Protease Activity Assay in Fly Intestines
苍蝇肠道蛋白酶活性测定   

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Mar 2017

 

Abstract

The intestine is a central organ required for the digestion of food, the absorption of nutrients and for fighting against aggressors ingested along with the food. Impairment of gut physiology following mucosal damages impacts its digestive capacities that consequently will affect growth, wellbeing or even survival of the individual. Hence, the assessment of intestinal functions encompasses, among others, the monitoring of its integrity, its cellular renewing, its immune defenses, the production of enteroendocrine hormones and its digestive capacities. Here, we describe in detail how to assess the activity of the proteases secreted in the intestinal lumen of adult Drosophila melanogaster flies. This method can also be used for larval intestines. The present protocol is adapted and improved from the Sigma-Aldrich’s protocol proposed in the ‘Protease Fluorescent Detection Kit’ (Product code PF0100).

Keywords: Drosophila melanogaster (黑腹果蝇), Intestine (肠道), Opportunistic bacteria (机会性细菌), Protease activity (蛋白酶活性), Protein metabolism (蛋白质代谢)

Background

The intestine is subjected to many stresses such as feasting, fasting, chemicals, pathogens, injuries etc. The gut is able to overcome such stresses by maintaining its physiological equilibrium named homeostasis. To perceive the incoming stress and to yield an adapted answer to maintain gut functions, the intestine has developed robust and conserved mechanisms such as local innate immune defenses and tissue regeneration (Royet and Charroux, 2013; Bonfini et al., 2016). However, the maintenance of gut homeostasis can be compromised in certain cases. For example, during aging, there is an overall decline in tissue homeostasis maintenance with the presence of numerous immature or misdifferentiated cells (Jasper, 2015; Hu and Jasper, 2017). Another case where homeostasis can also be disrupted is upon exposure to xenobiotic or pathogens (such as opportunistic bacteria) that damage or kill cells impairing their functions (Bonfini et al., 2016). Hence, during the above cited examples, the digestive capacities of the gut are reduced. Moreover, during the process of tissue regeneration itself that produces many precursor cells the digestive capacities are also reduced (Loudhaief et al., 2017). Therefore, the assessment of the digestive capacities of the gut are of prime importance to evaluate the potential impact that can have an aggression on the gut physiology. Importantly, gut digestive function disruption may have both local and systemic metabolic consequences that will affect growth, immune defenses, reproduction, wellbeing, longevity…. Dietary proteins are essential for many (if not all) physiological functions (Soultoukis and Partridge, 2016). Imbalanced amino-acid absorption by the intestine can have dramatic consequences on growth for example. Protein digestion being essential to generate absorbable amino-acids by the enterocytes, the measurement of luminal protease activity appears a good readout to evaluate the physiological state of the intestine and its capacity to fulfill its digestive functions.

Materials and Reagents

  1. Drosophila rearing
    1. 6oz Drosophila stock bottles (Genesee Scientific, catalog number: 32-130 )
    2. Cotton balls for stock bottles (Genesee Scientific, catalog number: 51-102B )
    3. CantonS flies (Bloomington Drosophila Stock Center, catalog number: 64349 ) (flystocks.bio.indiana.edu)
    4. Agar (VWR, BDH®, catalog number: 20768-361 )
    5. Sugar (Carrefour or any other supermarket)
    6. Cornflour (AB, Celnat - NaturDis)
    7. Yeast (Biospringer, catalog number: BA10/0-PW )
    8. Tegosept (Apex, Fly Food preservative, Genesee Scientific, catalog number: 20-258 )
    9. Standard nutrient medium for Drosophila (see Recipes)

  2. Bacterial culture
    1. Petri dishes
    2. Sterile tip
    3. 15 ml tubes (Corning, Falcon®, catalog number: 352096 )
    4. Graduated test tube
    5. Bacillus thuringiensis var. kurstaki (Btk) strain identified under the code 4D22 at the Bacillus Genetic Stock Center (http://www.bgsc.org/) and described by (Gonzalez et al., 1982)
    6. Erwinia carotovora carotovora (Ecc) was kindly provided by Bruno Lemaitre’s laboratory (École Polytechnique Fédérale, Lausanne, Switzerland)
    7. Escherichia coli (Ec) (One ShotTM TOP10 Chemically Competent E. coli) (Thermo Fisher Scientific, InvitrogenTM, catalog number: C404003 )
    8. Luria broth powder (Conda, catalog number: 1551 )
    9. Agar bacteriological (Euromedex, catalog number: 1330 )
    10. LB medium (see Recipes)
    11. LB-agar medium (see Recipes)

  3. Intoxication
    1. Cotton balls for narrow vials 25 mm (Genesee Scientific, catalog number: 51-101 )
    2. Spectrophotometry cuvettes (Ratiolab, catalog number: 2712120 )
    3. 2 ml microtubes (Paul Bottger, catalog number: 02-043 )
    4. 20 mm filter disks (Chromatography paper 3MM Chr) (GE Healthcare, catalog number: 3030-917 )
    5. 50 ml tube
    6. Drosophila narrow vials 25 mm (Genesee Scientific, catalog number: 32-109RL )
    7. Sucrose (Euromedex, catalog number: 200-301-B )
    8. 10% sucrose (see Recipes)

  4. Dissection
    1. 1.5 ml microtubes (Paul Bottger, catalog number: 02-063 )
    2. Graduated test tube
    3. Watch glass (Steriplan Petri dishes, DWK Life Sciences, catalog number: 237554008 )
    4. Ice
    5. Ethanol 70% (VWR, catalog number: 83801.360 )
    6. 10x PBS (Euromedex, catalog number: ET330 )
    7. 1x phosphate-buffered saline (PBS) (see Recipes)

  5. Sample preparation
    1. Microtube pestle 1.5 ml (Argos Technologies, catalog number: P7339-901 )
    2. 0.5 ml microtubes (Paul Bottger, catalog number: 02-053 )
    3. 1x phosphate-buffered saline (PBS) (see Recipes)

  6. Assay
    1. 1.5 ml microtubes (Paul Bottger, catalog number: 02-063 )
    2. 96-well black microplates (Greiner Bio One International, catalog number: 655076 )
    3. 2 ml microtube
    4. 15 ml tube
    5. Aluminum foil
    6. Trypsin from bovine pancreas (Sigma-Aldrich, catalog number: T1005 )
    7. 1 mM HCl
    8. Casein Fluorescein IsoThioCyanate from bovine milk (Sigma-Aldrich, catalog number: C0528 )
    9. Distilled water
    10. cOmplete tablets EDTA-free (Roche Diagnostics, catalog number: 04693132001 )
    11. Trichloroacetic acid (TCA) (Sigma-Aldrich, catalog number: T6399 )
    12. Tris base
    13. Trypsin solution (see Recipes)
    14. Casein-FITC (see Recipes)
    15. 25x cOmplete (see Recipes)
    16. 10% TCA (see Recipes)
    17. 0.5 M Tris/HCl pH 8.5 (see Recipes)

Equipment

  1. Drosophila rearing
    Refrigerated oven at constant temperature of 25 °C and with a 12 h/12 h light/dark cycle (Fisher Scientific, catalog number: 11857552). Humidity has to be maintained between 40% and 70%
    Manufacturer: LMS, model: Model 240 .

  2. Bacterial culture
    1. 100 ml flasks (Fisher Scientific, catalog number: 15409103 )
    2. 30 °C/37 °C shaking incubator (Infors, model: AK 82 )

  3. Intoxication
    1. Spectrophotometer (Aqualabo, Secomam, model: Prim Light & Aduanced)
    2. Droso-sleeper (Inject-Matic)

  4. Dissection
    1. Stereomicroscope (Leica Microsystems, model: Leica M60 )
    2. Dumont forceps #5 (Fine Science Tools, catalog numbers: 11251-20 and 11252-20 )

  5. Sample preparation
    1. Pestle motor (Heidolph Instruments, model: RZR 2100 )
    2. Refrigerated microfuge (Eppendorf, model: 5430 R )

  6. Assay
    1. Vortex (Scientific Industries, model: Vortex-Genie 2 )
    2. Automated pipette (Eppendorf, model: Multipette® plus)
    3. 37 °C solid-door incubator (Jouan)
    4. Fluorimeter (Agilent Technologies, model: Cary Eclipse )

Software

  1. Kyplot
  2. Excel

Procedure

  1. Drosophila rearing
    1. CantonS flies are reared on standard medium for Drosophila melanogaster (see Recipes) at 25 °C.
    2. Only virgin five-days-old females are used in our experiments. To obtain synchronized five-day-old females, we empty bottles from adult flies, then we wait for the emergence of new flies from pupae. Among the emergent flies, males are rapidly discarded and females are transferred in new bottles for five days at 25 °C.

  2. Bacterial culture
    In the experiments presented below, we use three different bacterial strains (Btk, Ecc and Ec, see Materials and Reagents) and water as negative control (Ctrl). Spread bacteria from the stocks on LB agar Petri dishes and let grow overnight at the required temperature (30 °C for 4D22 and Ecc and 37 °C for Ec).
    1. Pick a single colony using a sterile tip and initiate a 10 ml starter culture of LB (see Recipes) in a 15 ml tube. Let grow for 8 h shaking (220 rpm) at 30 °C for 4D22 and Ecc and at 37 °C for Ec.
    2. Next, use the small starter culture to inoculate a second, larger culture: 50 µl of starter culture + 50 ml LB in a 100 ml flask and allow to grow overnight at the required temperature.

  3. Intoxication
    1. On the day of infection, measure the optic density (OD) of the culture at a dilution of 1/5 (200 µl culture + 800 µl LB). A reliable OD must be between 0.2 and 0.8.
    2. Depending on the cultured bacteria, the required ODs to intoxicate flies with 106 Colony Forming Unit (CFU) per Drosophila are presented in the Table 1:

      Table 1. Required bacterial concentrations


    3. Dilute the overnight bacterial culture with LB to obtain the required OD. For example: to obtain an OD 1.8 of 4D22 bacteria from an overnight culture at OD 9, you need to dilute the overnight culture by 5 (9/1.8 = 5). Use LB medium to make the dilution.
    4. Then mix at 1:1 the diluted culture with 10% sucrose (see Recipes). For practical reasons mix 1 ml of diluted culture with 1 ml of 10% sucrose. This mixture will constitute the intoxication solution.
    5. 5-day-old virgin females are allowed to fast for 2 h at 25 °C: flies are placed in empty Drosophila bottles at 25 °C for 2 h.
    6. Starved flies are then transferred onto a Drosophila narrow vials containing fly medium covered with filter disks soaked with 50 µl of the intoxication solution (Figure 1).
      Note: Before to deposit your flies onto the soaked filter disk in the vials, wait for a few minutes until the filter disk becomes dry, otherwise the flies will be wet and will probably remain stuck on the filter disk.
    7. Flies are kept to feed on the contaminated media at 25 °C until dissection.


      Figure1. Intoxication procedure. A. Annotate the Drosophila vials and place the filter disks inside on the top of the medium; B. Zoom up on a filter disk on the top of the medium; C. Add 50 µl of the intoxication solution in the vials; D. Sort by 10 the starved female flies using the Droso-sleeper; E. Deposit carefully 10 flies in each contaminated vial.

  4. Dissection
    1. Forceps and dissecting watch glasses have to be washed with a 70% ethanol solution.
    2. The flies (10 virgin females per condition) are anesthetized using the Droso-sleeper.
    3. Place one fly in a watch glass pre-filled with 1 ml 1x PBS (see Recipes) (Figure 2A).
    4. Using forceps, hold the fly by the head and pull gently on the posterior part of the abdomen to carefully detach the abdomen from the thorax (Figure 2B).
    5. Then, the intestine is carefully stretched (Figure 2C).
    6. Cut the head and untie very gently the intestine first from its anterior part and then from its posterior part (Figures 2D-2F).
      Note: Here, we need only the midgut so we cut the foregut and the hindgut. We also removed Malpighian tubules that can still be attached at the midgut/hindgut boundary (Figures 2D-2F).


      Figure 2. Dissection procedure. A. Place one fly in a watch glass pre-filled with 1 ml 1x PBS under a stereomicroscope; B. Hold the fly by the head and pull gently on the posterior part of the abdomen to carefully detach the abdomen from the thorax; C. Carefully stretch the intestine; D. Cut the head and untie very gently the intestine first from its anterior part and then from its posterior part. The whole intestine (foregut/midgut/hindgut) must be separated from the rest of the abdomen. E. Remove the crop; F. Remove the proventriculus, the hindgut and the Malpighian tubules. See also http://www.bio-protocol.org/e2079 (Chen et al., 2016)

    7. The dissected midguts are then immediately placed in 1x PBS on ice waiting for the dissection of the other intestines.
    8. Only 8 guts (over the 10) are transferred into 50 µl of 1x PBS in a 1.5 ml microtube for sample preparation.

  5. Sample preparation
    1. Recover gut content by pressing them with a microtube pestle fixed on a motor set to 1,000 rpm: put the motor on and perform 10 up and down movements with the microtube to allow the pestle to reach the bottom of the microtube (Video 1).
      Note: Keep the tubes on ice to avoid any protein degradation. The goal is to recover most of lumen contents, so do not strongly crush the guts and do not perform more than 10 up and down movements.

      Video 1. Crushing intestines. The video describes the procedure to gently crush intestines using a microtube pestle. Note at the end of the video that to avoid to waste gut content, we take off the pestle and press it against the edge of the microtube

    2. Centrifuge for 5 min at 10,000 x g and 4 °C.
    3. Dilute at 1:10 a part of the supernatant: take 20 µl of the supernatant and transfer it in a 1.5 ml microtube containing 180 µl of 1x PBS.
    4. Take another 20 µl of the supernatant in a 0.5 ml microtube and place it at 4 °C waiting for protein assay if one wants to normalize the results per mg of protein.

  6. Assay
    Note: For each sample, we assay 3 volumes to be sure to be in the fluorimeter’s measurement range and to have reproducible results. As negative control, we use the maximal sample volume (i.e., 20 µl) complemented with a cocktail of protease inhibitors (25x cOmplete). As positive control, we use trypsin. The Table 2 gives an example of a 96-well plate’s scheme according to these rules. This is a plane of an independent experiment with two technical replicates (one from columns 1 to 4 lanes B to E and one from columns 6 to 9 lanes B to E).

    Table 2. 96-well plate’s plane. Volume (µl) for: 10x PBS/Distilled water/Sample/Trypsin/25x cOmplete.

    aTo be sure to stay in the reading range of the spectrophotometer, carry out two assays with a dilution of trypsin to 1:1

    1. Prepare the trypsin solution (see Recipes) as described below.
    2. Annotate 1.5 ml microtubes with the letter and the number corresponding to their position in the 96-well plate.
    3. Add the indicated volume of distilled water in each tube.
    4. Add the 10x PBS.
    5. Finally add the indicated volume of sample or trypsin.
    6. When necessary add the protease inhibitors (25x cOmplete) (see Recipes).
      Note: From this point, it is important to work with the least of light as possible.
    7. Prepare the Casein-FITC (see Recipes) as described below.
    8. Add 20 µl Casein-FITC in each tube and vortex few seconds.
      Note: Be careful, do not vortex too strongly to avoid fluorescence background.
    9. Incubate for 1.5 h in a 37 °C solid-door incubator (samples have to be in the dark).
      Note: Samples can be incubated until 24 h to increase the signal. Beyond one day, Casein-FITC can be degraded leading to high fluorescence background.
    10. Add 300 µl 10% TCA (see Recipes) and vortex for a few seconds.
    11. Incubate for 0.5 h in a 37 °C solid-door incubator.
    12. Centrifuge for 10 min at 10,000 x g, 4 °C.
      Note: Whole proteins and big fragments, which underwent a few or no proteolytic cleavage, precipitate with the TCA and are centrifuged down in the pellet. Casein-FITC small fragments that underwent a lot of proteolytic cleavage are in the supernatant. That’s what will be measured.
    13. In a black 96-well plate, put 50 µl of supernatant according to the Table 2.
    14. Add 150 µl 0.5 M Tris/HCl (see Recipes) pH 8.5.
    15. Read the fluorescence on a spectrofluorimeter set on 485 nm excitation wavelength and 535 nm emission wavelength.

Data analysis

  1. FITC-intensity measurement analysis
    1. We systematically perform at least 3 independent experiments. We defined ‘independent experiments’ when they are performed on different days.
    2. Each experiment is performed in duplicate meaning that for a given day for one condition of intoxication, there are two different pool of 10 Drosophila contaminated by the same batch bacteria.
    3. Below we present results for 4 conditions of intoxication (Ctrl, 4D22, Ecc and Ec). For each condition there are 3 independent experiments and for each experiment two replicates.
    4. For each replicate, we have assayed 3 different volumes and a negative control (5 µl, 10 µl or 20 µl of samples and 20 µl of samples + 25x cOmplete).
    5. In each replicate, 8 intestines were used, therefore we have in total 48 intestines/each condition.
    6. We have calculated the mean of results obtained in the 3 independent experiments (i.e., six replicates) for each condition and for each volume.
    7. Table 3 and the Figure 3 give an example of raw data obtained after FITC fluorescence measurement.
      Note: Below are presented all the raw data without excluding any value. However, the experimenter can remove some replicates if he has performed more than 3 independent experiments and if he judges that one of the replicates failed.

      Table 3. FITC intensity measurements



      Figure 3. Graph of average FITC-intensity. The data of the Table 3 are reported in this graph. Only the means were used to draw the graph.

    8. For result presentation and statistical analyses, we selected the data obtained with the volume 20 µl of samples (more reliable and reproducible).
      1. Results are presented in % of FITC intensity (reflecting protease activity) where Ctrl is considered at 100% (Table 4).
      2. We used SEM (Standard Error of the Mean) to present error bars in the graph (Figure 4).A. FITC-intensity measurement analysis

      Table 4. Mean and percentage of FITC-intensity reflecting the protease activity



      Figure 4. Percentage of protease activity. Protease activity (proportional to FITC intensity) shows a reduction of about 20% following 4D22 or Ecc treatment. No difference is observed between the Ctrl and Ec ingestion. *P ≤ 0.05.

  2. Statistical analysis
    1. Effects of treatments are analyzed using a pair wise comparison test (Tukey’s test).
    2. Samples are compared to the control.
    3. Differences are considered significant when P < 0.05 (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001) (Figure 4).
    4. The used software is Kyplot.

Notes

The protocol described here can easily be transposed to Drosophila larval midgut and even to any other insect midguts.

Recipes

  1. Standard nutrient medium for Drosophila melanogaster
    Note: All the reagents are mixed in distilled water.
    8 g/L agar
    25 g/L sugar
    80 g/L cornflour
    20 g/L yeast
    6 g/L tegosept (stock solution at 100 g/L in 95% ethanol. Store at 4 °C)
  2. LB medium
    1. Weigh out 25 g of Luria broth medium powder
    2. Adjust to 1 L with distilled water in a graduated test tube
    3. Adjust pH to 7.2 if necessary
    4. Autoclave
  3. LB-agar medium
    1. Weigh out 25 g of Luria broth medium powder
    2. Adjust to 1 L with distilled water in a graduated test tube
    3. Adjust pH to 7.2 if necessary
    4. Add 15 g agar powder
    5. Autoclave
  4. 10% sucrose
    1. Weigh out 2 g of sucrose in a 50 ml tube
    2. Add 20 ml distilled water and vortex
    Note: Do not keep this solution.
  5. 1x phosphate-buffered saline (PBS)
    1. Add 100 ml of 10x PBS solution to 900 ml of distilled water in a graduated test tube
    2. Filter and store at 4 °C if you want to keep this solution
  6. Trypsin solution
    Note: This solution has to be prepared immediately before use, do not keep it because of trypsin self-digest.
    1. Weigh out 10 mg of trypsin powder in a 2 ml microtube and add 1 ml 1 mM HCl
    2. Vortex to dissolve powder and keep the tube on ice
  7. Casein-FITC
    1. Weigh out 10 mg of Casein Fluorescein IsoThioCyanate powder in a 15 ml tube
    2. Add 10 ml of distilled water
    3. Wrap the tube with aluminum foil and vortex thoroughly until total dissolution
    4. Keep Casein-FITC on ice and in the dark until use
    Note: This solution can be stored at -20 °C up to 6 months but do not expose it to the light.
  8. 25x cOmplete
    1. Dissolve one tablet of cOmplete tablets EDTA free in 2 ml of distilled water in a 5 ml tube
    2. Vortex strongly
    3. Divide it into two 1.5 ml microtubes and store one at -20 °C
      Note: Keep the other one tube containing cOmplete on ice until use.
  9. 10% TCA
    1. Make a stock solution with 100 mg of trichloroacetic acid powder in 100 ml of distilled water
    2. Dilute at 1:10 this stock solution by adding 5 ml of TCA stock solution to 45 ml of distilled water
    Note: Do not keep the diluted solution.
  10. 0.5 M Tris/HCl pH 8.5
    1. Weigh out 60.57 g of Tris base powder for 1 L of distilled water
    2. Adjust pH to 8.5 with 12 N HCl
    3. Filter if you want to store this solution

Acknowledgments

We would like thank David Pauron for his help at the beginning of the protocol setup. MPNE was supported by INRA. RL was supported by the Fondation pour la Recherche Médicale (FRM) and Université Côte d'Azur (UCA) and AG was supported by the CNRS.

References

  1. Bonfini, A., Liu, X. and Buchon, N. (2016). From pathogens to microbiota: How Drosophila intestinal stem cells react to gut microbes. Dev Comp Immunol 64: 22-38.
  2. Chen, J., Li, J., Huang, H. and Xi, R. (2016). Gene expression analysis of sorted cells by RNA-seq in Drosophila intestine. Bio Protoc 6(24): e2079.
  3. Gonzalez, J. M., Jr., Brown, B. J. and Carlton, B. C. (1982). Transfer of Bacillus thuringiensis plasmids coding for delta-endotoxin among strains of B. thuringiensis and B. cereus. Proc Natl Acad Sci U S A 79(22): 6951-6955.
  4. Hu, D. J. and Jasper, H. (2017). Epithelia: Understanding the cell biology of intestinal barrier dysfunction. Curr Biol 27(5): R185-R187.
  5. Jasper, H. (2015). Exploring the physiology and pathology of aging in the intestine of Drosophila melanogaster. Invertebr Reprod Dev 59(sup1): 51-58.
  6. Loudhaief, R., Brun-Barale, A., Benguettat, O., Nawrot-Esposito, M. P., Pauron, D., Amichot, M. and Gallet, A. (2017). Apoptosis restores cellular density by eliminating a physiologically or genetically induced excess of enterocytes in the Drosophila midgut. Development 144(5): 808-819.
  7. Royet, J. and Charroux, B. (2013). Mechanisms and consequence of bacteria detection by the Drosophila gut epithelium. Gut Microbes 4(3): 259-263.
  8. Soultoukis, G. A. and Partridge, L. (2016). Dietary protein, metabolism, and aging. Annu Rev Biochem 85: 5-34.

简介

肠是消化食物所需的中枢器官,吸收营养物质,并与食物一起摄入的侵略者作斗争。 粘膜损伤后的肠道生理损伤影响其消化能力,从而影响个体的生长,健康甚至生存。 因此,肠功能的评估包括监测其完整性,细胞更新,免疫防御,肠内分泌激素的产生及其消化能力。 在这里,我们详细描述如何评估分泌在成年果蝇的肠腔中的蛋白酶的活性。 这种方法也可以用于幼虫肠。 本协议由“蛋白酶荧光检测试剂盒”(产品代码PF0100)中提出的Sigma-Aldrich公司的方案进行了改进和改进。
【背景】肠道受到诸如斋戒,禁食,化学物质,病原体,损伤等诸多压力的影响。肠道能够通过保持其体内平衡的生理平衡来克服这种压力。为了感知进入的压力并产生适应性的维持肠功能的答案,肠已经开发出强健和保守的机制,如局部先天免疫防御和组织再生(Royet和Charroux,2013; Bonfini等,2016)。然而,在某些情况下,肠道稳态的维持可能受到影响。例如,在老化期间,随着许多未成熟或分化不良的细胞的存在,组织稳态维持总体下降(Jasper,2015; Hu和Jasper,2017)。体内平衡也可能被破坏的另一种情况是暴露于损害或杀死损害其功能的细胞的异种生物或病原体(如机会性细菌)(Bonfini等,2016)。因此,在上述实施例中,肠的消化能力降低。此外,在组织再生本身产生许多前体细胞的过程中,消化能力也降低(Loudhaief等,2017)。因此,肠道消化能力的评估对于评估侵袭肠道生理学的潜在影响至关重要。重要的是,肠道消化功能紊乱可能具有影响生长,免疫防御,繁殖,健康,长寿的局部和全身代谢后果。膳食蛋白质对许多(如果不是全部)生理功能(Soultoukis和Partridge,2016)是必不可少的。肠道不平衡的氨基酸吸收可能会对生长产生重大影响。蛋白质消化对于由肠细胞产生可吸收的氨基酸是必不可少的,对肠道蛋白酶活性的测量似乎是评估肠道生理状态及其消化功能的能力的良好读数。

关键字:黑腹果蝇, 肠道, 机会性细菌, 蛋白酶活性, 蛋白质代谢

材料和试剂

  1. 果蝇饲养
    1. 6盎司果蝇储存瓶(Genesee Scientific,目录号:32-130)
    2. 用于储存瓶子的棉球(Genesee Scientific,目录号:51-102B)
    3. 广州 苍蝇(布卢明顿果蝇 Stock Center,目录号:64349)( flystocks.bio.indiana.edu
    4. 琼脂(VWR,BDH ®,目录号:20768-361)
    5. 糖(家乐福或任何其他超市)
    6. Cornflour(AB,Celnat - NaturDis)
    7. 酵母(Biospringer,目录号:BA10 / 0-PW)
    8. Tegosept(Apex,Fly Food preservative,Genesee Scientific,目录号:20-258)
    9. 用于果蝇的标准营养培养基(参见食谱)

  2. 细菌培养
    1. 培养皿
    2. 无菌提示
    3. 15 ml管(Corning,Falcon ®,目录号:352096)
    4. 刻度试管
    5. 苏云金芽孢杆菌 var。在芽孢杆菌遗传库存中心( ke-insertfile“href =”http://www.bgsc.org/“target =”_blank“> http://www.bgsc.org/ )并由(Gonzalez等人)描述,1982)
    6. 欧文氏carotovora carotovora ( Ecc )由Bruno Lemaitre的实验室(瑞士洛桑ÉcolePolytechniqueFédérale)提供
    7. (Thermo Fisher Scientific,Invitrogen)(大肠埃希氏菌)(Thermo Fisher Scientific,Invitrogen) TM ,目录号:C404003)
    8. Luria肉汤粉(Conda,目录号:1551)
    9. 琼脂细菌(Euromedex,目录号:1330)
    10. LB培养基(见食谱)
    11. LB-琼脂培养基(参见食谱)

    1. 窄小瓶的棉球25毫米(Genesee Scientific,目录号:51-101)
    2. 分光光度计比色皿(Ratiolab,目录号:2712120)
    3. 2 ml微管(Paul Bottger,目录号:02-043)
    4. 20 mm过滤盘(色谱纸3MM Chr)(GE Healthcare,目录号:3030-917)
    5. 50ml管
    6. 果蝇窄小瓶25毫米(Genesee Scientific,目录号:32-109RL)
    7. 蔗糖(Euromedex,目录号:200-301-B)
    8. 10%蔗糖(见食谱)

  3. 解剖
    1. 1.5毫升微管(Paul Bottger,目录号:02-063)
    2. 刻度试管
    3. 手表玻璃(Steriplan Petri dish,DWK Life Sciences,目录号:237554008)

    4. 乙醇70%(VWR,目录号:83801.360)
    5. 10x PBS(Euromedex,目录号:ET330)
    6. 1x磷酸缓冲盐水(PBS)(参见食谱)

  4. 样品制备
    1. Microtube杵1.5 ml(Argos Technologies,目录号:P7339-901)
    2. 0.5 ml微管(Paul Bottger,目录号:02-053)
    3. 1x磷酸盐缓冲盐水(PBS)(见食谱)

  5. 化验
    1. 1.5毫升微管(Paul Bottger,目录号:02-063)
    2. 96孔黑色微孔板(Greiner Bio One International,目录号:655076)
    3. 2 ml微管
    4. 15毫升管子
    5. 铝箔
    6. 来自牛胰腺的胰蛋白酶(Sigma-Aldrich,目录号:T1005)
    7. 1 mM HCl
    8. 酪蛋白荧光素来自牛奶的异硫氰酸酯(Sigma-Aldrich,目录号:C0528)
    9. 蒸馏水
    10. cOmplete片剂不含EDTA(Roche Diagnostics,目录号:04693132001)
    11. 三氯乙酸(TCA)(Sigma-Aldrich,目录号:T6399)
    12. 三碱基
    13. 胰蛋白酶溶液(参见食谱)
    14. 酪蛋白FITC(参见食谱)
    15. 25x cOmplete(见配方)
    16. 10%TCA(见配方)
    17. 0.5 M Tris / HCl pH 8.5(参见食谱)

设备

  1. 果蝇饲养
    在25℃恒温和12小时/ 12小时光照/暗循环的冷藏烘箱中(Fisher Scientific,目录号:11857552)。湿度必须保持在40%到70%之间 制造商:LMS,型号:Model 240。

  2. 细菌培养
    1. 100ml烧瓶(Fisher Scientific,目录号:15409103)
    2. 30°C / 37°C振荡培养箱(Infors,型号:AK 82)

    1. 分光光度计(Aqualabo,Secomam,型号:Prim Light&amp; Aduanced)
    2. Droso-sleeper(Inject-Matic)

  3. 解剖
    1. 立体显微镜(Leica Microsystems,型号:Leica M60)
    2. Dumont镊子#5(精细科学工具,目录号:11251-20和11252-20)

  4. 样品制备
    1. 蹄电机(Heidolph Instruments,型号:RZR 2100)
    2. 冷冻微型离心机(Eppendorf,型号:5430 R)

  5. 化验
    1. 涡旋(科学工业,型号:Vortex-Genie 2)
    2. 自动移液器(Eppendorf,型号:Multipette ® plus)
    3. 37°C固体培养箱(Jouan)
    4. 荧光计(Agilent Technologies,型号:Cary Eclipse)

软件

  1. Kyplot
  2. Excel

程序

  1. 果蝇饲养
    1. 在25°C下, />
    2. 在我们的实验中只使用五岁的女性。为了获得同步的五岁女性,我们从成年苍蝇身上清空瓶子,然后等待蛹出现新的苍蝇。在紧急苍蝇中,雄性被迅速丢弃,女性在25℃下转入新瓶中五天。

  2. 细菌培养
    在下面的实验中,我们使用三种不同的细菌菌株( Btk ,ecc 和 E c 和试剂)和水作为阴性对照(Ctrl)。从LB琼脂培养皿上的股票中分散细菌,并在所需温度(对于4D22 30℃)和Ecc <30℃生长过夜,对于Ec < / em>的)。
    1. 使用无菌尖端挑取单个菌落,并在15 ml管中启动10ml初始培养LB(见食谱)。让我们在30℃下振荡(220rpm)持续8小时,对于4D22和Ecc,并在37℃下Ec 生长8小时。 >
    2. 接下来,使用小发酵培养物接种第二个更大的培养物:将50μl起始培养物+ 50ml LB置于100ml烧瓶中,并允许在所需温度下生长过夜。

    1. 在感染当天,以1/5(200μl培养物+800μlLB)的稀释度测量培养物的视觉密度(OD)。可靠的OD值必须介于0.2和0.8之间。
    2. 根据培养的细菌,表1中给出了每只每只果蝇具有10个/ 6个菌落形成单位(CFU)的所需OD值,如表1所示:

      表1.所需细菌浓度


    3. 用LB稀释过夜的细菌培养物以获得所需的OD。例如:要从OD 9的过夜培养物中获得4×10 22细菌的OD 1.8,您需要用5(9 / 1.8 = 5)稀释过夜培养。使用LB培养基稀释。
    4. 然后以1:1稀释培养物与10%蔗糖混合(参见食谱)。出于实际的原因,将1ml稀释的培养物与1ml 10%蔗糖混合。这种混合物将构成中毒溶液
    5. 5日龄的处女女性在25℃下可以快速停留2小时:将苍蝇放在空的果汁瓶中,25℃下2小时。
    6. 然后将饥饿的苍蝇转移到含有用50μl中毒溶液浸泡的过滤盘的飞行培养基的狭窄小瓶中(图1)。
      注意:在将您的苍蝇存放在小瓶中浸泡的过滤盘上之前,请等待几分钟直到过滤器盘变干,否则苍蝇会变湿,并可能会停留在过滤盘上。
    7. 苍蝇保持在25摄氏度的污染媒介上进食,直到解剖

      的如图一。中毒程序。 A.注释果蝇小瓶,将过滤盘放在培养基的顶部; B.放大介质顶部的过滤盘; C.在瓶中加入50μl中毒溶液; D.使用Droso-sleeper排除饥饿的女性苍蝇10; E.在每个污染的小瓶中仔细存放10只苍蝇。

  3. 解剖
    1. 镊子和解剖眼镜必须用70%乙醇溶液洗涤。
    2. 使用Droso-sleeper将苍蝇(每个条件10只处女女)麻醉。
    3. 将一个飞行物放在预先装满1ml 1x PBS的手表玻璃板(参见食谱)(图2A)。
    4. 使用镊子,握住头部的苍蝇,轻轻地拉在腹部后部,以小心地从胸部分离腹部(图2B)。
    5. 然后,仔细拉伸肠(图2C)
    6. 切开头部,先从肠前部轻轻取出肠,然后从其后部解剖(图2D-2F)。
      注意:在这里,我们只需要中肠,所以我们切割前肠和后肠。我们还删除了仍然可以在中肠/后肠边界附着的马鞭状小管(图2D-2F)。


      图2.解剖程序 A.将一个飞行物放置在预先装有1ml 1x PBS的表玻璃下,在立体显微镜下; B.握住头部的飞翼,轻轻地拉在腹部的后部,小心地从腹部拔出腹部; C.小心伸展肠D.切开头部,先从肠前部轻轻取出肠,然后从后部切开肠。整肠(前肠/肠肠/肠胃肠)必须与腹部的其余部分分开。 E.取下作物; F.取出前肠,后肠和马鞭状小管。另见 http://www.bio-protocol.org/e2079
    7. 然后将解剖的中肠立即置于1x PBS中,等待其他肠道的解剖。
    8. 只有8个肠道(10个以上)被转移到1.5μl的微量管中的50μl1x PBS中,用于样品制备。

  4. 样品制备
    1. 通过将固定在设定为1,000rpm的电动机上的微型管杵压入内容物来恢复肠内容物:将电动机放在上面,并用微管进行10次上下运动,以允许杵到达微管的底部(视频1)。 br /> 注意:将管保持在冰上以避免任何蛋白质降解。目标是恢复大部分流明内容,所以不要强烈地粉碎胆量,不要执行超过10次的上下移动。

      Video 1. Crushing intestines. The video describes the procedure to gently crush intestines using a microtube pestle. Note at the end of the video that to avoid to waste gut content, we take off the pestle and press it against the edge of the microtube

      To play the video, you need to install a newer version of Adobe Flash Player.

      Get Adobe Flash Player


    2. 在10,000 x g和4℃下离心5分钟。
    3. 以1:10稀释一部分上清液:取20μl上清液,并将其转移到含有180μl1x PBS的1.5ml微量管中。
    4. 将另外20μl的上清液加入0.5ml微量管中,并将其置于4℃等待蛋白质测定,如果想使每mg蛋白质的结果标准化。

  5. 测定
    注意:对于每个样品,我们测定3个体积以确保在荧光计的测量范围内并具有可重现的结果。作为阴性对照,我们使用补充有蛋白酶抑制剂(25倍完全)的混合物的最大样品体积(即20μl)。作为阳性对照,我们使用胰蛋白酶。表2给出了根据这些规则的96孔板方案的示例。这是一个独立实验的平面,具有两项技术重复(一列从第一列至第四栏,第B至第E栏,第1至第6列至第9栏,第B至第5列)。
    #E53333;“>蒸馏水 / 样本 / >胰蛋白酶 / 25x cOmplete 。

    a 要确保留在分光光度计的阅读范围内,进行两次测定,将胰蛋白酶稀释至1:1 />
    1. 准备胰蛋白酶溶液(参见食谱),如下所述。
    2. 注明1.5ml微管,字母和数字对应于其在96孔板中的位置。
    3. 在每个管中加入指示体积的蒸馏水。
    4. 加入10x PBS。
    5. 最后添加指定体积的样品或胰蛋白酶。
    6. 必要时添加蛋白酶抑制剂(25x完全)(参见食谱) 注意:从这一点来看,尽可能少的工作是很重要的。
    7. 如下所述准备酪蛋白FITC(参见食谱)。
    8. 在每个管中加入20μl酪蛋白FITC,并旋转几秒钟。
      注意:注意,不要太强烈地涡旋以避免荧光背景。
    9. 在37℃的固体培养箱中孵育1.5小时(样本必须在黑暗中)。
      注意:样品可以孵育至24小时以增加信号。超过一天,酪蛋白FITC可以降解,导致高荧光背景。
    10. 加入300μl10%TCA(见食谱),旋涡数秒钟。
    11. 在37℃的固体培养箱中孵育0.5小时。
    12. 以10,000 x g,4℃离心10分钟。
      注意:经过几次或无蛋白水解切割的全蛋白质和大片段与TCA一起沉淀,并在沉淀中离心。经过大量蛋白水解切割的酪蛋白-FITTC小片段在上清液中。这将是衡量的。
    13. 在黑色96孔板中,加入50μl上清液,如表2所示
    14. 加入150μl0.5 M Tris / HCl(见配方)pH 8.5。
    15. 读取在485nm激发波长和535nm发射波长上设置的分光荧光计上的荧光

数据分析

  1. FITC强度测量分析
    1. 我们系统地执行至少3次独立实验。我们在不同的日子执行“独立实验”
    2. 每个实验都是重复进行的,这意味着对于一个中毒条件的给定日,有两个不同的10只果蝇潜水被相同的批次细菌污染。
    3. 下面我们将介绍4种中毒条件(Ctrl, 4D22 ,ecc 和 Ec )的结果。对于每个条件,有3个独立实验,每个实验两个重复。
    4. 对于每个重复,我们已经测定了3种不同体积和阴性对照(5μl,10μl或20μl样品和20μl样品+ 25×cOmplete)。
    5. 在每次复制中,使用8个肠,因此我们共有48个肠/每个条件。
    6. 我们已经计算了每个条件和每个卷在3个独立实验(,即6个重复)中获得的结果的平均值。
    7. 表3和图3给出了FITC荧光测量后获得的原始数据的实例 注意:下面列出所有原始数据,而不排除任何值。然而,如果他执行了3次以上的独立实验,并且如果他判断出其中一个重复失败,那么实验者可以删除一些重复。

      表3. FITC强度测量



      图3.平均FITC强度的图。表3的数据在该图中报告。只用这些手段画图。

    8. 对于结果呈现和统计分析,我们选择了体积为20μl的样品(更可靠和可重复)获得的数据。
      1. 结果以FITC强度(反映蛋白酶活性)的百分比表示,其中Ctrl被认为是100%(表4)
      2. 我们使用SEM(平均值的标准误差)在图表中显示误差条(图4).A。FITC-强度测量分析

      表4.反映蛋白酶活性的FITC强度的平均值和百分比



      图4.蛋白酶活性的百分比蛋白酶活性(与FITC强度成比例)显示出在4D22或Ecc治疗后减少约20%。在Ctrl和 Ec 摄入之间没有观察到差异。 * P ≤0.05
  2. 统计分析
    1. 使用成对比较试验(Tukey's test)分析治疗效果。
    2. 样本与对照进行比较。
    3. 当P 时,差异被认为是重要的, 0.05(* P <0.05,** P <0.01,*** <0.001)(图4)。
    4. 所使用的软件是Kyplot 。

笔记

这里描述的方案可以很容易地转置到果蝇幼虫中肠,甚至可以转移到任何其他昆虫中肠。

食谱







  1. 注意:所有试剂都在蒸馏水中混合。
    8 g / L琼脂
    25克/升糖
    80克/升玉米粉
    20克/升酵母
    6g / L tegosept(在95%乙醇中以100g / L储存的溶液,在4℃下储存)
  2. LB培养基
    1. 称取25克Luria肉汤中等粉末
    2. 在分级试管中用蒸馏水调节至1升
    3. 如有必要,请将pH调至7.2
    4. 高压灭菌器
  3. LB-琼脂培养基
    1. 称取25克Luria肉汤中等粉末
    2. 在分级试管中用蒸馏水调节至1升
    3. 如有必要,请将pH调至7.2
    4. 加入15克琼脂粉末
    5. 高压灭菌器
  4. 10%蔗糖
    1. 在50ml管中称量2克蔗糖
    2. 加入20毫升蒸馏水并涡旋
    注意:请勿保留此解决方案。
  5. 1x磷酸缓冲盐水(PBS)
    1. 在刻度试管中加入100毫升10倍PBS溶液至900毫升蒸馏水
    2. 如果要保持此解决方案,请过滤并存储在4°C
  6. 胰蛋白酶溶液
    注意:此解决方案必须在使用前立即准备,不要因为胰蛋白酶自我消化而保留。
    1. 称取10毫升胰蛋白酶粉末在2毫升微量管中,并加入1毫升1毫升的盐酸盐
    2. 涡旋溶解粉末并将管保持在冰上
  7. 酪蛋白-FITC
    1. 称取10毫克酪蛋白荧光素异硫氰酸酯粉末在15毫升管中
    2. 加入10ml蒸馏水
    3. 用铝箔包裹管,彻底涡旋直到完全溶解
    4. 将酪蛋白FITC放在冰上,黑暗中直至使用
    注意:该解决方案可以保存在-20°C至多6个月,但不要将其暴露在光线下。
  8. 25倍cOmplete
    1. 在一个5ml的管中,将一片含完整片的EDTA溶于2ml蒸馏水中
    2. 涡流强烈
    3. 将其分成两个1.5毫升的微量管,并储存在-20°C 注意:将另一只含有cOmplete的管放在冰上,直到使用。
  9. 10%TCA
    1. 用100毫升三氯乙酸粉末在100ml蒸馏水中制备储备溶液
    2. 通过向45ml蒸馏水中加入5ml TCA储备溶液在1:10稀释该储备溶液
    注意:不要保留稀释溶液。
  10. 0.5M Tris / HCl pH8.5
    1. 称取60.57克Tris基粉末1升蒸馏水
    2. 用12N HCl将pH调节至8.5
    3. 如果要存储此解决方案,请过滤

致谢

我们要感谢David Pauron在协议设置开始时的帮助。 MPNE得到INRA的支持。 RL由科技基金会(FRM)和蔚蓝海岸大学(UCA)支持,AG得到CNRS的支持。

参考

  1. Bonfini,A.,Liu,X.和Buchon,N。(2016)。从病原体到微生物群:果蝇如何肠干细胞对肠道微生物发生反应。 Dev Comp Immunol 64:22-38。 />
  2. Chen,J.,Li,J.,Huang,H. and Xi,R。(2016)。&nbsp; 肠杆菌中RNA-seq的分选细胞的基因表达分析。 Bio Protoc 6(24):e2079。 />
  3. Gonzalez,JM,Jr.,Brown,BJ和Carlton,BC(1982)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/6294667”靶标=“_ blank”>转移编码B型菌株中δ-内毒素的苏云金芽孢杆菌质粒。 thuringiensis 和。 cereus 。 Proc Natl Acad Sci U S A 79(22):6951-6955。
  4. Hu,DJ和Jasper,H.(2017)。&nbsp; 上皮:了解肠屏障功能障碍的细胞生物学。 Curr Biol 27(5):R185-R187。
  5. Jasper,H.(2015)。&nbsp; 探索黑腹果蝇肠道老化的生理学和病理学。 59(sup1):51-58。
  6. Loudhaief,R.,Brun-Barale,A.,Benguettat,O.,Nawrot-Esposito,MP,Pauron,D.,Amichot,M.and Gallet,A.(2017)。&lt; a class =插入文件“href =”http://www.ncbi.nlm.nih.gov/pubmed/28246211“target =”_ blank“>细胞凋亡通过消除果蝇中肠中的生理或遗传诱导的肠细胞过剩来恢复细胞密度 开发 144(5):808-819。
  7. Royet,J.和Charroux,B。(2013)。&nbsp; 果蝇肠上皮细菌检测的机理和后果 肠微生物 4(3):259-263。
  8. Soultoukis,GA和Partridge,L.(2016)。&nbsp; 膳食蛋白质,新陈代谢和老化。 Annu Rev Biochem 85:5-34。
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引用:Nawrot-Esposito, M., Loudhaief, R. and Gallet, A. (2017). Protease Activity Assay in Fly Intestines. Bio-protocol 7(18): e2560. DOI: 10.21769/BioProtoc.2560.
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