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A Reliable Assay to Evaluate the Virulence of Aspergillus nidulans Using the Alternative Animal Model Galleria mellonella (Lepidoptera)
以大蜡螟(鳞翅目)为动物替代模型评估构巢曲霉毒力的可靠实验方法   

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Abstract

The greater wax moth Galleria mellonella has emerged as an effective heterologous host to study fungal pathogenesis and the efficacy of promising antifungal drugs (Mylonakis et al., 2005; Li et al., 2013). Here, a methodology describing the Aspergillus nidulans infection in G. mellonella larvae, along with insect survival analysis, is reported. This protocol allowed the distinction between virulent A. nidulans strains (such as TNO2A3), which induced high larval mortality rates, to those in which gene deletion was accompanied by reduced pathogenicity such as ∆gcsA and ∆sdeA (Fernandes et al., 2016).

Keywords: Aspergillus nidulans(小巢状曲菌), Galleria mellonella(大蜡螟), Fungal pathogenicity( 真菌致病性), Fungal virulence( 真菌毒力), Alternative models(模型替代)

Background

G. mellonella is an inexpensive model, easy to handle and its innate immune response shares functional similarities with the mammalian immune system. Additionally, larvae and mice infected with fungal mutant strains exhibited similar survival rates (Brennan et al., 2002). Therefore, larvae constitute a convenient animal host to substitute the use of vertebrates in fungal pathogenesis analysis. Despite all the advantages of the insect model, only a few reports have shown the effect of Aspergillus infection in G. mellonella. This protocol describes an efficient methodology to analyze Aspergillus nidulans pathogenesis in G. mellonella larvae.

Materials and Reagents

  1. Sterile toothpick
  2. Inoculation loop (microstreaker) (Thermo Fischer Scientific, Thermo ScientificTM, catalog number: SL1S )
  3. 200-1,000 µl pipette tips (Corning, Axygen®, catalog number: T-1000-B )
  4. 2-20 µl pipette tips (Corning, Axygen®, catalog number: T-200-Y )
  5. 15 ml conical tube (Greiner Bio One International, catalog number: 188271 )
  6. Miracloth filter, pore size 22-25 µm (EMD Millipore, catalog number: 475855 )
  7. 20 ml syringe (BD, catalog number: 302830 )
  8. 1.5 ml microcentrifuge tube (Corning, Axygen®, catalog number: MCT-150-R )
  9. Gloves
  10. Weighing paper
  11. Glass wool (Sigma-Aldrich, catalog number: 18421 )
  12. 280 ml plastic boxes
  13. Galleria mellonella larvae
  14. Aspergillus nidulans strains (TNO2A3 strain is available commercially in Fungal Genetics Stock Center as #A1149; ∆sdeA and ∆gcsA mutants can be provided by us upon request)
  15. Sterile deionized water
  16. Yeast extract (BD, BactoTM, catalog number: 212750 )
  17. Glucose (Sigma-Aldrich, catalog number: G8270 )
  18. Agar (BD, BactoTM, catalog number: 214010 )
  19. Uridine (Sigma-Aldrich, catalog number: U3750 )
  20. Uracil (Sigma-Aldrich, catalog number: U0750 )
  21. Zinc sulfate heptahydrate (ZnSO4·7H2O) (Sigma-Aldrich, catalog number: V000283 )
  22. Boric acid (H3BO3) (Sigma-Aldrich, catalog number: V000263 )
  23. Manganese(II) chloride tetrahydrate (MnCl2·4H2O) (Sigma-Aldrich, catalog number: 221279 )
  24. Iron(II) sulfate heptahydrate (FeSO4·7H2O) (Sigma-Aldrich, catalog number: V000119 )
  25. Cobalt(II) chloride hexahydrate (CoCl2·6H2O) (Sigma-Aldrich, catalog number: V000213 )
  26. Copper(II) sulfate pentahydrate (CuSO4·5H2O) (Sigma-Aldrich, catalog number: V000118 )
  27. Ammonium molybdate tetrahydrate (Sigma-Aldrich, catalog number: V000122 )
  28. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: V000114 )
  29. 10 N sodium hydroxide (NaOH) solution
  30. Ethanol
  31. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: V000106 )
  32. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
  33. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: V000317 )
  34. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: V000225 )
  35. Hydrogen chloride (HCl)
  36. Honey (Local supermarket)
  37. Glycerol (Sigma-Aldrich, catalog number: V000123 )
  38. Milk (Local supermarket)
  39. Wheat germ (Local supermarket)
  40. Wheat flour (Local supermarket)
  41. Wheat bran (Local supermarket)
  42. Complete media (YUU media) for A. nidulans growth (see Recipes)
  43. Trace elements solution for Aspergillus (see Recipes)
  44. 70% ethanol solution (see Recipes)
  45. PBS buffer (see Recipes)
  46. Artificial diet for Galleria mellonella (see Recipes)

Equipment

  1. 100 x 10 mm glass Petri dishes (Corning, PYREX®, catalog number: 3160-100 )
  2. Autoclave (Prismatec, model: CS-18 )
  3. Laminar flow cabinet (TROX Technik, model: TLF-A1 )
  4. 250 ml beaker (Roni Alzi, catalog number: 2-570 )
  5. Blunt tipped tweezer (EMD Millipore, catalog number: XX6200006P )
  6. Bunsen burner/lighter
  7. Hemocytometer (Hausser Scientific, catalog number: 3500 )
  8. Optical microscope with a 100x objective (American Optical Corporation)
  9. 10 µl Hamilton 700 series cemented syringe (Hamilton, catalog number: 80300 )
  10. Analytical balance (Denver Instrument, model: XL-410 , catalog number: 8515.1)
  11. Spatula (Sigma-Aldrich, catalog number: Z282774 )
  12. Graduated cylinder (Roni Alzi, catalog number: 2-950 )
  13. Glass bottle (Corning, PYREX®, catalog number: 1395-1L )
  14. Manual adjustable pipette (200-1,000 µl, Pipetman P1000) (Gilson, catalog number: F123602 )
  15. Manual adjustable pipette (2-20 µl, Pipetman P20) (Gilson, catalog number: F123600 )
  16. Thermostatic incubators (Fanem®, model: 515 )

Software

  1. GraphPad Prism 7.0 program (GraphPad Software; https://www.graphpad.com/)

Procedure

  1. Fungal growth and conidia isolation
    1. Prepare the culture media (solid YUU media) for A. nidulans growth and heat-sterilize by autoclaving at 121 °C for 20 min. Cool the media at room temperature until it turns pleasant to touch.
    2. Transfer approximately 15 ml of the melted culture media to a sterile Petri dish. Let the agar plate solidify and air-dry for 20 min at room temperature.
      Note: From this step to the end of Procedure B, it is recommended to work in a laminar flow cabinet.
    3. From a stock culture (usually stored in slant culture tubes or Petri dishes containing solid media), pick a small quantity of fungal mycelia using a sterile inoculation loop or a sterile toothpick. Transfer the loop/toothpick content to solid YUU media (prepared in step A2) by gently streaking back and forth. Make sure that mycelium is equally distributed throughout the plate, instead of performing streak plating procedure. Incubate the culture at 37 °C for 2 days or until colonies are grown.
    4. Transfer 1 ml of PBS buffer to the surface of A. nidulans culture grown in solid YUU media (after step A3). Using the pipette tip, suspend the fungal cells and transfer the suspension to a sterile 15 ml conical tube. To obtain enough conidia, repeat this step with fresh PBS buffer at least 5 times, increasing the suspension volume to ≥ 5 ml.
    5. Fill a 250 ml beaker with approximately 200 ml of 70% ethanol solution. Immerse the tweezer in ethanol solution and carefully flame it in a Bunsen burner or in a lighter. Repeat this procedure 3 x and cool the tweezer for 2-3 min at room temperature.
    6. Using the flamed tweezer, insert a Miracloth filter or a small amount of the glass wool into the end of a sterile 20 ml syringe and isolate A. nidulans conidia by filtering the fungal suspension. Collect the filtrate in a new and sterile 15 ml conical tube.
      Note: The steps A3-A6 are shown in the Video 1 (Aspergillus growth and conidia isolation). 

      Video 1. Manipulation of A. nidulans for growth and isolation of conidia. This video shows step-by-step the manipulation of A. nidulans for growth and recovery in YUU (solid media), and isolation of the conidia by filtration using a syringe containing Miracloth filter or glass wool.

    7. Transfer 10 µl of the conidial suspension to a hemocytometer and, under a 100x objective of an optical microscope, count the number of A. nidulans conidia. The concentration of conidia/ml can be estimated through the sum of cells x 10,000/number of counted squares.
      Notes:
      1. Conidia should be uniformly distributed throughout the hemocytometer and the suspension should be diluted enough in order that cells do not overlap on the grids.
      2. The fungal suspension should contain at least 108 conidia/ml, so the volume of fungal suspension used is ≤ 10 µl. This is required to maintain the volume of G. mellonella hemolymph nearly to constant, as fungal suspension will be injected in larva body cavity (as described in Procedure B). If the conidial concentration obtained is inferior to 108 conidia/ml, harvest more A. nidulans mycelia (described in step A4) and repeat the filtration and counting.
    8. Use 106 A. nidulans conidia to each larva infection. Transfer the volume of conidial suspension containing 106 conidia x the number of larvae in the experimental group to a new 1.5 ml microcentrifuge tube. Reserve an equivalent volume of PBS to the control group.

  2. Injection of Aspergillus nidulans conidia in Galleria mellonella larvae
    1. Obtain the G. mellonella larvae after oviposition of the adult moths reared at the insectarium. Insects should be maintained inside plastic boxes, with small holes on its cover. Provide enough artificial diet (ad libitum) for the larvae to be able to dig their tunnels within the diet (Figures 1A and 1B). The development of instars (shown in Figure 1C) should occur after 20-30 days at 21 °C and in the dark.


      Figure 1. Maintenance and development of G. mellonella instars include incubation in aerated plastic boxes containing ad libitum artificial diet

    2. Using gloves to avoid bacterial contamination and a blunt tipped tweezer, manually select 10-20 healthy G. mellonella larvae, from the last instar with similar size (15-20 mm) and weight (approximately 0.2 g) for each experimental group. Place up to 10 larvae into a sterile 100 x 10 mm Petri dish without any food.
      Notes:
      1. Creamy colored insects, which do not exhibit any grey pigmentation and are responsive to the tweezer touch, can be defined as healthy. Grey colored larvae should be discarded.
      2. Provide enough larvae for the experimental conditions and for two different control groups. The first control group may include larvae that are inoculated with PBS buffer, to monitor deaths provoked by physical injury. The insects of the second control group should not receive any injection.
    3. Wash the 10 µl Hamilton syringe by aspirating and discarding the 70% ethanol solution. Repeat this procedure at least 3 times.
    4. Remove the residual ethanol by washing the syringe with room temperature sterile water.
    5. Using the Hamilton syringe, aspirate 10 µl of fungal suspension, which contains 106 A. nidulans conidia (obtained in the step A8).
    6. Gently, pick a G. mellonella larva with one hand and firmly hold it by its back using your index and thumb fingers (Figure 2).


      Figure 2. Handling G. mellonella larva for A. nidulans infection

    7. Carefully insert the needle of the Hamilton syringe into the larva last left pro-leg and slowly inject the volume corresponding to 106 A. nidulans conidia in the insect hemolymph (Figure 3).
      Notes:
      1. Avoid using too much force not to puncture the larva and leak the hemolymph. In such situation, select another larva.
      2. The steps B3-B7 are shown in the Video 2 (Injecting A. nidulans conidia in G. mellonella larvae)


        Figure 3. Inoculation of G. mellonella by injection of fungal suspension in the larval last left pro-leg

        Video 2. G. mellonella infection with A. nidulans conidia. This video shows all steps, from the washing of Hamilton syringe to inoculation by A. nidulans conidia injection. In all experiments, inoculation was performed in the last left pro-leg.

    8. Place the larvae of each experimental group in a new sterile Petri dish. After manipulating all larvae, incubate them in the dark at 28 °C.
      Note: Larvae can also be incubated at 37 °C to mimic human body temperature.
    9. After 24 h of infection, gently touch the larvae with the blunt tipped tweezers to evaluate survival. Alive larvae (shown in Figure 4A) should respond quickly to touch, while absence of movement associated with a grey/dark pigmentation characterize dead larvae (present in 4B). Record the number of alive and dead larvae every 24 h in each experimental group. After approximately 10 days, larvae turn into pupa.
      Notes:
      1. Disposal of all animals used in the experiment should be done after autoclaving/decontamination of the Petri dishes containing the larvae.
      2. This step is shown in the Video 3 (Analysis of larvae survival).


        Figure 4. Analysis of the larvae survival after A. nidulans infection. Alive instars (panel A) exhibit creamy pigmentation and show movement response under stimulation. Dead instars exhibit grey/dark pigmentation and are unresponsive to touch.

        Video 3. Evaluation of G. mellonella survival after A. nidulans infection. After A. nidulans inoculation, G. mellonella larvae were daily analyzed for unresponsiveness to stimulation (dead larvae).

Data analysis

  1. Data analysis of G. mellonella survival using GraphPad Prism 7.0.
    1. Open the GraphPad Prism 7.0 as a new project. A free trial of the software is available online (https://www.graphpad.com/).
    2. On the New Table and Graph window, select Survival table where each row tabulates the survival or censored time of a subject (Figure 5).


      Figure 5. The selection of ‘Survival table’ in GraphPad Prism initial menu

    3. Record the experimental data in ‘Data Tables’ by inserting, in the x-axis, time measurement of the experiment (days) and, in the y-axis, the number of dead larvae per day for each experimental group.
      Note: If no larva dies on a given time point, it is not necessary to add number ‘0’ in this respective time. Record only the days/hours in which larvae death occurred by inserting number ‘1’ for each dead animal. If more than one animal in the group died in a time point, repeat the same day in a subsequent row and insert ‘1’ as many as necessary. In the data table shown below (Figure 6), days 7, 8, 9 were not included since no larval death was observed.


      Figure 6. Table fills with the experimental data in survival data table

    4. In the last time point of the experiment, score 0 to each alive larva in the respective group. If more than one animal in the group remained alive, repeat the same day/hour in a subsequent row and insert ‘0’ as many as necessary (Figure 7).


      Figure 7. The registering of each alive larva by the end of the experiment

    5. GraphPad Prism automatically analyzes statistical significance by using Kaplan-Meier survival curves. Open the ‘Results Table’ in the same project to check statistical significance (Figure 8).


      Figure 8. Results table showing the analysis of statistical significance

    6. GraphPad Prism also automatically creates data graph, which can be easily edited (Figure 9).


      Figure 9. Illustration of a survival graph created by GraphPad Prism

    7. For project presentation or manuscript submission, data graph can be edited and exported in several formats. On project window, select ‘export’ on file attributions.

Recipes

  1. Complete media (YUU media) for A. nidulans growth
    0.5% yeast extract
    2% glucose
    2% agar
    0.1% trace elements solution
    5 mM uridine
    10 mM uracil
    1. Place a weighing paper on the balance and tare it. Using a spatula, transfer the required mass of each reagent
    2. Place the agar, uridine and uracil powders directly in the glass container where the culture media will be autoclaved
    3. Dissolve the yeast extract, glucose and trace elements solution in ¾ of the final volume with deionized water
    4. Using a graduated cylinder, complete with water to the final volume and transfer this solution to the glass bottle containing the agar, uridine and uracil. Heat-sterilize by autoclaving it at 121 °C for 20 min

      Note: Make sure that the total volume of the culture media represents less than 2/3 of the glass container capacity, to avoid spillage during the autoclaving process.

  2. Trace elements solution for Aspergillus
    75 mM zinc sulfate heptahydrate
    180 mM boric acid
    25 mM manganese(II) chloride tetrahydrate
    18 mM iron (II) sulfate heptahydrate
    6 mM cobalt(II) chloride hexahydrate
    6 mM copper(II) sulfate pentahydrate
    1 mM ammonium molybdate tetrahydrate
    140 mM ethylenediaminetetraacetic acid
    1. In the analytical balance, weigh each reagent. Dissolve the powders, following the order above, in 1/8 of the final volume with deionized water
    2. Heat the solution to 100 °C and then cool it to 60 °C
    3. Adjust the pH to 6.5-6.8 with a 10 N sodium hydroxide solution
    4. Chill the solution to room temperature and then add deionized water to the final volume
  3. 70% ethanol solution
    Each 100 ml contains 70 ml of ethanol diluted in 30 ml of distilled water
  4. PBS buffer (according to Sambrook et al., 2005)
    137 mM sodium chloride
    2.7 mM potassium chloride
    10 mM sodium phosphate dibasic
    1.8 mM potassium phosphate monobasic
    1. Weigh the reagents and dissolve them in ¾ of the final volume with deionized water
    2. Adjust the pH for 7.4 with hydrogen chloride and then add deionized water to the total volume
    3. Divide the solution in smaller aliquots and sterilize by autoclaving for 20 min
  5. Artificial diet for Galleria mellonella
    120 g of honey
    120 g of glycerol
    200 g of milk
    60 g of yeast extract
    100 g of wheat germ
    100 g of wheat flour
    120 g of wheat bran
    Using the analytical balance, weigh each ingredient. Mixture the artificial diet components and heat-sterilize by autoclaving at 121 °C for 20 min

Acknowledgments

Sources of funding for this work were from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo a Pesquisa do Rio de Janeiro (FAPERJ), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). This protocol is a detailed description of the methodology used in Fernandes et al., 2016.

References

  1. Brennan, M., Thomas, D. Y., Whiteway, M. and Kavanagh, K. (2002). Correlation between virulence of Candida albicans mutants in mice and Galleria mellonella larvae. FEMS Immunol Med Microbiol 34(2): 153-157.
  2. Fernandes, C. M., de Castro, P. A., Singh, A., Fonseca, F. L., Pereira, M. D., Vila, T. V., Atella, G. C., Rozental, S., Savoldi, M., Del Poeta, M., Goldman, G. H. and Kurtenbach, E. (2016). Functional characterization of the Aspergillus nidulans glucosylceramide pathway reveals that LCB Δ8-desaturation and C9-methylation are relevant to filamentous growth, lipid raft localization and Psd1 defensin activity. Mol Microbiol 102(3): 488-505.
  3. Li, D. D., Deng, L., Hu, G. H., Zhao, L. X., Hu, D. D., Jiang, Y. Y. and Wang, Y. (2013). Using Galleria mellonella-Candida albicans infection model to evaluate antifungal agents. Biol Pharm Bull 36(9): 1482-1487.
  4. Mylonakis, E., Moreno, R., El Khoury, J. B., Idnurm, A., Heitman, J., Calderwood, S. B., Ausubel, F. M. and Diener, A. (2005). Galleria mellonella as a model system to study Cryptococcus neoformans pathogenesis. Infect Immun 73(7): 3842-3850.
  5. Sambrook, J., Fristch, E. F. and Maniatis, T. (1989). Molecular cloning: a laboratory manual. (2nd edition). Cold Spring Harbor Laboratory Press.

简介

作为一种有效的异源宿主,越来越多的蜡蛾已经出现了一种有效的异源宿主,用于研究真菌发病机制和有希望的抗真菌药物的功效(Mylonakis等人,2005; Li& et al。2013)。这里,描述了一种描述构巢曲霉感染的方法。 mellonella 幼虫与昆虫存活分析一起报道。该协议允许区分有毒的 A。造成高幼虫死亡率的构巢组织菌株(如TNO2A3)与基因缺失伴随着致病性降低的病例如ΔGCA和ΔSAA(Fernandes 等人,2016)。

背景 -G。 mellonella 是一种廉价的模型,易于处理,其先天免疫反应与哺乳动物免疫系统分享功能相似性。此外,感染真菌突变菌株的幼虫和小鼠表现出相似的存活率(Brennan等人,2002)。因此,幼虫构成了一种方便的动物宿主,用于在真菌发病机理分析中代替脊椎动物的使用。尽管昆虫模型具有所有优点,但只有少数报告显示了G中曲霉菌感染的作用。蜡螟。该协议描述了一种有效的方法,用于分析G中的构巢曲霉发病机制。 mellonella 幼虫。

关键字:小巢状曲菌, 大蜡螟,  真菌致病性,  真菌毒力, 模型替代

材料和试剂

  1. 无菌牙签
  2. 接种环(微细纹)(Thermo Fischer Scientific,Thermo Scientific TM,目录号:SL1S)
  3. 200-1,000微升移液管吸头(Corning,Axygen ®,目录号:T-1000-B)
  4. 2-20μl移液器吸头(Corning,Axygen ®,目录号:T-200-Y)
  5. 15ml锥形管(Greiner Bio One International,目录号:188271)
  6. 微孔过滤器,孔径22-25μm(EMD Millipore,目录号:475855)
  7. 20 ml注射器(BD,目录号:302830)
  8. 1.5ml微量离心管(Corning,Axygen ,目录号:MCT-150-R)
  9. 手套
  10. 称重纸
  11. 玻璃棉(Sigma-Aldrich,目录号:18421)
  12. 280 ml塑料盒
  13. 幼虫
  14. 构巢曲霉菌株(TNO2A3菌株可以在真菌遗传学库中心商购获得,如#A1149;ΔscAA和ΔgcsA突变体可由我们提供根据要求)
  15. 无菌去离子水
  16. 酵母提取物(BD,Bacto TM ,目录号:212750)
  17. 葡萄糖(Sigma-Aldrich,目录号:G8270)
  18. 琼脂(BD,Bacto TM ,目录号:214010)
  19. 尿苷(Sigma-Aldrich,目录号:U3750)
  20. 尿嘧啶(Sigma-Aldrich,目录号:U0750)
  21. 硫酸锌七水合物(ZnSO 4·7H 2 O)(Sigma-Aldrich,目录号:V000283)
  22. 硼酸(H 3 O 3 BO 3)(Sigma-Aldrich,目录号:V000263)
  23. 四氢化锰(II)四水合物(MnCl 2·4H 2 O)(Sigma-Aldrich,目录号:221279)
  24. 硫酸铁(II)七水合物(FeSO 4·7H 2 O)(Sigma-Aldrich,目录号:V000119)
  25. 二氯化钴(II)六水合物(CoCl 2·6H 2 O)(Sigma-Aldrich,目录号:V000213)
  26. 硫酸铜(II)五水合物(CuSO 4·5H 2 O)(Sigma-Aldrich,目录号:V000118)
  27. 钼酸铵四水合物(Sigma-Aldrich,目录号:V000122)
  28. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:V000114)
  29. 10 N氢氧化钠(NaOH)溶液
  30. 乙醇
  31. 氯化钠(NaCl)(Sigma-Aldrich,目录号:V000106)
  32. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9541)
  33. 磷酸氢二钠(Na 2 HPO 4)(Sigma-Aldrich,目录号:V000317)
  34. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:V000225)
  35. 氯化氢(HCl)
  36. 蜂蜜(本地超市)
  37. 甘油(Sigma-Aldrich,目录号:V000123)
  38. 牛奶(当地超市)
  39. 小麦胚芽(本地超市)
  40. 小麦粉(本地超市)
  41. 麦麸(本地超市)
  42. 完整的媒体(YUU媒体)为构建成功者(见配方)
  43. 曲霉菌微量元素解决方案(见食谱)
  44. 70%乙醇溶液(见食谱)
  45. PBS缓冲液(参见食谱)









  46. 设备

    1. 100×10mm玻璃培养皿(Corning,PYREX ®,目录号:3160-100)
    2. 高压釜(Prismatec,型号:CS-18)
    3. 层流柜(TROX Technik,型号:TLF-A1)
    4. 250毫升烧杯(Roni Alzi,目录号:2-570)
    5. 钝头镊子(EMD Millipore,目录号:XX6200006P)
    6. 本生灯/打火机
    7. 血细胞计数器(Hausser Scientific,目录号:3500)
    8. 具有100x物镜的光学显微镜(美国光学公司)
    9. 10μlHamilton 700系列胶结注射器(Hamilton,目录号:80300)
    10. 分析天平(丹佛仪器,型号:XL-410,目录号:8515.1)
    11. Spatula(Sigma-Aldrich,目录号:Z282774)
    12. 量筒(Roni Alzi,目录号:2-950)
    13. 玻璃瓶(康宁,PYREX ®,目录号:1395-1L)
    14. 手动可调吸管(200-1,000μl,Pipetman P1000)(Gilson,目录号:F123602)
    15. 手动可调移液管(2-20μl,Pipetman P20)(Gilson,目录号:F123600)
    16. 恒温培养箱(Fanem ®,型号:515)

      软件

      1. GraphPad Prism 7.0程序(GraphPad Software; https://www.graphpad.com/

        程序

        1. 真菌生长和分生孢子分离
          1. 制备用于构巢曲霉的培养基(固体YUU培养基)生长并通过在121℃下高压灭菌20分钟进行热灭菌。在室温下冷却介质,直至接触愉快。
          2. 将大约15ml的熔融培养基转移到无菌培养皿中。让琼脂板在室温下固化并风干20分钟 注意:从本步骤到步骤B的最后,建议在层流柜中工作。
          3. 从储存培养物(通常储存在含有固体培养基的斜培养管或培养皿中)中,使用无菌接种环或无菌牙签挑取少量真菌菌丝体。通过轻轻地来回传送环/牙签内容到固体YUU介质(在步骤A2中制备)。确保菌丝体均匀分布在整个板上,而不是进行条纹电镀程序。将培养物在37℃孵育2天或直到菌落生长。
          4. 将1ml PBS缓冲液转移到A的表面。构建体培养在固体YUU培养基中生长(在步骤A3之后)。使用吸头,悬浮真菌细胞并将悬浮液转移到无菌的15毫升锥形管中。要获得足够的分生孢子,用新鲜PBS缓冲液重复此步骤至少5次,将悬浮液体积增加至≥5ml。
          5. 用约200ml 70%乙醇溶液填充250ml烧杯。将镊子浸入乙醇溶液中,并在本生灯或打火机中小心地点燃。重复此步骤3 x,并在室温下冷却镊子2-3分钟。
          6. 使用火焰镊子,将Miracloth过滤器或少量玻璃棉插入无菌20ml注射器的末端,并隔离A。通过过滤真菌悬浮液构成分生孢子。在新的无菌的15毫升锥形管中收集滤液。
            注意:视频1(曲霉生长和分生孢子分离)中显示了步骤A3-A6。 

          7. 将10μl的分生孢子悬浮液转移到血细胞计数器中,并在光学显微镜的100倍物镜下计数A的数目。构巢体分生孢子。可以通过细胞总数×10,000 /计数方格数来估计分生孢子/ ml的浓度。
            注意:
            1. 分生孢子应均匀分布在整个血细胞计数器中,悬浮液应足够稀释,以使细胞在网格上不重叠。
            2. 真菌悬浮液应含有至少10分/分,因此使用的真菌悬浮液体积≤10μl。由于真菌悬浮液将注入幼虫体腔(如方法B所述),所以需要保持G.mellonella血淋巴的体积几乎恒定。如果获得的分生孢子浓度低于分生孢子/ ml的10分钟,则收获更多的构巢曲霉菌丝体(在步骤A4中描述),并重复过滤和点数。
          8. 使用10 6 A。结核分枝杆菌分生孢子对每只幼虫感染。将实验组中含有10 sup 6分生孢子x的分生孢子体积转移到新的1.5ml微量离心管中。保留等量的PBS给对照组。

        2. 注射构巢曲霉分生孢子中的分枝杆菌幼虫(Galleria mellonella)幼虫
          1. 在昆虫蛹后饲养的成年蛾产卵后,获得。ella on。。昆虫应保持在塑料盒内,盖子上有小孔。为幼虫提供足够的人工饮食(),以便能够在饮食中挖掘他们的隧道(图1A和1B)。时代的发展(如图1C所示)应在21°C和黑暗中20-30天后发生

            图1.维护和开发G。 mellonella instars包括在含有自由口头人工饮食的充气塑料盒中孵育

          2. 使用手套避免细菌污染和钝头镊子,手动选择10-20健康的G。来自具有相似尺寸(15-20mm)的最后一龄的幼虫,并且每个实验组的体重(约0.2g)。将最多10只幼虫放入无菌的100 x 10毫米培养皿中,无任何食物。
            注意:
            1. 乳白色的昆虫,不显示任何灰色色素沉着和对镊子的触感,可以被定义为健康。灰色幼虫应丢弃。
            2. 为实验条件和两个不同的对照组提供足够的幼虫。第一对照组可以包括用PBS缓冲液接种的幼虫,以监测由身体损伤引起的死亡。第二个对照组的昆虫不应该接受任何注射。
          3. 通过吸取和丢弃70%乙醇溶液清洗10μlHamilton注射器。重复此过程至少3次。
          4. 通过用室温无菌水洗涤注射器去除残留的乙醇。
          5. 使用汉密尔顿注射器,吸出10μl真菌悬浮液,其中含有10μg/ ml的真菌悬浮液。构巢体分生孢子(在步骤A8中获得)。
          6. 轻轻一点,选一个。 mellonella 幼虫用一只手牢牢握住它的背部,使用你的索引和拇指(图2)。


            图2.处理 G。 mellonella 幼虫为 A。 nidulans 感染

          7. 小心地将哈密尔顿注射器的针插入幼体最后一个左前腿,并缓慢注射相当于10 6 的体积。构巢体分生孢子在昆虫血淋巴中(图3) 注意:
            1. 避免使用太多的力量不刺穿幼虫并泄漏血淋巴。在这种情况下,请选择另一只幼虫。
            2. 步骤B3-B7显示在视频2(注入A.mellonella幼虫中的构巢曲霉分生孢子中)


              图3. G的接种。 mellonella 通过注射真菌悬浮液在幼虫最后一个左前腿


          8. 将每个实验组的幼虫置于新的无菌培养皿中。在操作所有幼虫后,在28°C的黑暗中孵育。
            注意:幼虫也可以在37℃下孵育以模拟人体体温。
          9. 感染24小时后,用钝头镊子轻轻触摸幼虫以评估存活。活着的幼虫(如图4A所示)应该快速反应,而没有与灰色/深色素沉着相关的运动表征死亡的幼虫(存在于4B中)。在每个实验组中每24小时记录一次活的和死亡的幼虫数量。约10天后,幼虫变成蛹。
            注意:
            1. 实验中使用的所有动物的处理应在含有幼虫的培养皿的高压灭菌/去污后进行。

            2. 这个步骤显示在视频3(幼虫生存分析)中

              图4. A后的幼虫生存分析。构巢体感染。活组织(图A)显示出乳白色色素沉着,并显示刺激下的运动反应。死亡时期表现出灰色/深色色素沉着,无反应。

        数据分析

        1. G的数据分析。使用GraphPad Prism 7.0的mellonella 生存。
          1. 打开GraphPad Prism 7.0作为新项目。可以在线获得该软件的免费试用版( https://www.graphpad.com/ < / a>)。
          2. 在“新建表和图表”窗口中,选择“生存”表,其中每一行列出主体的生存或审查时间(图5)。


            图5. GraphPad Prism初始菜单中的“生存表”选择

          3. 在“数据表”中记录实验数据,通过在x轴上插入实验的时间测量(天),并在y轴上插入每个实验组每天的死亡幼虫数量。
            注意:如果在给定的时间点没有幼虫死亡,则不需要在此时间内添加数字“0”。只记录幼虫死亡的天数/小时,为每只死亡动物插入数字“1”。如果该组中多于一只的动物在一个时间点中死亡,请在随后的一天重复同一天,并根据需要插入“1”。在下面所示的数据表(图6)中,没有包括第7,8,9天,因为没有观察到幼虫死亡。


            图6.表填写生存数据表中的实验数据

          4. 在实验的最后一个时间点,对各组中的每个活着的幼虫进行0分。如果该组中有多只动物仍然存活,请在随后的一行重复相同的日/小时,并根据需要插入“0”(图7)。


            图7.在实验结束之前注册每个活的幼虫

          5. GraphPad棱镜通过使用Kaplan-Meier生存曲线自动分析统计学显着性。在同一个项目中打开“结果表”,以检查统计学意义(图8)。


            图8.显示统计学意义分析的结果表

          6. GraphPad Prism还自动创建数据图,可以轻松编辑(图9)

            图9. GraphPad Prism创建的生存图的图示

          7. 对于项目演示或稿件提交,数据图可以以多种格式进行编辑和导出。在项目窗口中,选择“导出”文件归属。

        食谱

        1. 完整的媒体(YUU媒体)为 A。构建者增长
          0.5%酵母提取物
          2%葡萄糖
          2%琼脂
          0.1%微量元素溶液
          5 mM尿苷
          10 mM尿嘧啶
          1. 在天平上放置称重纸,称重。使用刮铲,转移所需质量的每种试剂
          2. 将琼脂,尿苷和尿嘧啶粉末直接放入玻璃容器中,培养基将被高压灭菌
          3. 用去离子水溶解酵母提取物,葡萄糖和微量元素溶液至最终体积的3/4
          4. 使用量筒,完成水至最终体积,并将该溶液转移到含有琼脂,尿苷和尿嘧啶的玻璃瓶中。通过在121℃高压灭菌20分钟进行热灭菌

            注意:确保培养基的总体积小于玻璃容器容量的2/3,以避免在高压灭菌过程中溢出。

        2. 曲霉菌
          的微量元素溶液 75毫升七水硫酸锌七水合物 180 mM硼酸
          25mM氯化锰(II)四水合物
          18 mM硫酸铁(II)七水合物
          6mM氯化钴(II)六水合物
          6 mM硫酸铜(II)五水合物
          1mM钼酸铵四水合物
          140mM乙二胺四乙酸
          1. 在分析天平中称量每种试剂。按照上述顺序将粉末溶解在最终体积的1/8中,用去离子水进行
          2. 将溶液加热至100°C,然后将其冷却至60°C
          3. 用10 N氢氧化钠溶液调节pH至6.5-6.8
          4. 将溶液冷却至室温,然后加入去离子水至最终体积
        3. 70%乙醇溶液
          每100ml含有稀释在30ml蒸馏水中的70ml乙醇
        4. PBS缓冲液(根据Sambrook等人,2005)
          137 mM氯化钠
          2.7 mM氯化钾
          10 mM磷酸氢二钠
          1.8mM磷酸二氢钾
          1. 称取试剂并用去离子水将其溶解至最终体积的3/4
          2. 用氯化氢调节pH值为7.4,然后加入去离子水至总体积
          3. 将溶液分成较小的等分试样,并通过高压灭菌消毒20分钟







        5. 120克蜂蜜
          120克甘油
          200克牛奶
          60克酵母提取物 100克小麦胚芽
          100克小麦面粉
          120克麦麸 使用分析天平,称量每种成分。混合人造饮食成分,并在121℃高压灭菌20分钟进行热灭菌

        致谢

        这项工作的资金来源是来自国家科技促进委员会(CNPq),安巴卢基金会,里约热内卢(FAPERJ),巴塞罗那高等教育协调中心(CAPES)。该协议是对Fernandes等人,2016年的使用方法的详细描述。

        参考

        1. Brennan,M.,Thomas,DY,Whiteway,M.and Kavanagh,K。(2002)。小鼠中的白色念珠菌突变体和幼虫的毒力之间的相关性。FEMS Immunol Med Microbiol / em> 34(2):153-157。
        2. Fernandes,CM,de Castro,PA,Singh,A.,Fonseca,FL,Pereira,MD,Vila,TV,Atella,GC,Rozental,S.,Savoldi,M.,Del Poeta,M.,Goldman,GH和Kurtenbach,E.(2016)。功能表征构巢曲霉葡萄糖神经酰胺途径揭示了LCBΔ8-去饱和和C9-甲基化与丝状生长,脂筏定位和Psd1防御素活性相关。分子微生物3):488-505。
        3. Li,DD,Deng,L.,Hu,GH,Zhao,LX,Hu,DD,Jiang,YY and Wang,Y。(2013)。使用环孢菌素 - 白色念珠菌感染模型评估抗真菌药物。 Bull 36(9):1482-1487。
        4. Mylonakis,E.,Moreno,R.,El Khoury,JB,Idnurm,A.,Heitman,J.,Calderwood,SB,Ausubel,FM和Diener,A.(2005)。&lt; a class = insertfile“href =”http://www.ncbi.nlm.nih.gov/pubmed/15972469“target =”_ blank“> 作为模型系统研究新型隐球酵母< / em>发病机制。感染免疫 73(7):3842-3850。
        5. Sambrook,J.,Fristch,EF和Maniatis,T。(1989)。&lt; a class =“ke-insertfile”href =“http://www.cell.com/cell/abstract/0092-8674(90 )90210-6“target =”_ blank“>分子克隆:实验室手册。 (2 nd edition)冷泉港实验室出版社。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Fernandes, C. M., Fonseca, F. L., Goldman, G. H., Pereira, M. D. and Kurtenbach, E. (2017). A Reliable Assay to Evaluate the Virulence of Aspergillus nidulans Using the Alternative Animal Model Galleria mellonella (Lepidoptera). Bio-protocol 7(11): e2329. DOI: 10.21769/BioProtoc.2329.
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