neubauer血细胞计数器具有双重计数室(Gizmo Supply,德国)

    注意:设置在37°C的孵化器对于与大肠杆菌一起工作是必要的,但如果使用其他细菌,生长温度条件当然应该相应地进行修改。
    注意:在实验过程中,倒置显微镜对于培养瓶和细胞培养板的检查非常有用。

程序

注意:

  1. 所有的工作都是在无菌条件下使用层流罩进行的。
  2. 实验用盘基网柄菌的液体无菌培养物进行。 D. discoideum菌株通常设在板上(例如,从Dicty股票中心)。在开始实验之前,应将变形虫带入液体培养。有关培养盘根杆菌的详细和有用的信息,请参见Dicty股票中心的首页, http: //dictybase.org/ (Fey et al。,2007)。
  3. 细胞培养瓶中的盘牙菌丝的先前培养具有允许直接检测培养物的优点,但也可以在摇动液体培养物例如125ml锥形瓶中培养培养物。这可以使得在培养物中更容易获得高细胞密度。
  4. 如果提供洗涤的热杀菌细菌,则可以改善盘根杆菌的生长。我们以前已经使用大肠杆菌的过夜培养物,其通过离心洗涤,重悬于SorC中并在65℃下热灭活10分钟。
  5. 在这个协议中,我们使用不同的大肠杆菌菌株。该程序可用于其他细菌;在这种情况下,细菌特异性生长条件,细菌细胞密度等估计的光密度(OD)应相应修改。
  1. 第一天开始。 discoideum 文化
    1. 加入500μl2-3天的老鼠。在无菌培养瓶中培养至10ml新鲜无菌HL5。
    2. 将培养瓶在22℃孵育(不摇动)
  2. 第2天(-3)。开始细菌培养
    1. 通过接种具有1%接种物E的25ml无菌LB培养基来开始细菌培养。大肠杆菌培养物(过夜生长)在125ml锥形瓶中
    2. 在旋转振荡器上在37℃,200rpm下孵育过夜。

  3. 第3天(-4)。生存检测
    1. 收获细菌
      1. 通过以12,000×g离心2分钟收获1ml细菌。
      2. 通过小心地移取液体去除上清液,而不会干扰沉淀。
      3. 将细菌沉淀重悬于1 ml的1:4 HL5:SorC溶液中
      4. 以12,000 x g离心2分钟。
      5. 去除上清液并将细菌沉淀重悬于1ml的1:4 HL5:SorC溶液中
      6. 将细菌密度调节至0.1(约10 CFU ml -1)的OD 600。
      7. 收获D.单核细胞
        1. 在50毫升Falcon管中加入亚麻培养物,并以500克xg离心4分钟。
        2. 通过小心地移取液体去除上清液,而不会干扰沉淀。
        3. 将阿米巴重悬于25 ml的1:4 HL5:SorC溶液中
        4. 以500 x g离心4分钟。
        5. 重新使用1:4 HL5:SorC溶液中的变形虫。
        6. 通过在血细胞计数器中计数来估计悬浮液中的变形虫密度。
        7. 将变形虫密度调整至1×10 6细胞ml -1 。
      8. 开始生存测定
        1. 添加900μl的阿米巴悬浮液(来自步骤C2g)到24孔培养板中的许多孔。
          注意:我们每个细菌菌株至少使用6个重复。
        2. 加入调节至约4.5×10 7细胞ml -1的100μl细菌悬浮液,以获得多重感染(MOI)为5.
        3. 为了获得其他水平的MOI,细菌细胞密度可以相应调整。
        4. 准备一组重复使用900μl混合悬浮液和100μl无菌1:4 HL5:SorC。
          注意:这种治疗作为一种阴性对照,以确保最初的亚麻培养物免于细菌。
        5. 对于每个细菌菌株,用100μl细菌悬浮液和900μl1:4 HL5:SorC溶液(无变形虫)制备一组重复。
          注意:这种治疗方法可用于控制庆大霉素杀死细胞外细菌的效率。
        6. 在22℃下孵育6小时(不摇动)。
      9. 加入庆大霉素
        1. 将24孔细胞培养板中的每个孔的内容物转移到1.5ml Eppendorf管中
        2. 以500 x g离心4分钟。
        3. 通过小心地移取液体去除上清液,而不会干扰沉淀。
        4. 加入1ml SorC缓冲液
        5. 以500 x g离心4分钟。
        6. 通过小心地移取液体去除上清液,而不会干扰沉淀。
        7. 重复步骤C4d-C4f。
        8. 加入含有400μg/ ml庆大霉素的1ml SorC。
        9. 在室温下孵育1小时。
      10. Lyse变形细胞并计数内在细菌
        1. 以500 x g离心4分钟。
        2. 通过小心地移取液体去除上清液,而不会干扰沉淀。
        3. 加入1ml SorC缓冲液
        4. 以500 x g离心4分钟。
        5. 通过小心地移取液体去除上清液,而不会干扰沉淀。
        6. 重复步骤C5c-C5e。
        7. 加入1ml用SorC缓冲液稀释0.4%Triton X-100的溶液 注意:这将溶解变形细胞。
        8. 混合并制备10倍稀释系列的裂解液。稀释液使用涡旋混合器在SorC缓冲液中进行 注意:添加Triton-X后,应立即进行稀释系列和电镀的制备,因为长时间接触溶剂会导致细菌死亡。最重要的是,不同的重复在大约相同的时间内暴露于溶剂。
        9. 在LB琼脂平板上平板培养100μl相关稀释液 注意:我们建议每个稀释水平使用至少两个重复琼脂平板,以减少此步骤的变异性。在我们的实验中,我们的细菌密度相对较低,所以对于我们最低的稀释度,我们直接从实验孔中平铺100μl样品。
        10. 在37°C孵育16小时。
        11. 在琼脂平板上计数菌落。
  4. 数据分析

    使用约20-200个菌落的琼脂平板计算每个重复孔的每ml的菌落形成单位数(CFU)。通过单因素方差分析CFU在不同治疗方法中的数量,然后进行对比手段的检验。我们使用了邓肯的测试(郝等人,2016),但其他测试也同样适用。在上面的例子中,我们只比较了两个菌株,这里我们使用了一个 t -test(图1)。
    注意:两种对照处理(步骤C3d和C3e)应不含细菌。细菌在没有细菌的阴性对照中的存在(步骤C3d)表明,变形虫培养物被细菌污染,实验将不得不用新建立的无污染细菌的无菌亚麻培养物重复。没有变形虫的对照处理中的细菌存在(步骤C3e)表明细菌没有被庆大霉素有效杀死,数据不能用于评估细菌的存活。


    图1.与 的野生型菌株共培养的 <强>电子。大肠杆菌(W3110)和具有三重基因缺失的突变体E345 。以MOI 1进行测定。> -test,并且两个菌株在1%水平(每个菌株的p = 0.0045,n = 6)显着不同。更多的细节和例子可以在昊等人的中找到。

    食谱

    1. 无菌修饰HL5
      14.0克胰蛋白胨(Oxoid)
      7.0克酵母提取物(Oxoid)
      0.35g Na 2 HPO 4 <7H 2 O
      1.2g KH 2 PO 4
      将组分溶解在937.5ml蒸馏水中,用HCl调节pH至6.4-6.7,高压灭菌20分钟消毒
      通过0.2μm无菌膜过滤器过滤,制备无菌的葡萄糖溶液(224 g L -1)。加入62.5毫升此溶液至937.5毫升高压灭菌的溶液上方 (0.05g L 二氢链霉素硫酸盐*)
      注意:当培养物开始或被细菌污染时,添加二氢硫酸链霉素 - 硫酸盐可能是有用的。然而,对于实际实验的预培养,我们避免使用抗生素,因为抗生素可以与我们的细菌生存测定相互作用。
    2. SorC缓冲区
      2.0g L -1 KH 2 4
      0.29g L Na 2 HPO 4
      溶解在蒸馏水中,检查pH值为6.0±0.1,如果不用HCl调整,则不能 向1升SorC溶液中加入1毫升50毫摩尔CaCl 2,高压釜
    3. 无菌Luria肉汤(LB)
      10.0 g L -1 胰蛋白胨
      5.0g L -1 酵母提取物
      10.0g L -1 NaCl
      将组分溶于蒸馏水中,用HCl调节pH至6.8-7.2,高压灭菌20分钟
    4. 琼脂板与Luria琼脂
      向Luria Broth添加15.0 g L -1 琼脂 高压釜和倒板
      注意:确保计算实验所需的琼脂平板数量。请记住,对于每种细菌菌株,您需要从添加细菌的复制品(步骤C3a-C3b)和没有变形虫杆菌(步骤C3e)的对照板中平铺样品。此外,除了用细菌菌株进行处理之外,还应制备用于使用变形虫控制处理但没有添加细菌的平板(步骤C3d)。

    致谢

    研究实验室由福建农林大学创业基金和福建省百人计划资助。作者要感谢沙特阿拉伯国王沙特阿拉伯国际博士后研究奖学金计划(第20050079号)和沙特阿拉伯国王沙特阿拉伯大学的特拉索尔研究优秀计划“TRE计划”。哥伦比亚大学富兰布赖特大学荣获富布莱特学者奖学金。这个协议是由郝等人于2016年出版发行的。

    参考

    1. Fey,P.,Kowal,AS,Gaudet,P.,Pilcher,KE和Chisholm,RL(2007)。&nbsp; 生物发酵方案 2(6):1307-1316 。
    2. Gong,J.,Qing,Y.,Zou,S.,Fu,R.,Su,L.,Zhang,X. and Zhang,Q.(2016)。&nbsp; 抗原细菌协会:Gammaproteobacteria 和 Alphaproteobacteria 是普遍的作为纤毛原生动物中的消化性细菌。前微生物7:498.
    3. Hao,X.,Luthje,F.,Ronn,R.,German,NA,Li,X.,Huang,F.,Kisaka,J.,Huffman,D.,Alwathnani,HA,Zhu,YG and Rensing,C 。(2016)。铜在原生动物放牧中的作用 - 细菌铜电阻选择二十亿年。分子微生物 102(4):628-641。
    4. Hodgkinson,V. and Petris,MJ(2012)。&nbsp; 主体 - 病原体界面中的铜稳态。 J Biol Chem 287(17):13549-13555。
    5. Kaneko,M.,Emoto,Y。和Emoto,M。(2016)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/26847277” target =“_ blank”>一种简单,可重复,便宜,但老式的确定巨噬细胞吞噬和杀菌活性的方法。 Yonsei Med J 57(2):283-290。
    6. Matz,C.和Kjelleberg,S.(2005)。脱钩 - 细菌如何在原生动物放牧中生存。 趋势微生物 13(7):302-307。
    7. Rønn,R.,Vestergård,M.和Ekelund,F。(2012)。&lt; a class =“ke-insertfile”href =“http://www.ejournals.eu/Acta-Protozoologica/Tom-51( 2012)/ Issue-3 / art / 756 /“target =”_ blank“>土壤中细菌,原生动物和线虫之间的相互作用。 Acta Protozool 51:223-235。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Rønn, R., Hao, X., Lüthje, F., German, N. A., Li, X., Huang, F., Kisaka, J., Huffman, D., Alwathnani, H. A., Zhu, Y. and Rensing, C. (2017). Bacterial Survival in Dictyostelium. Bio-protocol 7(13): e2376. DOI: 10.21769/BioProtoc.2376.
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