Protease Activity Assay in Fly Intestines

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. Canton^S 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 Shot^TM TOP10 Chemically Competent E. coli) (Thermo Fisher Scientific, Invitrogen^TM, 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. Canton^S 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

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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.