Infection of the Developing Central Nervous System of Drosophila by Mammalian Eukaryotic and Prokaryotic Pathogens

Materials and Reagents

1. Fly strain and Drosophila larval culture

   1. mdr65-mtd: Tomato (Benmimoun et al., 2020 and Figure 1)

      
      

      
      Figure 1. Confocal image of top view (A) and cross-section with orthogonal views (B) of the Drosophila BBB in a portion of the ventral nerve cord

      
      

   2. Egg laying plates (50 mm, Deep) (Thermo Scientific, catalog number: 124-17), see Figure 2A

   3. Fly egg laying cages (homemade, see Figure 2B)

   4. Larval collection plates (60 mm Petri dishes) (Corning-Falcon, catalog number: 353004), see Figure 2C

      
      

      
      Figure 2. Fly larval collection and culture materials

      
      

   5. Active dry yeast (Genesee Scientific, catalog number: 62-103)

   6. Corn flour (Limagrain, catalog number: WFMZ01HS)

   7. Agar (Sigma-Aldrich, catalog number: A7002-1KG)

   8. Sugar (Dominique Dutscher, catalog number: 067508B)

   9. Organic grape juice

   10. Propionic acid (Sigma-Aldrich, catalog number: P5561-1L)

   11. Methyl-4-hydroxybenzoate (Sigma-Aldrich, catalog number: H3647)

   12. Ethanol (Sigma-Aldrich, catalog number: 24105)

   13. Egg laying medium (see Recipes)

   14. Fly food (see Recipes)

       
       

2. Pathogen preparation

   1. Pathogen growth media [Brain Heart Infusion broth (BHI), Yeast extract Peptone Dextrose (YPD), or de Man Rogosa Sharpe (MRS)] (BD Difco, Millipore, catalog numbers: 237500, 242820 or 288130)

   2. Round-bottom tubes with cap (Fisher Scientific, Falcon, catalog number: 352059)

   3. Micro cuvette for spectrophotometer (Dutscher, Greiner Bio-One, catalog number: 613101)

   4. Microcentrifuge tubes (Fisher Scientific, Eppendorf, catalog number: 10708704)

   5. Dulbecco’s phosphate-buffered saline (PBS) (Sigma-Aldrich, catalog number: D1408)

   6. Drosophila Schneider’s medium (Fisher Scientific, Gibco, catalog number: 21720-024)

      
      

3. Drosophila dissection

   1. Fine forceps (Fine Science Tools, Dumont #5, catalog number: 11252-20)

   2. Tissue culture dishes (Fisher Scientific, Corning, Falcon, catalog number: 3530001)

   3. Glass cavity dish (Atom Scientific, catalog number: SDCE4040-1)

   4. Dulbecco’s phosphate-buffered saline (PBS) (Sigma-Aldrich, catalog number: D1408)

   5. Ethanol (Sigma-Aldrich, catalog number: 493511)

   6. Drosophila Schneider’s medium (Fisher Scientific, Gibco, catalog number: 21720-024)

      
      

4. CNS explant culture

   1. 24-well cell culture plate (Fisher Scientific, Corning Falcon, catalog number: 353047)

   2. Drosophila Schneider’s medium (Fisher Scientific, Gibco, catalog number: 21720-024)

   3. l-Glutamine (Fisher Scientific, Gibco, catalog number: 25030-032)

   4. Sodium l-ascorbate (Sigma-Aldrich, catalog number: A4034)

   5. Fetal bovine serum (Sigma-Aldrich, catalog number: F4135)

   6. CNS culture medium I (see Recipes)

   7. CNS culture medium II (see Recipes)

      
      

5. In vivo CNS infection

   1. Fine forceps (Fine Science Tools, Dumont #5, catalog number: 11252-20)

   2. Pipette Drummond Scientific 3.5’’ (Dutsher, Drummond Scientific, catalog number: 3-000-203-G/X)

   3. Nano-injector Nanoject III (Dutsher, Drummond Scientific, catalog number: 075250)

   4. Mineral oil (Sigma-Aldrich, catalog number: M5904)

   5. Microscope slides (Fisher Scientific, Fisherbrand, catalog number: 10219280)

      
      

6. Permeability assay

   1. Drosophila Schneider’s medium (Fisher Scientific, Gibco, catalog number: 21720-024)

   2. Dextran, Texas Red, lysine fixable 10 KDa (ThermoFisher, catalog number: 11530236, Invitrogen D1863)

   3. Dulbecco’s phosphate-buffered saline (PBS) (Sigma-Aldrich, catalog number: D1408)

   4. 4% methanol-free formaldehyde (ThermoFisher Scientific, catalog number: 28908)

      
      

7. Immunohistochemistry

   1. 4% methanol-free formaldehyde (ThermoFisher Scientific, catalog number: 28908)

   2. Bouin’s solution (Sigma-Aldrich, catalog number: HT10132)

   3. Dulbecco’s phosphate-buffered saline (PBS) (Sigma-Aldrich, catalog number: D1408)

   4. Triton X-100 (Sigma-Aldrich, catalog number: T9284)

   5. Bovine serum albumin (Sigma-Aldrich, catalog number: A3608)

   6. Normal goat serum (Invitrogen, catalog number: 10000C)

   7. Mowiol mounting medium (homemade), or equivalent

   8. Microscope slides (Fisher Scientific, Fisherbrand, catalog number: 10219280)

   9. Coverslips (Fisher Scientific, Fisherbrand, catalog number: 15707592)

   10. Anti-GBS antibody (Benmimoun et al., 2020)

       
       

8. Gene expression

   1. Fine forceps (Fine Science Tools, Dumont #5, catalog number: 11252-20)

   2. Dulbecco’s phosphate-buffered saline (PBS) (Sigma-Aldrich, catalog number: D1408)

   3. Glass cavity dish

   4. RNeasy Mini kit (QIAGEN, catalog number: 74104)

   5. QuantiTect Rev. Transcription Kit (QIAGEN, catalog number: 205311)

   6. TaqMan gene expression assay (ThermoFisher, catalog number: 4331182)

   7. TaqMan Universal PCR Master Mix II (ThermoFisher, catalog number: 4440042)

   8. 96-well PCR plates (e.g., Bio-Rad, catalog number: MLL9601)

   9. Seals for PCR plate (e.g., Bio-Rad, catalog number: MSB1001)

Equipment

1. Incubator with regulated temperature and humidity (Memmert, HPP110, or equivalent)

2. Agitator Titramax 100 (Heidolph Instruments, catalog number: 544-11200-00)

3. Spectrophotometer (Pharmacia, Novaspec II)

4. Centrifuge (Sigma Bioblock scientific, 1-15K)

5. Microbiological Safety Post (HeraSafe KS9)

6. Flaming/Brown micropipette puller (Sutter Instrument Model P-97)

7. Nano-injector Nanoject III (Dutsher, Drummond Scientific, catalog number: 075250)

8. Laser scanning confocal microscope [Zeiss LSM 880 with Zen software (2012 S4)]

9. Real-Time PCR machine (e.g., ThermoFisher, StepOne Real-Time PCR System, catalog number: 4376357)

Software

1. Zen software (2012 S4)

2. Fiji (ImageJ version 2020 2.1.0/1,53c and version 1.52p; available at: https://imagej.net/software/fiji/)

3. GraphPad Prism software [version 7 and version 2020 8.4.2 (464)]

4. R, SPSS, or comparable software

Procedure

1. Drosophila larval culture

   1. Set fly crosses for 2–3 h in cages with grape juice egg-laying plates (supplemented with a bit of yeast paste, see Figure 2A and 2B) at appropriate temperature in order to collect fly embryos (Figure 3A).

   2. After 21 h at 25 °C (or 48 h at 18 °C), remove the already hatched larvae from the plate. Then, at each hour, collect and transfer equivalent numbers (approximately 100) of hatching first instar larvae to standard food plates at appropriate temperature (29 °C for RNAi knockdown, for example) (Figures 2C and 3B-C).

   3. Let the larvae grow and then collect them from the appropriate developmental stage to perform experiments. Of note, pathogenic infection has so far been done with third instar larvae (+72 h at 25 °C) (Figure 3D).

      
      

      
      Figure 3. Schematic representation of Drosophila larval culture procedure

      
      

2. Pathogen preparation

   1. Set an overnight bacteria or a 36 h yeast preculture from -80 °C glycerol stocks on appropriate growth medium (BHI, YPD, MRS, …) and at adequate temperature (30 °C or 37 °C) (Figure 4A).

   2. a) For bacteria, dilute the overnight preculture at 1/20 in corresponding growth medium, and allow them to grow till the mid-exponential phase (~2 h 30 min at 37 °C for GBS) (to adapt depending on the bacterial strains) (Figure 4B).

      b) For fungi, dilute the overnight preculture at OD₆₀₀ = 0.2, and allow them to grow at 30 °C for 5–6 h until OD₆₀₀ of 1 (to adapt depending on the strains) (Figure 4B).

   3. Pellet the pathogens, after OD₆₀₀ measurement (Figure 4C), by centrifugation at 3,500 × g and 4 °C for 5 min.

   4. Wash pathogenic culture in PBS, then pellet the pathogens by centrifugation at 3,500 × g and 4 °C for 5 min. Do this step twice (Figure 4D).

   5. Wash pathogenic culture in Drosophila Schneider’s medium, then pellet the pathogens by centrifugation at 3,500 × g and 4 °C for 5 min (Figure 4E).

   6. a) For the ex vivo CNS infection protocol: Resuspend the pathogens in 750 µL of Drosophila Schneider’s medium (Figure 4F), to prepare a 10× infectious dose and keep them at 4 °C until use (for example: 10 × 10⁸ CFU/mL for Streptococcus agalactiae, Streptococcus pneumoniae, Listeria innocua, and Listeria monocytogenes; 10 × 10⁷ CFU/mL for Neisseria meningitidis, Candida albicans, and Candida glabrata, and 10 × 10⁵ CFU/mL for Cryptococcus neoformans).

      b) For the in vivo CNS infection protocol: Resuspend the pathogens in 100 µL of Drosophila Schneider’s medium (Figure 4F), to reach the infectious dose in the hemolymph (estimated at 2 μL) by injecting 20 nL of concentrated pathogen (for example: 4.4 × 10¹⁰ GBS/mL Streptococcus agalactiae).

      Note: Pathogen concentration was calculated by OD₆₀₀ correlation (Streptococcus agalactiae, Streptococcus pneumoniae, Listeria innocua, and Listeria monocytogenes: 1 OD₆₀₀ = 8.8 × 10⁸ CFU/mL; Neisseria meningitidis: 1 OD₆₀₀ = 10⁹ CFU/mL; Candida albicans and Candida glabrata: 1 OD₆₀₀ = 3 × 10⁷ CFU/mL; Cryptococcus neoformans: 1 OD₆₀₀ = 6 × 10⁷ CFU/mL).

      
      

   
   Figure 4. Schematic representation of pathogen preparation procedure

   
   

3. CNS explant culture and ex vivo CNS infection

   1. Wash staged larvae successively in PBS and ethanol 70% v/v in water and transfer them into cold Drosophila Schneider’s medium inside dissection wells.

   2. Dissect larvae by (a) cutting at approximately a quarter from the posterior spiracle, to minimise damage to motor nerves, (b) turning the anterior part inside-out to expose the CNS [Figure 5A and see video (Hafer and Schedl, 2006)]. Keep all larval tissues except for the gut (to avoid contamination with intestinal symbiotic pathogens).

   3. Transfer eight larvae to one well (24-well cell culture plate) and culture them under gentle rotary agitation (275 rpm) in 750 μL of CNS medium I at 30 °C and 60% humidity (Figure 5B).

      Note: A temperature of 30 °C was chosen as a compromise temperature, to allow Drosophila development while culturing mammalian pathogens closer to their usual environment.

   4. Dilute 1/10 of the prepared 10× infectious dose of each pathogen in the CNS culture medium I (Figure 5C), to reach the appropriate infectious dose (see step B6a).

   5. Replace the infected culture medium with Drosophila Schneider’s medium supplemented with 2 mM l-Glutamine, 0.5 mM sodium l-ascorbate, and 1% fetal bovine serum. Place the culture at 30 °C and 60% humidity under gentle rotary agitation (275 rpm) for a defined time (3 h for GBS; the same time must be kept for the control condition without pathogenic inoculum).

   6. Replace the medium after 3 h and every 10 h, with a fresh culture medium (Drosophila Schneider’s medium supplemented with 2 mM l-Glutamine, 0.5 mM sodium l-ascorbate, and 1% fetal bovine serum)

      Note: In these conditions, CNS explants can be kept for up to 48 h, at 30 °C.

   7. Proceed with the treatment required for each different analysis (D, E, and F).

      
      

   
   Figure 5. chematic representation of the ex vivo CNS infection procedure

   
   

4. Pathogenic microinjection and in vivo CNS infection

   1. Pull micropipettes for injection (Heat: 350–360, Pull: 60–80, Velocity: 100, Time: 150). This step can be done in advance.

   2. Fill the completely prepared micropipette with mineral oil, using a needle and syringe (Figure 6A).

   3. Attach the micropipette filled with mineral oil to the injector head (Figure 6A).

   4. Eject part of the mineral oil and replace it by filling the micropipette with prepared concentrated pathogens (Figure 6A) (this will avoid contamination of the injector head with pathogens, by forming two distinct phases in the micropipette).

   5. Wash staged larvae successively in PBS and ethanol 70% v/v in water and transfer them into cold Drosophila Schneider’s medium inside dissection wells.

   6. One by one, dry the larvae, place them on one slide, and gently inject 20 nL of Drosophila Schneider’s medium (mock control) or prepared pathogen in its posterior part, using the nanoinjector (Figure 6B). This injection should be done very gently to only puncture the cuticle without affecting other tissues. We have chosen to inject the posterior part as it is far from the CNS.

   7. Keep injected larvae on standard fly food plates in an incubator at 30 °C and 60% humidity until analysis.

      Notes:

      1. All injected larvae will present a melanization spot, which seals and heals the cuticle punctured by the injection.

      2. Mock injection results in lethality per se, due to a combination of unsuccessful healing of the punctured cuticle, potential damage to tissues neighbouring the injection point, and potential temperature-induced stress (30 °C). In our hands, approximately 10% of GBS-injected larvae survive 4 h post-injection (all animals died between 4 and 6 h post-injection), while approximately 40% of mock-injected larvae survive 4 h post-injection (Benmimoun et al., 2020).

         
         

   
   Figure 6. Schematic representation of the in vivo CNS infection procedure

   
   

5. Blood–brain barrier permeability assay

   1. Prepare a solution of 10 KDa dextran Texas Red (50 mM) in CNS culture medium II.

   2. Change CNS culture medium and incubate CNS explants with this solution under agitation (275 rpm) for 30 min.

   3. CNS explants are immediately fixed 4 × 5 min (to wash out excess dextran) in 4% methanol-free formaldehyde.

   4. Wash 3 × 10 min with PBS.

   5. Dissect the CNS and mount samples in mounting medium.

   6. Visualise with a laser scanning confocal microscope, with an optimal distance between each slice of 0.38 μm.

      
      

6. Immunohistochemistry

   This protocol is a classic, general protocol to perform immunochemistry on the Drosophila larval CNS. For infections, we have used it to assess pathogen localization inside the CNS, by using specific antibodies against the strains of interest (Benmimoun et al., 2020), as well as cell fate (anti-Deadpan, anti-Repo, and anti-ElaV antibodies), morphology, and proliferation (anti-phosphohistone 3). It can also detect fusion proteins (anti-GFP antibody). A variation (using Bouin’s solution, see below) exists for antigens and antibodies, which do not work well with the classical formaldehyde fixation (for example, anti-Neurexin-IV antibody, Babatz et al., 2018).

   1. Fix (inside-out larvae ex vivo, or dissect and turn inside-out in vivo injected larvae).

      1. In 4% methanol-free formaldehyde at room temperature for 30 min, or overnight at 4 °C.

      2. In Bouin’s solution at room temperature for 3 min.

   2. Wash 3 × 10 min in PBS.

   3. Permeabilize 3 × 10 min in PBS-Triton 0.3%.

   4. Incubate with primary antibodies at 4 °C in blocking solution (PBS-Triton 0.3%, bovine serum albumin 5%, normal goat serum 2%) for 18–36 h.

      Of note: medium acidification could occur upon infection for some pathogenic strains (e.g., lactic pathogens, such as GBS or S. pneumoniae). If so, all GFP fusions should be detected with an anti-GFP antibody.

   5. Wash 3 × 10 min with PBS-Triton 0.3%.

   6. Incubate with secondary antibodies in blocking solution at room temperature for 3 h, or at 4 °C for 18–24 h.

   7. Wash 3 × 10 min with PBS-Triton 0.3%.

   8. Dissect the CNS and mount samples in Mowiol mounting medium.

   9. Visualise with a laser scanning confocal microscope, with an optimal distance between each slice of 0.38 μm (Figure 7).

      
      

   
   Figure 7. Confocal images showing GBS (attached to the BBB or inside the brain) using a specific GBS antibody

   
   

7. Pathogenic load (CFU quantifications)

   1. Without tissue fixation, wash five injected flies (by condition) on paper with ethanol 70%.

   2. Open and bleed the larvae in 10 μL of PBS, and pool their hemolymph (keep on ice).

   3. Dissect, transfer, and homogenise the CNS in 10 μL of PBS (keep on ice).

   4. Transfer and homogenise the rest of the larval carcass (other tissues except for the gut) in 10 μL of PBS (keep on ice).

   5. Prepare seven serial tenfold dilutions with 2 μL in 20 μL of PBS for each tissue tested (hemolymph, CNS, carcasses).

   6. Plate 2 x 4 μL for each dilution on pathogen culture medium agarose plate(s).

   7. Incubate the plates at 37 °C for 16 h prior to the quantifications.

      Note: While these steps are described for the in vivo infection, only CNS bacterial load is determined for ex vivo infection. For this, follow steps 3 and 5–6 only.

      
      

8. qPCR for gene expression

   1. Dissect 9–12 CNS with fine forceps at the desired time (3 h or 6 h post infection for GBS) and transfer each dissected CNS directly into the lysis buffer of the RNeasy Mini kit on ice.

   2. Isolate RNA according to the manufacturer’s instructions.

   3. Directly proceed with the cDNA synthesis. Use 450–600 ng RNA per reaction. Store the remaining isolated RNA at -80 °C.

   4. Set up qPCR reactions with a final volume of 10 µL.

   5. Seal plates carefully and spin them down in a centrifuge at 3,000 rpm for approximately 2 min.

   6. Run qPCR.

   7. Analyse qPCR data (see Data analysis).

      Notes:

      1. 50 ng of cDNA for each reaction is generally sufficient to get reasonable cycle threshold (Ct) values.

      2. Rpl32 can be used as a housekeeping gene.

Data analysis

1. Pathogen quantifications in infected Drosophila CNS

   After imaging (immunohistochemistry)

   1. Manually determine the exact number of pathogens for each analysed CNS by counting each individual bacterium contained within the boundary of the BBB (mdr65-mtd:: Tomato) through confocal acquisition.

   2. Compare between pathogen entry into the CNS using Student’s t-test (two conditions) or one-way ANOVA test followed by Tukey’s post-hoc analysis (more than two conditions), when values follow a normal distribution (assessed by Shapiro–Wilk normality test). Otherwise, using non-parametric Mann–Whitney tests (two conditions) or Kruskal–Wallis tests (more than two conditions).

      
      

   After pathogenic load (CFU quantifications)

   1. Count the bacterial colonies (CFUs) for each dilution (when possible, i.e., when isolated single colonies are detectable).

   2. Calculate an average of CFU/µL (bacterial load) by condition.

   3. Represent the bacterial loads per larva in a log10 scale.

   4. For in vivo infection, calculate the ratios from raw counting and represent them on a log10 scale:

      1. Ratio CNS/hemolymph = log10 (cfu per CNS/cfu per hemolymph)

      2. Ratio CNS/other tissues = log10 (cfu per CNS/cfu per other tissues)

      3. Ratio CNS/(hemolymph + other tissues) = log10 [cfu per CNS/(cfu per hemolymph + cfu per other tissues)].

         
         

2. Blood–brain barrier permeability assay

   1. Calculate Texas Red pixel intensity of three selected equal-sized areas (300 × 300 µm) from each CNS.

   2. Calculate the average of the mean pixel intensity.

   3. Calculate background intensity (empty equal-sized area 300 × 300 µm).

   4. Calculate permeability index by subtracting background intensity from the average of the mean pixel intensity.

   5. Compare between permeability index using Student’s t-test (two conditions) or one-way ANOVA test followed by Tukey’s post-hoc analysis (more than two conditions) when values follow a normal distribution (assessed by Shapiro–Wilk normality test). Otherwise, using non-parametric Mann–Whitney tests (two conditions) or Kruskal–Wallis tests (more than two conditions).

      
      

3. Quantification of gene expression levels

   1. Perform qPCR with at least three biological and three technical replicates.

   2. Calculate the average for the technical replicates for each sample.

   3. Calculate the ∆Ct, by subtracting the average Ct of the housekeeping gene from the gene of interest.

   4. Calculate the ∆∆Ct, by calculating the average of the ∆Ct of the control group (uninfected) as a calibrator. Then, subtract the calibrator from each calculated ∆Ct of each biological replicate.

   5. For easier representation of the expression level, the data can be normalised to the uninfected control, by calculating the fold gene expression levels. For this, calculate two to the power of negative ∆∆Ct (2^-∆∆Ct) for each biological replicate.

   6. For statistical analysis, first test the calculated fold gene expression levels for normal distribution (e.g., by using the Shapiro–Wilk test). When normally distributed, perform a Student’s t-test; if not normally distributed, perform a Mann–Whitney U Test. Represent data in a box plot.

      
      

4. Survival of in vivo infected Drosophila

   1. Score survival overtime (each 30 min) and compare survival curves using the log-rank test. When more than two conditions are considered, p-values should be adjusted by determining their statistical significance (alpha = 0.05) through stacked p-values analysis, through the Holm–Sidak method.

   2. Represent data as Kaplan–Meier curves with error bars corresponding to standard errors (SE).

Recipes

1. Egg laying medium

   Reagent	Final concentration	Amount
   Agar	22.2 g/L	22.2 g
   Sugar	25 g/L	25 g
   Organic grape juice	25% v/v	250 mL
   Methyl-4-hydroxybenzoate (20% in ethanol)	0.25% v/v	12.5 mL
   Purified water (reverse osmosis)	n/a	900 mL
   Total	n/a	~1 L

   
   

2. Fly food

   Reagent	Final concentration	Amount
   Active dry yeast	n/a	300 g
   Corn flour	n/a	211 g
   Agar	n/a	46 g
   Sugar	n/a	300 g
   Propionic acid	0.40% v/v	25 mL
   Methyl-4-hydroxybenzoate (20% in ethanol)	0.25% v/v	75 mL
   Purified water (reverse osmosis)	n/a	6.3 L
   Total	n/a	6 L (after cooking)

   
   

3. CNS culture medium I

   Reagent	Final concentration	Amount
   l-Glutamine (200 mM)	2 mM	100 μL
   Sodium l-ascorbate (500 mM)	0.5 mM	10 μL
   Drosophila Schneider’s medium	n/a	9.89 ml
   Total	n/a	10 mL

   
   

4. CNS culture medium II

   Reagent	Final concentration	Amount
   l-Glutamine (200 mM)	2 mM	100 μL
   Sodium l-ascorbate (500 mM)	0.5 mM	10 μL
   Fetal bovine serum	1%	100 μL
   Drosophila Schneider’s medium	n/a	9.79 mL
   Total	n/a	10 mL

Acknowledgments

We thank S. Dramsi, C. d’Enfert, G. Janbon, F. Leulier, M.-K. Taha, O. Disson, M. Lecuit, and F. Schweisguth for reagents and strains. We thank S. Liégeois for his help on hemolymph injection of Drosophila larvae and CFU counting. We thank D. Briand for his technical help on qPCR. This work has been funded by a starting package from Institut Pasteur/ LabEx Revive and a JCJC grant from Agence Nationale de la Recherche (NeuraSteNic, ANR- 17-CE13-0010-01) to P.S. B.B. has been supported by a Roux-Cantarini (Institut Pasteur) and a LabEx Revive post-doctoral fellowships.

This protocol was adapted from previous work (Benmimoun et al., 2020; Winkler et al., 2021).

Competing interests

The authors declare no conflicts of interest.

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