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Jan 2019

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Zebrafish Bacterial Infection Assay to Study Host-Pathogen Interactions
斑马鱼细菌感染检测用于研究宿主-病原相互作用    

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Abstract

The study of host-pathogen interactions has improved our understanding of both pathogenesis and the response of the host to infection, including both innate and adaptive responses. Neutrophils and macrophages represent the first line of innate host defense against any infection. The zebrafish is an ideal model to study the response of these cells to a variety of pathogens. Zebrafish possess both neutrophils and macrophages exhibiting similar defense mechanisms to their human counterparts. The transparency of zebrafish embryos greatly facilitates in vivo tracking of infection dynamics in a non-invasive manner at high-resolution using labelled pathogens, while immune cells can also be labelled transgenically to enable even more in-depth analysis. Here we describe a procedure for performing a bacterial infection assay in zebrafish embryos using fluorescently-labelled E. coli bacteria and demonstrate the monitoring and quantification of the infection kinetics. Of note, this procedure helps in understanding the functional role of genes that are important in driving the innate immune response.

Keywords: Infection (感染), Fluorescent bacteria (荧光细菌), Innate immunity (先天性免疫), Zebrafish (斑马鱼), Imaging (成像)

Background

Host-pathogen interaction studies are important to understand disease pathogenesis and also for the development of effective treatments. Multiple aspects of both the host and pathogen need to be considered to determine potential risk factors in the host and key virulence factors in the pathogen. Innate immunity represents the first line of defense against infections, triggering a cascade of responses, including inflammation, neutralization and recruitment of components of the adaptive immune system (Akira et al., 2006). Neutrophils are rapidly recruited to the site of bacterial infection in response to chemoattractant gradients of host chemokines released from damaged cells as well as bacterial products themselves, where they initiate the immune response by phagocytosis and netolysis (Renshaw and Trede, 2012; Deng et al., 2013). Macrophage recruitment follows, with these cells focused on removing dead cells, remodeling injured tissue and coordinating adaptive immune cells (Weiss and Schaible, 2015).

The development and function of innate immune cells are controlled by a range of specific genes, the dysregulation of which can lead to a number of pathological states, including enhanced susceptibility to infection and chronic inflammation. Being short-lived, neutrophils are depleted rapidly in response to exposure to a pathogen, with so-called ‘emergency granulopoiesis’ being initiated to generate additional neutrophils (Hall et al., 2012; Manz and Boettcher, 2014). Granulocyte colony-stimulating factor (G-CSF) acting via its receptor, G-CSFR, is a key mediator of emergency granulopoiesis through its action promoting the proliferation and differentiation of relevant hematopoietic progenitor cells (Panopoulos and Watowich, 2008; Liongue et al., 2009). Understanding the role of factors that regulate the innate immune response during bacterial infection in an appropriate in vivo model can provide unique insights into infection and immunity.

Zebrafish represents a very attractive in vivo model to perform host-pathogen studies, with its transparent embryos allowing in vivo imaging at high-resolution to track infection in real-time, by tagging the pathogens with fluorescent markers (Basheer et al., 2019). In addition, using transgenic approaches in zebrafish, neutrophils and macrophages can also be monitored via fluorescent tags (Ellett et al., 2011; Gray et al., 2011). Importantly, these cells along with other components of the immune system share remarkable similarity with those of humans (Meeker and Trede, 2008). The zebrafish innate immune system forms early during their development with the generation of macrophages at 24 h post fertilization (hpf) and neutrophils by 32-48 hpf (Herbomel et al., 1999; Willett et al., 1999; Bennett et al., 2001). The zebrafish adaptive immune system develops later, allowing the innate immune system to be studied independently. Zebrafish infection models of different pathogen–bacterial, viral and fungal–have been established (Gratacap and Wheeler 2014; Masud et al., 2017; Varela et al., 2017).

Our research has employed a zebrafish bacterial infection model to understand the role of key genes involved in innate immunity in defense against infection. Escherichia coli (E. coli) bacteria expressing green fluorescent protein (GFP) were microinjected into 72 hpf larvae derived from either wild-type zebrafish or those mutant for the gene encoding G-CSFR (Figure 2). These were monitored by fluorescence microscopy over a time-course to determine the relative infection kinetics (Basheer et al., 2019). Overall, this procedure helps in understanding different host-pathogen interactions and unravel the functions of key immune genes involved in the process using zebrafish as an animal model.

Materials and Reagents

  1. Transfer pipette, 3 ml (Heinz Herenz, catalog number: 1131303 )
  2. Mesh strainer, 12-15 cm in diameter (pore size should be less than zebrafish embryo with chorion diameter, approximately 0.5 mm mesh)
  3. Petri plate (PLP, catalog number: S9014UV20 )
  4. Parafilm
  5. Thin-wall borosilicate with filament, 1.0 mm x 0.78 mm x 15 cm (SDR Scientific, catalog number: 30-0039 GC100TF-15)
  6. Microloader pipette tips (Point of Care Diagnostics Pty Ltd, catalog number: EPP5242 956.003)
  7. Corning square dish, 245 mm Non-Treated (Corning, catalog number: 38020 )
  8. Paper towel
  9. Multiwell tissue culture plates, 6-well and 12-well (Interpath, Greiner, catalog number: 657160 , 665180 )
  10. Cuvettes (Bio-Rad, catalog number: 2239950 )
  11. Breeding/mating tanks, 3 L (Techniplast)
  12. Zebrafish (Danio rerio), wild-type
  13. E. coli-GFP bacteria (ATCC, catalog number: 25922GFP )
  14. Agar bacteriological grade (Astral Scientific, catalog number: J637-500 )
  15. N-phenylthiourea, PTU (Sigma-Aldrich, catalog number: P7629-10G )
  16. Benzocaine (Sigma-Aldrich, catalog number: E1501-100G )
  17. NaCl (Astral Scientific, catalog number: 0241-5kg )
  18. KCl (Sigma-Aldrich, catalog number: P9541-500G )
  19. CaCl2·2H2O (Sigma-Aldrich, catalog number: C3306-250G )
  20. MgSO4·7H2O (Sigma-Aldrich, catalog number: 63138-1KG )
  21. Methylene blue (Sigma-Aldrich, catalog number: M9140-25G )
  22. NaOH
  23. Phosphate buffered saline-PBS tablets (Astral Scientific, catalog number: AME404-200TABS )
  24. Triton X-100 (Sigma-Aldrich, catalog number: X100-500ML )
  25. Blu-tack
  26. BD BactoTM Tryptic Soy Broth (Soybean-Casein Digest Medium) (Becton Dickinson, catalog number: 211825 )
  27. Ampicillin, 100 mg/ml, ready-made solution (Sigma-Aldrich, catalog number: A5354-10ML )
  28. Milli-Q or distilled water
  29. Phenol red, 0.5%, liquid, sterile-filtered (Sigma-Aldrich, catalog number: P0290 )
  30. Mineral oil (Sigma-Aldrich, catalog number: M5904-5ML )
  31. 60x E3 media (see Recipes)
  32. E3 media (see Recipes)
  33. Methylene blue stock solution (see Recipes)
  34. E3 containing 0.3 mg/L methylene blue (see Recipes)
  35. PTU stock, 0.3% (w/v) (see Recipes)
  36. E3 containing 0.003% (w/v) PTU (see Recipes)
  37. Benzocaine stock solution (see Recipes)
  38. 10x PBS stock solution (see Recipes)
  39. PBS (see Recipes)
  40. 1% Triton X-100 in PBS (see Recipes)

Equipment

  1. Glass bottles, 1 L and 2 L
  2. Magnetic stir bar–PTFE Cylindrical 15 mm length x 6 mm diameter (PLP, Cowie, catalog number: 001.115.6 )
  3. Eppendorf tube racks
  4. Pipettes, 100-1,000 μl, 20-200 μl, 2-10 μl, 0.5-2 μl
  5. Watch makers forceps, pointed tweezers, style 5, 114 mm (ProSciTech, catalog number: T65-SA )
  6. Incubator, 28 °C (Sanyo, catalog number: MIR-162 )
  7. Incubator, 37 °C (Thermo Scientific, Hereaus, catalog number: B6030 )
  8. Shaking incubator (200-225 rpm) (Infors HT Multitron Standard incubator)
  9. Spectrophotometer (Bio-Rad SmartSpec Plus Spectrophotometer, catalog number: 273BR03695 )
  10. Micropipette needle puller (Sutter Instrument, catalog number: P-97 )
  11. Bunsen burner
  12. Atherton Chipmunk Autoclave Sterilizer
  13. Pipette pump
  14. Mini centrifuge (Thermo Fisher Scientific Pty Ltd, catalog number: 75004061 )
  15. Microcentrifuge (Thermo Fisher Scientific Pty Ltd, catalog number: THR75002410 )
  16. Magnetic stirrer (Ratek Instruments Pty Ltd, catalog number: MS10 )
  17. Refrigerator, 4 °C
  18. Microinjector set up (SDR Clinical Technology, Nanoject II Nanoliter In Jecto, catalog number: 690131 , Footswitch, catalog number: 690140 , Support Base, catalog number: 690141 , Micropipette Holder Kit Option, catalog number: M-PIP-Kit )
  19. Stereo dissecting microscope with light base (Nikon, catalog number: SMZ745 )
  20. Fluorescence microscope (Olympus, model: MVX10 with DP72 camera)
  21. Tissue Lyser II (Qiagen, model: 85300 )
  22. Fume hood

Software

  1. GraphPad Prism 8 (http://www.graphpad.com)
  2. ImageJ (https://imagej.nih.gov/ij/index.html)

Procedure

  1. Preparing the embryos
    1. Transfer adult zebrafish (one male and two females) to a 3 L breeding tank in the evening to allow them to acclimatize before stimulation of mating the next morning using light.
    2. Collect embryos from the tanks using a mesh strainer and transfer them to a 10 cm Petri dish containing 25 ml E3 media with 0.3 mg/L methylene blue.
    3. Remove any dead or unfertilized eggs (embryos turn black as they die and need to be removed by using suction from a transfer pipette and then discarded) from the dish and transfer the remaining fertilized eggs to an incubator held at 28 °C.
    4. At 24 hpf, transfer embryos to E3 media with 0.3 mg/L methylene blue and 0.003% (w/v) 1-phenyl-2-thiourea (PTU) to prevent pigmentation.
    5. Replace E3 media in the dish daily until 72 hpf.
    6. Any embryos unhatched by 72 hpf (injection day) may be dissected out of the chorion membrane using pointed watch makers forceps.

  2. Tryptic soy broth preparation
    1. Weigh 15 g tryptic soy broth (TSB) powder and transfer to 1 L glass bottle.
    2. Add 500 ml of Milli-Q water and a magnetic stir bar and place the bottle on a magnetic stirrer to allow dissolution.
    3. Autoclave the media at 121 °C, pressure 400-500 kPa in an autoclave (Atherton Chipmunk Sterilizer) for 1 h 30 min to sterilize (make sure the lid of the glass bottle is loosened before autoclaving).
    4. Once sterilization is complete, remove the media and close the bottle lid tightly.
    5. Allow the media to completely cool down at room temperature before transferring to 4 °C for long term storage (3-4 weeks).

  3. Tryptic soy-agar-ampicillin plate preparation
    1. Weigh 6 g Agar-bacteriological grade and 15 g TSB powder to a 1 L glass bottle.
    2. Add 500 ml of Milli-Q water and a magnetic stir bar and place the bottle on a magnetic stirrer to allow dissolution.
    3. Autoclave the media at 121 °C in an autoclave, pressure 400-500 KPa (Atherton Chipmunk Sterilizer) for 1 h and 30 min to sterilize (make sure the lid of the glass bottle is loosened before autoclaving).
    4. Once sterilized, remove the media and close the bottle lid tightly.
    5. Allow the media to completely cool down at room temperature before adding 500 μl ampicillin solution (100 mg/ml), for a final concentration of 100 μg/ml ampicillin.
    6. Pour approximately 25 ml TSB-agar-ampicillin solution under flame into 10 cm Petri dishes without generating bubbles and allow them to completely cool down to room temperature (Figure 1).
    7. Once cooled, close the lid of the Petri dish, seal with parafilm and store at 4 °C for later use.


      Figure 1. Image of a Tryptic soy broth (TSB)-Agar-ampicillin (Amp) plate

  4. Bacterial cultivation and enumeration
    1. Inoculate E. coli-GFP bacteria into 10 ml of tryptic soy broth supplemented with 10 μl 100 mg/ml ampicillin.
    2. Grow this bacterial culture overnight at 37 °C in a shaking incubator at 1.1-1.4 g.
    3. Transfer 1 ml of bacterial culture into a cuvette and place in a spectrophotometer previously blanked against uninoculated TSB and use the absorbance as 600 nm to determine the colony forming units (CFU) according to Biorad SmartSpec Plus OD600 absorbance assay.
    4. Perform serial dilutions of the bacterial culture (ten-fold serial dilutions between 10-6 and 10-10, 1 ml of bacterial culture in 9 ml of uninoculated TSB), plating 50-100 μl diluted broth onto TSB-agar-ampicillin plates and incubating the plates overnight at 37 °C incubator to determine the exact number of CFU in the broth.
    5. Pellet the bacterial cells at 13,751 x g for 1-2 min and remove the supernatant.
    6. Resuspend the bacterial cells in sterile PBS at a concentration of ~9 x 1010 CFU/ml based on the spectrophotometry.

  5. Microinjection of bacterial cells into embryos
    1. Form a thin-walled precision borosilicate glass capillary tube into a fine needle using a micropipette needle puller (After loading the borosilicate glass capillary tube, pull the needle with settings, heat–365, pull–45, velocity–80, time–150 and pressure–500, and press the pull button to make needles). These variable settings are increments that differ with each equipment (https://www.sutter.com/manuals/P-87_OpMan.pdf).
    2. Place the needle onto a square plate/dish with Blu-tack to protect them from breakage before use.
    3. Prepare the injection material by mixing 4 μl ~9 x 1010 CFU bacteria-PBS suspension with 1 μl 2% (w/v) phenol red to allow visualization of the injectate.
    4. Using a microloader pipette, load the wide end of the needle with 3 μl injection material containing phenol red.
    5. Shake the needle to bring the injection material to the tip of the needle and to remove any air bubbles.
    6. Turn on the air source and the microinjector and transfer the needle into the microinjector source and secure tightly within the housing.
    7. Adjust the micromanipulator to the correct position to allow for fine adjustments while injecting, bringing the needle close to the stage and area of injection.
    8. Anesthesize 72 hpf embryos with 0.1 mg/ml of benzocaine in E3 media and transfer the Petri dish to the microinjection stage.
    9. Break the tip of the microinjection needle using sharp watch makers forceps to an extent that allows the bacterial cells to expel easily, releasing consistent amounts and able to pierce the embryos with minimal damage.
    10. The volume of injecting solution can be determined by injecting the material into a drop of mineral oil, measuring the diameter of the injected drop over a scale bar and calculating the volume. The volume can be adjusted by altering the injector pressure and/or the needle tip.
    11. Once the microinjector is setup completely, align the embryos close to the needle tip.
    12. Pierce the needle tip into the venous return of 72 hpf embryos (Figure 2) and use the foot pedal to inject 2-5 nano litres (nl) of injection solution into each embryo, repeating until the desired number is injected.
      Note: The quality of the needle tip is crucial for performing consistent injections that do not unnecessarily damage the embryos.


      Figure 2. Image of a 72 hpf embryo with E. coli-GFP bacteria injected into their venous return (shown by arrow head). Scale bars = 200 μm.

    13. Once injected, place the embryos in a Petri dish containing fresh E3 media with 0.3 mg/L of methylene blue and 0.003% PTU for 15 min to recover before they are transferred to a 28 °C incubator.
    14. In performing the injection assay, each experimental group should have a minimum of 30-60 embryos, including both sterile PBS injected and uninjected controls (Figure 3).


      Figure 3. Images of groups of uninjected embryos (A) and those injected with E. coli-GFP bacteria (B). Scale bars = 500 μm.

    15. After microinjection, remove any dead or deformed embryos and un-injected larvae from the plate.
    16. To determine the actual number of CFU injected per embryo, five injected embryos should be homogenized and plated directly onto TSB-Agar-Amp plates, followed by overnight growth at 37 °C prior to CFU counting.

  6. Imaging of injected embryos
    1. Transfer the injected embryos to 12-well or 6-well plates for imaging.
    2. Anesthetize the embryos with E3 media containing 0.1 mg/ml of benzocaine and orient them in the desired angle for imaging using Olympus Cell Sens Standard software with a Olympus MVX10 fluorescence microscope (Olympus, model: MVX10 with a LH100HG and a U-MGFPA/XL GFP mirror filter attached to a DP74 camera for photography).
    3. Repeat this at regular intervals, such as 0, 4, 8, 16, 24 and 48 h post infection (hpi), to monitor the rate of infection and pathogenesis of GFP positive E. coli bacteria (Figure 4).
      To determine relative survival, visually inspect embryos to determine the number of dead embryos based on the absence of their heart beat.


      Figure 4. Images of embryos injected at 72 hpf with E. coli-GFP bacteria and visualized at 0.1 hpi (A) and 48 hpi (B). Scale bars = 200 μm.

  7. Bacterial enumeration by plate counting
    1. For bacterial enumeration, collect 10 live injected larvae at each time point (for example 0, 4, 8, 16, 24 and 48 hpi) separately in Eppendorf tubes using a transfer pipette post euthanasia with an overdose of 50 μg/ml benzocaine in E3 media.
    2. Remove all the E3 media, rinse once in fresh E3 media to remove all the benzocaine.
    3. Transfer 1,000 μl 1% Triton X-100 in sterile PBS solution to the tube.
    4. Homogenize the samples in a Qiagen tissue lyser II for 5 min at a frequency of 30 s. If required, repeat homogenization for 5 min to ensure complete embryo disintegration.
    5. Plate 50-100 μl of the homogenate onto a TSB-agar-ampicillin plate and incubate at 37 °C for up to 48 h to determine the number of CFU per larvae (Figure 5) at each time point of infection to deduce the infection kinetics.
    6. Confirm the growth of E. coli-GFP bacteria on TSB-agar-ampicillin plates by observing them under fluorescence microscopy (Figure 6).


      Figure 5. Image of a TSB-Agar-Amp plate used to quantify E. coli-GFP CFU in embryos


      Figure 6. Fluorescent image of E. coli-GFP bacterial colonies on a TSB-Agar-Amp plate. Scale bars = 500 μm.

Data analysis

  1. Bacterial colonies from the plate could be either counted manually or using ImageJ plugin “analyze particles” as follows:
    1. Open the image of the bacterial plate with colonies.
    2. Select the region of interest by either selecting oval or elliptical shape or use the free hand tool to draw the region of interest.
    3. Clear the region outside of interest–Edit > Clear Outside.
    4. Convert them to 16 bit grey scale image–Image > Type > 16-bit.
    5. Adjust the image threshold–Image > Adjust > Threshold and click apply once the threshold is adjusted to the desired level.
    6. If the colonies are crowded and not separated well enough, follow–Process > Binary > Watershed.
    7. Count the colonies–Analyze > Analyze Particles, in the pop-up window select the size of the pixels (10-10,000) and circularity (0.00-1.00) depending on the size of the bacterial colonies, check the boxes “Display results”, “Clear results”, “Summarize” and “Add to manager”. This will provide the number of colonies and an overview of the colonies selected.
  2. Analyse the bacterial load enumerated by plate counting and survival of the infected embryos using GraphPad Prism 8 software.
  3. The bacterial load data can be analysed using non-parametric unpaired Student’s t-test and survival of the infected embryos can be displayed as a Kaplan-Meier curve, with statistical significance determined using a log-rank (Mantel-Cox) test.

Recipes

  1. 60x E3 media
    34.8 g NaCl
    1.6 g KCl
    5.8 g CaCl2·2H2O
    9.78 g MgCl2·6H2O
    To prepare a 60x stock, dissolve the ingredients in Milli-Q water, to a final volume of 2 L
    Adjust the pH to 7.2 with NaOH and autoclave them (temperature: 121 °C, pressure: 400-500 KPa, duration: 1.5 h)
    Store at room temperature for up to 1 year
  2. E3 media
    Dilute 16.5 ml of the 60x stock to 990 ml with Milli-Q water
    Store at room temperature for up to 1 year
  3. Methylene blue stock solution
    Add 0.1 g of methylene blue powder to 100 ml of Milli-Q water and mix them
    Store at room temperature for up to 1 year
  4. E3 containing 0.3 mg/L of methylene blue
    Add 600 μl methylene blue stock solution to 1 L E3 media
    Store at room temperature for up to 1 year
  5. PTU stock [0.3% (w/v)]
    Add 0.3 g PTU to 100 ml E3 media
    Dissolve at 65 °C for overnight
    Store at room temperature for up to 1 year
  6. E3 containing 0.003% (w/v) of PTU
    Add 10 ml of 0.3% (w/v) PTU stock solution to 990 ml of E3 media
    If crystals are present in 0.3% PTU stock, incubate solution at 65 °C in oven until dissolved
  7. Benzocaine stock solution [10% (w/v)]
    Add 10 g of benzocaine powder to 100 ml 100% ethanol ( prepare in fume hood)
    Store in the dark, wrap in foil
  8. 10x PBS stock
    Dissolve 100 PBS tablets in 1 L Milli-Q water and autoclave (temperature: 121 °C, pressure: 400-500 KPa, duration: 1.5 h)
    Store at room temperature for up to 1 year
  9. PBS
    Add 100 ml of 10x PBS to 900 ml Milli-Q water and autoclave (temperature: 121 °C, pressure: 400-500 KPa, duration: 1.5 h)
    Store at room temperature for up to 1 year
  10. 1% Triton X-100 in PBS
    Add 1 ml Triton X-100 to 99 ml sterile PBS
    Store at 4 °C for up to 1 year

Acknowledgments

The authors recognize the support of a Deakin University International Research Scholarship (FB). The authors would like to thank the Deakin University Animal House staff for superb aquarium management that underpins the work described in this publication. This protocol was adapted from previous work (Fehr et al., 2015; Basheer et al., 2019).

Competing interests

The authors have no competing interests.

Ethics

All studies involving animals were approved by the Deakin University Animal Ethics Committee (G23/2016, 31/10/16-31/1/2020)

References

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  3. Bennett, C. M., Kanki, J. P., Rhodes, J., Liu, T. X., Paw, B. H., Kieran, M. W., Langenau, D. M., Delahaye-Brown, A., Zon, L. I., Fleming, M. D. and Look, A. T. (2001). Myelopoiesis in the zebrafish, Danio rerio. Blood 98(3): 643-65
  4. Deng, Q., Sarris, M., Bennin, D. A., Green, J. M., Herbomel, P. and Huttenlocher, A. (2013). Localized bacterial infection induces systemic activation of neutrophils through Cxcr2 signaling in zebrafish. J Leukoc Biol 93(5): 761-769.
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  6. Fehr, A., Eshwar, A. K., Neuhauss, S. C., Ruetten, M., Lehner, A. and Vaughan, L. (2015). Evaluation of zebrafish as a model to study the pathogenesis of the opportunistic pathogen Cronobacter turicensis. Emerg Microbes Infect 4(5): e29.
  7. Gratacap, R. L. and Wheeler, R. T. (2014). Utilization of zebrafish for intravital study of eukaryotic pathogen-host interactions. Dev Comp Immunol 46(1): 108-115.
  8. Gray, C., Loynes, C. A., Whyte, M. K., Crossman, D. C., Renshaw, S. A. and Chico, T. J. (2011). Simultaneous intravital imaging of macrophage and neutrophil behaviour during inflammation using a novel transgenic zebrafish. Thromb Haemost 105(05): 811-819.
  9. Hall, C. J., Flores, M. V., Oehlers, S. H., Sanderson, L. E., Lam, E. Y., Crosier, K. E. and Crosier, P. S. (2012). Infection-responsive expansion of the hematopoietic stem and progenitor cell compartment in zebrafish is dependent upon inducible nitric oxide. Cell Stem Cell 10(2): 198-209.
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  15. Panopoulos, A. D. and Watowich, S. S. (2008). Granulocyte colony-stimulating factor: molecular mechanisms of action during steady state and ‘emergency’ hematopoiesis. Cytokine 42(3): 277-288.
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  19. Willett, C. E., Cortes, A., Zuasti, A. and Zapata, A. G. (1999). Early hematopoiesis and developing lymphoid organs in the zebrafish. Dev Dyn 214(4): 323-336.

简介



[ 文摘动作 ] 宿主-病原体相互作用的研究已经提高了我们既发病机制和宿主对INFE响应的理解ction,包括先天性和适应性应答。中性粒细胞和巨噬细胞是抵抗任何感染的先天宿主防御的第一线。斑马鱼是研究这些细胞对多种病原体反应的理想模型。斑马鱼同时具有嗜中性粒细胞和巨噬细胞表现出荷兰国际集团类似的防御机制,它们人的相应。斑马鱼胚胎的透明性极大地促进了体内 使用标记的病原体以非侵入性的方式以高分辨率跟踪感染动态,同时还可以通过转基因方式标记免疫细胞,以进行更深入的分析。在这里,我们描述了一种方法,用于执行一个在斑马鱼中的细菌感染测定胚胎使用荧光标记的大肠杆菌细菌和演示感染动力学的监测和定量。值得注意的是,该程序有助于理解对驱动先天免疫应答至关重要的基因的功能作用。

[ 背景 ] 宿主-病原体相互作用的研究是重要的是了解疾病的发病机理,也为有效治疗药物的开发。需要确定宿主和病原体的多个方面,以确定宿主中的潜在危险因素和病原体中的关键毒力因素。先天性免疫是抵抗感染的第一道防线,它会引发一连串的反应,包括炎症,中和和适应性免疫系统各组分的募集(Akira et al。,2006)。嗜中性粒细胞被迅速招募到细菌感染部位,以响应从受损细胞释放的宿主趋化因子的化学趋化梯度以及细菌产物本身,在那里它们通过吞噬作用和网络分解来引发免疫反应(Renshaw和Trede ,2012; Deng 等。,2013 )。随后巨噬细胞募集,这些细胞专注于去除死细胞,重塑受损组织并协调适应性免疫细胞(Weiss 和Schaible ,2015)。

先天免疫细胞的发育和功能是由一系列特定基因的控制,其中所述失调可导致许多病理状态,包括增强的对感染的易感性和慢性炎症的。被短暂,嗜中性粒细胞迅速耗尽响应于暴露于病原体,与所谓的“紧急粒” 被启动,以产生附加的嗜中性粒细胞(霍尔等人,2012;曼茨和博彻,2014) 。通过其受体G-CSFR起作用的粒细胞结肠γ刺激因子(G-CSF)通过促进相关造血祖细胞的增殖和分化而发挥作用,是紧急粒细胞生成的关键介质(Panopoulos和Watowich ,2008 ; Liongue 等。,2009年)。了解合适的体内模型中细菌感染过程中调节先天性免疫应答的因素的作用,可以提供有关感染和免疫力的独特见解。

斑马鱼代表着进行宿主病原体研究的非常有吸引力的体内模型,它的透明胚胎可以通过用荧光标记物标记病原体来实现高分辨率的体内成像以实时追踪感染(Basheer 等人,2019)。另外,在斑马鱼中使用转基因方法,中性粒细胞和巨噬细胞也可以通过荧光标签监测(Ellett 等,2011; Gray 等,2011)。重要的是,这些细胞与免疫系统的其他组成部分与人类有着显着的相似性(Meeker 和Trede ,2008)。斑马鱼先天免疫系统的形式及其与在24的巨噬细胞的产生在开发过程中的早期ħ 受精后(HPF 由32-48)和嗜中性粒HPF (Herbomel 等人,1999;威利特。等人,1999 ;贝内特。等人,2001 )。斑马鱼适应性免疫系统发展较晚,可以独立研究先天性免疫系统。已经建立了不同病原体(细菌,病毒和真菌)的斑马鱼感染模型(Gratacap 和Wheeler 2014; Masud 等,2017; Varela 等,2017)。

我们的研究采用了斑马鱼的细菌感染模型,以了解涉及的关键基因的作用在天然免疫防御抗感染。将表达绿色荧光蛋白(GFP)的大肠杆菌(E. coli )显微注射到72 hpf 幼虫中,它们来自野生型斑马鱼或那些编码G-CSFR的基因的突变体(图2)。这些通过荧光显微镜监测ý Ô 版本时程来确定相对感染动力学(BASHEER 等人,2019) 。总体而言,该程序有助于了解不同的宿主-病原体相互作用,并阐明使用斑马鱼作为动物模型的过程中涉及的关键免疫基因的功能。

关键字:感染, 荧光细菌, 先天性免疫, 斑马鱼, 成像



材料和试剂


 


TR ansfer吸管,3米升(亨氏Herenz ,目录号:1131303)
网眼训练器,直径12-15厘米(孔径应小于斑马鱼胚绒毛直径,约0.5毫米网眼)
培养皿(PLP,目录号:S9014UV20 )
封口膜
带灯丝的薄壁硼硅酸盐,1.0毫米x 0.78毫米x 15厘米(SDR Scientific,目录号:30-0039 GC100TF-15)
Microloader 移液器吸头(Point of Care Diagnostics Pty Ltd,目录号:EPP5242 956.003)
康宁方菜,245 mm 未处理(Corning,目录号:38020 )
纸巾
多孔组织培养板,6 - 阱和12 - 阱(Interpath ,格雷纳,目录号小号:657160,665180)
Cuvettes(Bio -R a d,货号:2239950)
繁殖/交配槽,3 L(Techniplast )
野生型斑马鱼(Danio rerio )
大肠杆菌-GFP细菌(ATCC,目录号:25922GFP)
琼脂细菌学等级(Astral Scientific,目录号:J637-500)
N-苯基硫脲,PTU(Sigma - Aldrich,目录号:P7629-10G)
苯佐卡因(Sigma - Aldrich,目录号:E1501-100G)
氯化钠(Astral Scientific,目录号:0241-5kg)
氯化钾(Sigma - Aldrich,目录号:P9541-500G)
氯化钙2· 2H 2 O(Sigma - Aldrich,目录号:C3306-250G)
MgSO 4 · 7H 2 O(Sigma - Aldrich,目录号:63138-1KG)
亚甲蓝(Sigma - Aldrich,目录号:M9140-25G)
氢氧化钠
磷酸盐缓冲盐水-PBS 片剂(Astral Scientific,目录号:AME404-200TABS)
Triton X-100(Sigma - Aldrich,目录号:X100-500 ML )
蓝胶
BD 的Bacto TM 胰蛋白胨大豆肉汤(大豆-酪蛋白消化物培养基)(流式细胞迪克在儿子,目录号:211825)
氨苄西林,100 mg / ml ,成品溶液(Sigma- Aldrich,目录号:A5354-10ML)
Milli-Q或蒸馏水
酚红,0.5%,液体,无菌过滤(Sigma- Aldrich,目录号:P0290)
矿物油(Sigma- Aldrich,目录号:M5904-5ML)
60 x E3介质(请参阅食谱)
E3媒体(请参阅食谱)
亚甲蓝原液(请参阅食谱)
含有0.3 mg / L 亚甲蓝的E3 (请参阅食谱)
PTU库存,0.3%(w / v)(请参阅食谱)
包含0.003%(w / v)PTU的E3 (请参阅食谱)
苯佐卡因储备溶液(请参阅食谱)
10 x PBS储备溶液(请参阅食谱)
PBS (请参阅食谱)
PBS中的1%Triton X-100 (请参阅食谱)
 


设备


 


1 L和2 L玻璃瓶
磁力搅拌棒– PTFE圆柱形15 毫米长x 6 毫米直径(PLP,Cowie,目录号:001.115.6)
Eppendorf管架
移液器,100-1000 微升,20-200 微升,2-10 微升,0.5-2 微升
钟表制造商镊子,尖头镊子,样式5,114毫米(ProSciTech ,目录号:T65-SA)
孵化器,28 °C(Sanyo,目录号:MIR-162)
培养箱,37 °C(Thermo Scientific,Hereaus ,目录号:B6030)
摇动培养箱(200-225 rpm)(Infors HT Multitron 标准培养箱)
分光光度计(Bio -R ad SmartSpec Plus分光光度计,目录号:273BR03695)
微量移液器拔针器(Sutter仪器,目录号:P-97 )
本生灯
Atherton Chipmunk高压灭菌器
移液泵
微型离心机(Thermo F i s her Scientific Pty Ltd,目录号:75000461)
微量离心机(Thermo Fis her Scientific Pty Ltd,目录号:THR75002410)
MAGNETI Ç搅拌器(Ratek 仪器私人有限公司,目录号:MS10)
冰箱,4 °C
微量注射器设置(SDR临床技术,Nanoject II 纳升在Jecto ,目录号:690131,脚踏开关,目录号:690140,支座; 目录号:690141,微管支架套件选项,目录号:M-PIP-试剂盒)
带光源的立体解剖显微镜(尼康,目录号:SMZ745)
荧光显微镜(奥林巴斯,型号:MVX10,带DP72相机)
组织裂解仪II(Qiagen ,型号:85300)
油烟罩
 


软件


 


图形P 一个d P RI SM 8(http://www.graphpad.com)
ImageJ(https://imagej.nih.gov/ij/index.ht ml)
 


程序


 


准备胚胎
晚上将成年斑马鱼(一头雄性和两只雌性)转移至一个3 L的繁殖池中,以使其适应环境,然后在第二天早晨用光刺激交配。
收集胚胎从使用罐网状过滤器,并将其转移到10厘米的P 含有25 ETRI菜米升E3媒体用0.3毫克/ 大号亚甲蓝。
除去任何死亡或未受精的卵子(胚胎变黑,因为他们死和需要被利用吸力从移液管移出,然后弃去)从培养皿并传输其余的受精卵在28日举行的培养箱℃。
在24 hpf时,将胚胎转移至含有0.3 mg / L亚甲蓝和0.003%(w / v)1-苯基-2-硫脲(PTU)的E3培养基中,以防止色素沉着。
[R E放置E3媒体中的菜每日直至72 HPF 。
可以使用尖锐的钟表钳从绒毛膜上解剖出任何在72 hpf (注射日)之前未孵化的胚胎。
 


胰蛋白酶大豆肉汤制备
称重15克胰蛋白酶大豆肉汤(TSB)粉末,然后转移到1 L玻璃瓶中。
加入500 米升Milli-Q水中的和磁搅拌棒,并放置在瓶上磁力搅拌器,以允许溶解。
高压釜中加入培养基中于121 ℃,压力400-500 ķ 帕在高压釜(阿瑟顿花栗鼠灭菌)处理1小时30分钟以消毒(MAK Ë 确保玻璃瓶的盖子高压灭菌前被松开)。
灭菌完成后,取出培养基并盖紧瓶盖。
让介质在室温下完全冷却,然后再转移至4 °C进行长期存储(3-4 周)。
 


胰蛋白酶大豆琼脂-氨苄青霉素板的制备
称取6 g琼脂细菌级和15 g T SB粉到一个1 L玻璃瓶中。
加入500 米升Milli-Q水中的和磁搅拌棒,并放置在瓶上磁力搅拌器,以允许溶解。
高压釜媒体121 在高压釜中进行1℃,压力400-500千帕(阿瑟顿花栗鼠灭菌器)ħ 和30 分钟,以杀菌(MAK Ë 确保玻璃瓶的盖子高压灭菌前被松开)。
一旦消毒,删除媒体并盖紧瓶盖。
允许介质完全加入500之前在室温下冷却下来μ 升氨苄青霉素溶液(100毫克/ 米升),为100的终浓度微克/ 米升氨苄青霉素。
倾大约25 米升TSB琼脂氨苄青霉素下火焰溶液进入10厘米P ETRI菜肴而不产生气泡,并允许他们完全冷却到室温(图1)。
一旦冷却,接近盖P ETRI菜,密封用石蜡膜和储存在4 ℃下用于以后使用。




图1 。胰蛋白酶大豆肉汤(T SB)-琼脂-氨苄青霉素(Amp)板的图像


 


细菌培养与列举
接种大肠杆菌-GFP细菌进入10 米升补充有10胰蛋白酶大豆肉汤的μ 升100毫克/ 米升氨苄青霉素。
成长此细菌Ç 37 ulture过夜在振荡培养箱℃下1.1 - 1.4克。
传送1 米升的细菌培养物根据到试管,并装在先前针对未接种的TSB消隐分光光度计,并使用吸光度600nm到确定菌落形成单位(CFU)的Biorad SmartSpec 加OD 600 吸光度测定。
执行细菌培养物的系列稀释(10倍系列稀释10之间-6 10和-10 ,在9 1毫升细菌培养物的毫升TSB未接种的),电镀50-100 μ 升稀释液到TSB琼脂氨苄青霉素平板并将平板在37 °C的培养箱中孵育过夜,以确定肉汤中CFU的确切数量。
以13,751 x g f 或1-2 分钟沉淀细菌细胞,并除去上清液。
重悬STERI细菌细胞勒PBS在〜9 x的浓度10 10 CFU / 米升基于所述分光光度法。
将细菌细胞显微注射到胚胎中
使用微量移液器拔针器将薄壁精密硼硅酸盐玻璃毛细管制成细针(装入硼硅酸盐玻璃毛细管后,按以下设置拉动针头:h –365,–45,速度–80,时间–150和压力- 500 ,然后按下拉按钮,使针)。这些变量设置的增量因每种设备而异(https://www.sutter.com/manuals/P-87_OpMan.pdf)。
使用前,将针头放在带有Blu-tack的正方形板/盘上,以防止其破裂。
通过混合4准备注入材料μ 升〜9× 10 10 CFU的细菌-PBS悬浮液用1 μ 升2%(W / V)的酚红以允许注射物可视化。
使用微加载吸管,装入3针的宽端μ 升含有酚红的注射材料。
摇动针头,将注射材料带到针头的尖端,并清除气泡。
打开空气源和微型注射器,然后将针头转移到微型注射器源中,并牢固地固定在外壳内。
将微操纵器调整到正确的位置,以便在注射时进行微调,使针头靠近注射阶段和注射区域。
Anesthesize 72个HPF 胚胎用0.1mg / 米升在E3媒体苯佐卡因和转移P ETRI菜显微注射阶段。
使用锋利的钟表制造商的镊子将显微注射针的尖端弄破,使细菌细胞易于排出,释放出稳定的量,并能以最小的伤害刺穿胚胎。
可以通过将材料注入矿物油滴中,在比例尺上测量注入液滴的直径并计算体积来确定注入溶液的体积。可以通过更改注射器压力和/或针尖来调节体积。
微型注射器完全安装后,将胚胎对准针尖。
将针尖刺入72个hpf 胚胎的静脉回流中(图2),并使用脚踏板将2-5 纳升(nl )的注射溶液注射到每个胚胎中,重复进行直到注射所需数量。
注意:针尖的质量对于进行持续注射不会造成不必要的损害至关重要。


 






图2. 72 hpf 胚胎的图像,其中大肠杆菌-GFP细菌被注入静脉回流(箭头所示)。刻度条S = 200 微米。


 


一旦注入,放置在一个胚胎P 含有新鲜培养基E3用0.3 mg / L的亚甲基蓝和0.003%的PTU 15分钟,以恢复它们转移到一个28之前ETRI菜℃培养箱中。
在进行注射测定时,每个实验组应至少有30-60个胚胎,包括无菌PBS注射和未注射的对照(图3)。
 






图3 。组的图像的未注射的胚胎(A)和那些与注射大肠杆菌-GFP细菌(B) 。刻度条S = 500 微米。


 


显微注射后,除去任何死或变形的胚胎和未- 从板注射的幼虫。
为了确定每个胚胎注射的CFU的实际数量,应将五个注射的胚胎匀浆并直接铺在TSB-Agar-Amp板上,然后在37 °C 下过夜生长,然后计数CFU。
 


注射胚胎的成像
转移注射的胚胎至12 - 井或6 - 孔板成像。
甲nesthetize 胚胎用含有0.1M E3媒体克/ 米升苯佐卡因和定向它们在所需的角度进行成像使用奥林巴斯C ELL 小号ENS 标准软件用奥林巴斯MVX10荧光显微镜ë (奥林巴斯,型号:MVX10用LH100HG和将U-MGFPA / XL GFP镜滤镜连接到DP74相机进行摄影)。
ř EPEAT 此以规则的时间间隔,如0,4,8,16,24和48 ħ 感染后(HPI ),以监测GFP阳性的感染和发病率的大肠杆菌细菌(图4)。
要确定相对存活率,请根据无心跳的目视检查胚胎,以确定死亡的胚胎数量。


 






图4.在72注射的胚胎的照片HPF 与大肠杆菌-GFP细菌和在0.1可视化HPI (A)和48 HPI (B)。刻度条S = 200 微米。


 


通过平板计数进行细菌计数
用于细菌计数,收集10个住在每个时间点(例如0,4,8,16,24和48注入幼虫HPI )分别使用移液管后安乐死与50的过量的Eppendorf管微克/ 米升苯佐卡因在E3媒体。
取出所有E3介质,在新鲜的E3介质中冲洗一次以除去所有苯佐卡因。
转让1 ,000 微升1%的Triton X-100在无菌PBS溶液到管上。
在Qiagen 组织裂解仪II中以30 s的频率均质5分钟。如果需要,重复匀浆5分钟以确保胚胎完全分解。
板50-100 微升匀浆到TSB琼脂氨苄青霉素平板孵育在37 ℃下进行长达48小时,在感染的每个时间点,以确定每幼虫CFU(图5)的数量推导出感染动力学。
通过在荧光显微镜下观察它们,确认TSB-琼脂-氨苄青霉素平板上大肠杆菌-GFP细菌的生长(图6)。
 






图5 。TSB-琼脂-Amp 平板图像,用于量化胚胎中的大肠杆菌-GFP CFU


 






图6 。TSB-Agar-Amp平板上的大肠杆菌-GFP细菌菌落的荧光图像。刻度条S = 500 微米。


 


数据分析


 


可以手动计算板中的细菌菌落,也可以使用ImageJ插件“分析颗粒”进行计数,如下所示:
用菌落打开细菌平板的图像。
通过选择椭圆形或椭圆形或使用徒手工具绘制感兴趣区域来选择感兴趣区域。
清除感兴趣的外部区域– 编辑> 清除外部。
将它们转换为16位灰度图像– 图像> 类型> 16位。
调整图像阈值– 图像> 调整> 阈值,然后将阈值调整到所需水平后单击应用。
如果菌落很拥挤,并且分离得还不够好,请遵循– 处理> 二元> 分水岭。
算colonies- 分析> 分析颗粒,在弹出窗口中选择像素(10-10的大小,这取决于细菌菌落的大小000)和圆度(0.00-1.00),检查框“显示结果”,“清除结果”,“汇总”和“添加到管理员”。这将提供菌落数量和所选菌落的概述。
使用GraphPad Prism 8软件分析通过平板计数和感染的胚胎存活所列举的细菌载量。
细菌负荷数据可以分析,采用非参数配对学生牛逼-测试和受感染的胚胎存活率可以显示为的Kaplan-Meier曲线,具有统计学意义采用对数秩(曼特尔-Cox)检验确定。
 


菜谱


 


60 x E3介质
34.8 g NaCl
1.6克KCl


5.8克CaCl 2 ·2H 2 O


9.78 g氯化镁2 ·6H 2 O


为了制备60 X 股票,溶解在Milli-Q成分瓦特亚特,向2升的最终体积


用NaOH将pH调节至7.2并对其进行高压灭菌(温度:121 °C,压力:400-500 KPa,持续时间:1.5 h )


在室温下储存长达一年


E3媒体
d ilute 16.5 米升60的X 股票至990 米升用Milli-Q水


在室温下储存长达一年


亚甲蓝原液
将0.1 g亚甲蓝粉末添加到100 ml l Milli-Q水中并混合


在室温下储存长达一年


含有0.3 mg / L亚甲蓝的E3
添加600 μ 升亚甲蓝原液至1L E3媒体


在室温下存放最多1年


PTU库存[ 0.3%(w / v )]
将0.3 g PTU 添加到100 ml E3介质中                                                                                                                                           


在65 °C 溶解过夜


在室温下储存长达一年


E3包含0.003%(w / v)的PTU
将990 万升E3介质添加10 万升0.3%(w / v)PTU储备液                           


              如果晶体含量为0.3 %PTU,则在烤箱中于65 °C 孵育溶液直至溶解


奔驰ocaine储备溶液[ 10%(w / v的)]
10克苯佐卡因粉末添加到100 米升100%乙醇(在通风准备罩)                                                                     


存放在黑暗中,用铝箔纸包裹


10x PBS股票
将100片PBS片剂溶解在1升Milli-Q水和高压釜中(温度:121 °C,压力:400-500 KPa,持续时间:1.5 h )


在室温下储存长达一年


PBS
100添加米升10的X PBS至900 米升Milli-Q水和高压釜(温度:121 ℃,PRESSUR E:400-500千帕,持续时间:1.5小时)


在室温下储存长达一年


PBS中含1%Triton X-100
加入1 米升的Triton X-100至99 米升无菌PBS


在4 °C下存储长达1年


 


致谢


 


笔者小号识别支持一个的迪肯大学国际研究奖学金(FB)。作者要感谢迪肯大学动物馆的工作人员出色的水族馆管理工作,这些工作是本出版物中所述工作的基础。该方案改编自以前的工作(Fehr 等人,2015;Basheer 等人,2019 )。


 


利益争夺


 


作者没有竞争利益。


 


伦理


 


迪肯大学动物伦理委员会(G23 / 2016,31 /10 / 16- 31/1/20 20 )批准了所有涉及动物的研究。


 


参考文献


 


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引用:Basheer, F., Liongue, C. and Ward, A. C. (2020). Zebrafish Bacterial Infection Assay to Study Host-Pathogen Interactions. Bio-protocol 10(5): e3536. DOI: 10.21769/BioProtoc.3536.
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