Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

Summary

In vivo spatio-temporal interactions of pathogen and immune defenses at the mucosal level are not easily imaged in existing vertebrate hosts. The method presented here describes a versatile platform to study mucosal candidiasis in live vertebrates using the swimbladder of the juvenile zebrafish as an infection site.

Abstract

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their intercommunication, are paramount for the protection against infections. The interactions of epithelial and innate immune cells with a pathogen are best investigated in vivo, where complex behavior unfolds over time and space. However, existing models do not allow for easy spatio-temporal imaging of the battle with pathogens at the mucosal level.

The model developed here creates a mucosal infection by direct injection of the fungal pathogen, Candida albicans, into the swimbladder of juvenile zebrafish. The resulting infection enables high-resolution imaging of epithelial and innate immune cell behavior throughout the development of mucosal disease. The versatility of this method allows for interrogation of the host to probe the detailed sequence of immune events leading to phagocyte recruitment and to examine the roles of particular cell types and molecular pathways in protection. In addition, the behavior of the pathogen as a function of immune attack can be imaged simultaneously by using fluorescent protein-expressing C. albicans. Increased spatial resolution of the host-pathogen interaction is also possible using the described rapid swimbladder dissection technique.

The mucosal infection model described here is straightforward and highly reproducible, making it a valuable tool for the study of mucosal candidiasis. This system may also be broadly translatable to other mucosal pathogens such as mycobacterial, bacterial or viral microbes that normally infect through epithelial surfaces.

Introduction

Mucosal infections can lead to life threatening bloodstream infections due to the damage of the epithelial barrier, which allows pathogens access to the systemic environment^1,2. In addition, mucosal infections can also cause significant immunopathology even when contained externally^3-5. The commensal unicellular fungus Candida albicans is present in the majority of the population in the oral cavity and other mucosal sites^6-9. Although normally contained by innate and adaptive immune responses, innate immune defects and medical interventions can lead to severe mucosal candidiasis. The assault on the epithelial barrier results in an increased risk of life threatening disseminated disease as well as immunopathology, as in the case of vulvo-vaginal candidiasis, additionally C. albicans colonization has been linked with lung immune homeostasis^10,11. Disseminated candidiasis is now the fourth most common bloodstream infection in intensive care units¹² and mortality as high as 40% makes it a major concern. Due to the increase in immunomodulatory treatments for patients with autoimmune diseases, cancer or organ transplants, it is imperative to understand the interaction between this pathogen and the mucosal immune compartment.

The majority of cell biological advances regarding C. albicans-cell interactions at the mucosal level come from in vitro^13-15 and murine models^16-18. Both these approaches have distinct advantages, but the ability to image live cells at high resolution in an intact host has limited the temporal and spatial characterization of the infection. For these studies, there is the need for an in vivo model where the interaction of pathogen, innate immune and epithelial cells can be visualized in an intact vertebrate host.

The zebrafish has emerged as an invaluable tool for the understanding of human disease, mainly due to its transparency and amenability to genetic manipulation. Cell and organ development have been imaged in exquisite detail, which has led to the description of novel immune cell behaviors, such as T cell behavior in the developing thymus¹⁹ or the battle between intracellular mycobacteria and phagocytes^20-22. Recent work has described intestinal microbe-host interactions in zebrafish and shown that microbial colonization of the intestinal tract affects host intestinal physiology and resistance to other infections^23,24. Furthermore, infection through the gut epithelium has been described for several pathogens.

In contrast to the intestinal tract, the swimbladder represents a more isolated and complementary mucosal model. This organ is an extension of the developing gut tube and forms anteriorly to the liver and pancreas^25,26. It produces surfactant, mucus and antimicrobial peptides^27,28 and anatomically, as well as ontogenetically, this organ is considered a homologue of the mammalian lung^29,30. Since the pneumatic duct remains connected to the gut in the zebrafish, this allows for immersion infection to occur naturally. Remarkably, the only known naturally occurring infections of fish with Candida species are C. albicans infections in the swimbladder³¹. We recently described an experimental immersion infection model where C. albicans infects the swimbladder, and found that this infection recapitulates some of the hallmarks of C. albicans-epithelial interaction in vitro^32,33.

In the method presented here, the original immersion infection model is improved by directly injecting C. albicans into the swimbladder of 4 days post fertilization (dpf) zebrafish. This allows for precise temporal control of infection as well as a highly reproducible inoculum. It permits detailed intravital imaging, coupled with the versatility of the zebrafish model. As an example of what can be done with this method, we present the spatio-temporal dynamics of C. albicans growth along with neutrophil recruitment to the site of infection. Because zebrafish swimbladder tissue is challenging to image intravitally, we also present a rapid swimbladder dissection technique that improves fluorescence signal and microscopic resolution. These methods expand the toolbox for fungal, immunological, and aquaculture research as well as describing a novel infection route that may be translated to model other fungal, bacterial or viral infections of mucosal surfaces.

Protocol

NOTE: All zebrafish care protocols and experiments were performed in accordance with NIH guidelines under Institutional Animal Care and Use Committee (IACUC) protocol A2012-11-03.

1. Zebrafish Rearing to 4 Days Post Fertilization

1. Collect AB zebrafish, or any other transgenic lines, within the first 3 hr post fertilization, as shown in another video³⁴.
2. Incubate 120 eggs in a 15 cm Petri dishes containing 150 ml of E3 media (5 mM NaCl; 0.17 mM KCl; 0.33 mM CaCl₂; 0.33 mM MgCl₂; 2 mM HEPES; pH 6.8) with methylene blue (0.3 μg/ml final concentration) in a 33 °C incubator with 12/12 light/dark photoperiod.
3. Replace the media after 6 hr with E3 + PTU (1-phenyl-2-thiourea, 10 μg/ml final concentration) and remove the dead eggs.
4. Incubate for 4 days at 33 °C, exchanging the media with fresh E3 + PTU after 2 days.

2. Injection Micro-needle Preparation

1. Pull borosilicate capillary (OD 1.2 mm; ID 0.69 mm) into a micro-needle using a needle puller with the following settings (550 Heat; 0 Pull; 130 Velocity; 110 Time).
   NOTE: The settings will vary from filament to filament to obtain similarly shaped micro-needles (see Figure 1) and will need to be readjusted whenever a new filament is installed.
2. Fill a micro-needle with 3 μl of 0.4 μm-filtered phosphate buffer saline (PBS; 137 mM NaCl; 2.7 mM KCl; 10 mM Na₂HPO₄; 1.8 mM KH₂PO₄; pH 7.4) using a 20 μl microloader pipette tip.
3. Set up the micro-needle on a micropipette holder on a micromanipulator attached to a pressure injection system. Set the injection pressure to 30 PSI and the pulse duration at 30 msec.
4. Using a Petri dish with distilled water, lower the micro-needle until 1/5 of the needle is in the water. Clip the micro-needle with fine tweezers just below the water level. Bring the micro-needle out of the water and push the pedal several times until a bolus appears.
5. Measure the diameter of the bolus by lowering the micro-needle on a hemocytometer layered with one drop of type A immersion oil. Adjust the pulse duration to obtain the desired volume (see Table 1).
6. Set the backpressure for the bolus to remain stable in oil (lower the pressure if the bolus increases volume and increase the backpressure if the bolus volume decreases). Record the pulse duration for each micro-needle.
7. Remove the PBS from the micro-needle by aspirating it using a microloader pipette tip and expel the left-over PBS by placing it back on the injector and flipping the continuous pulse switch. Reserve each micro-needle.

3. Candida Preparation

1. Streak Candida albicans transformed with codon-optimized dTomato, GFP, BFP, EOS or Far Red from a frozen stock (-80 °C stock in 25% glycerol) using a sterile wooden dowel onto a yeast peptine dextrose (YPD) agar plate (10 g/L yeast extract, 20 g/L peptone, 20 g/L dextrose, 20 g/L agar; autoclaved and poured 25 ml in Petri dishes) and incubate overnight at 30 °C.
2. Pick one colony using a sterile wooden dowel and inoculate to 5 ml of YPD liquid (10 g/L yeast extract, 20 g/L peptone, 20 g/L dextrose, autoclaved and aseptically aliquotted in 5 ml in 16 x 150 mm glass culture tubes).
3. Incubate overnight at 30 °C in a roller drum at 60 rpm.
4. Collect 1 ml of C. albicans culture and transfer into a 1.7 ml sterile centrifuge tube.
5. Centrifuge at 5,000 x g for a couple of sec. Empty the supernatant and add 1 ml of sterile PBS, vortex thoroughly. Repeat this twice.
6. Count the colonies using a hemocytometer by diluting the 1 ml stock 1:1,000 in sterile PBS.

4. Injecting Zebrafish in the Swimbladder

1. Warm an injection dish (2% agarose) for 30 min in a 33 °C incubator.
2. At the desired time, remove 100 of the 150 ml of E3 + PTU from the 15 cm Petri dish containing the fish to inject using a 25 ml pipette without aspirating any zebrafish.
3. Anesthetize 4 dpf zebrafish by adding 2 ml of a 4 mg/ml buffered tricaine methane sulfonate stock (TR) to the 50 ml media remaining (final concentration 200 μg/ml) and wait for 15 min.
4. Under a dissecting microscope, select fish with an inflated swimbladder. Fish can also be screened at that time for homogenous phenotype, such as neutrophil distribution in mpo:GFP line using an epifluorescence dissecting microscope.
5. Dilute the C. albicans stock from its original concentration (step 3.8) to 1.5 x 10⁷ colony forming units per ml (cfu/ml) in PBS.
6. Transfer 30 zebrafish onto the injection dish using a plastic transfer pipette and remove the excess media.
7. Using a fishing-wire tool (0.012 inch diameter fishing line super-glued into a borosilicate capillary), make 3 lines of 10 fish, by holding the injection dish vertically and orientate the fish vertically.
8. Remove the excess media.
9. Thoroughly vortex the tube with 1.5 x 10⁷ cfu/ml C. albicans.
10. Fill one micro-needle with 3 μl of C. albicans using a new microloader pipette tip and set the pulse duration to the appropriate setting (see step 2.9).
11. Rotate the injection dish to make an angle of 20° between the micro-needle and the head of the fish, aiming toward the back of the swimbladder (Figure 3A).
12. Push the micro-needle into the swimbladder, making sure the bore of the needle is in the lumen, and press the pedal once.
13. Repeat for each fish and transfer to a recovery dish (25 ml E3 + PTU) by flooding the dish with E3 and emptying it into the recovery dish.
14. Transfer another 30 fish to the injection dish.
15. Repeat injection with a new micro-needle filled with thoroughly vortexed C. albicans.
16. Finish by injecting the PBS control, if necessary, using a different injection dish to avoid contamination and transfer to a separate recovery dish (step 4.10).

5. Screening the Fish for Desired Inoculum

1. Prepare 40 ml of 0.4% low-melt agarose solution (LMA) by adding 160 mg of low melt agarose to 40 ml of E3, boiling by microwaving until dissolved, and cooling to 37 °C. Add 400 μl of TR (final concentration 200 μg/ml) once cooled.
2. After 30 min recovery, anaesthetize the fish as described (step 4.3 using only 1 ml of TR into 25 mL E3 + PTU, final concentration 200 μg/ml).
3. Transfer 30 fish (with as little media as possible) to 2 ml of LMA in a weight boat.
4. Aspirate individual fish with a transfer pipette and plate the fish with 1 drops of LMA into a 96-well imaging plate, using only the 10 x 6 central wells.
5. Repeat until all the fish to screen are loaded to the imaging plate.
6. Let the media solidify at room temperature for 5 min.
7. Position the fish on their side, making sure they are in contact with the glass bottom.
8. Using the 20X objective on an inverted epifluorescence/confocal microscope and the appropriate filter, record the number of C. albicans yeast cells in each fish’s swimbladder.
9. Select the fish with 15-25 yeast cells in the swimbladder (or desired inoculum), euthanize non-selected fish (800 μg/ml of TR in E3) and return selected fish to a deep Petri dish with 50 ml of E3 + PTU by adding 3 drops of E3 to the imaging plate selected wells and aspirating the fish with a plastic transfer pipette.
10. Incubate at 33 °C for the desired duration.

6. Imaging the Fish Post-screening

1. At the desired time, remove 25 of the 50 ml of E3 + PTU media from the deep Petri dish containing the fish to image.
2. Anaesthetize the fish as described (step 4.3 using only 1 ml of TR into 25 ml E3 + PTU remaining, final concentration 200 μg/ml).
3. Plate the fish into a 96-well imaging plate and position appropriately (steps 5.3 - 5.7).
4. Image by scanning confocal microscopy using the appropriate lasers and emission filters. First focus on the fish using the 4X objective under differential interference contrast (DIC) and acquire images of the region of interest using the 20X objective with the confocal lasers in scanning mode with 2 μsec/pixel speed and 1024 x 600 pixel aspect ratio. Acquire Z-stacks with 1 μm step size, applying Kalman filter mode of 3 frames.
5. Return to a deep Petri dish with 50 ml E3 + PTU or into individual wells (24-well culture dish, 3 ml E3 + PTU) and incubate at 33 °C.

7. Dissecting the Swimbladder

1. Fill a 10 ml syringe with high vacuum grease.
2. Prepare a 2% LMA (step 5.3 with 160 mg of low melting agarose in 8 ml of E3).
3. At the desired time, anaesthetize the fish (step 5.2).
4. Transfer 10 fish to a 1.7 ml centrifuge tube with 1 ml of E3 and euthanize by adding 200 μl of 4 mg/ml Tricaine (final concentration 800 μg/ml) to the centrifuge tube and incubating for 15 min at room temperature.
5. Prepare an imaging glass slide by placing 4 drops of vacuum grease in a square, 1 cm apart on a microslide (75 x 25 mm, Figure 2).
6. Place 2 drops of 2% LMA in the middle of the square.
7. Place one fish on a separate glass slide under a dissecting microscope and verify that the heart has stopped beating, remove the excess water.
8. Using fine tweezers dissect the swimbladder by maintaining the head in a fixed position with the left tweezers and pulling down the anterior gut with the right tweezers (Figure 4A).
9. Pick up the swimbladder by the pneumatic duct with the left tweezers, separating it from the digestive tract, and place it at the center of the 2% LMA before it solidifies.
10. Place a cover-slip (18 x 18 mm) on top of the imaging glass slide and push it until it touches the vacuum grease as well as the LMA (Figure 2).
11. Image within 10 min of dissection as described in step 6.4. Repeat step 7.7 - 7.11 with the other fish.

Results

Microinjection in the posterior swimbladder

The experimental method presented here describes the injection of a consistent dose of C. albicans yeast cells in the swimbladder of 4 dpf zebrafish. Previous work with the immersion model suggests that the swimbladder immune response to C. albicans is similar to mammalian mucosal candidiasis³². Here we demonstrate a modified infection method that is more straightforward, reproducible and rapid; several hundreds of zebrafish...

Discussion

Advances and limitations of the swimbladder microinjection disease model

The model presented here is an extension of the mucosal candidiasis immersion model described in Gratacap et al. (2013); it adds the advantages of a controlled infection time, a highly reproducible infection dose, and therefore improved efficiency. We demonstrate here new methods that permit non-invasive temporal documentation of infection dynamics in great detail as well as higher resolution ex vivo imagin...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.7 ml tubes	Axygen	MCT-175-C
Deep Petri dishes	Fisher Scientific	89107-632
Transfer pipettes	Fisher Scientific	13-711-7M
Yeast Extract	VWR Scientific	90000-726
Peptone	VWR Scientific	90000-264
Dextrose	Fisher Scientific	D16-1
Agar	VWR Scientific	90000-760
Fine tweezers (Dumont Dumoxel #5)	Fine Science Tools	11251-30
Wooden dowels	VWR Scientific	10805-018
Low melt agarose	VWR Scientific	12001-722
Flaming Brown micropipette puller	Sutter Instruments	P-97
Borosilicate capillary	Sutter Instruments	BF120-69-10
MPPI-3 injection system	Applied Scientific Instrumentation	MPPI-3
Back pressure unit	Applied Scientific Instrumentation	BPU
Micropipette holder kit	Applied Scientific Instrumentation	MPIP
Foot switch	Applied Scientific Instrumentation	FSW
Micromanipulator	Applied Scientific Instrumentation	MM33
Magnetic base	Applied Scientific Instrumentation	Magnetic Base
Tricaine methane sulfonate	Western Chemical Inc.	MS-222
Dissecting microscope	Olympus	SZ61 top SZX-ILLB2-100 base
Confocal microscope	Olympus	IX-81 with FV-1000 laser scanning confocal system
20X microscope objective	Olympus	UPlanSApo 20x/0.75
Roller drum	New Brunswick Scientific	TC-7
Microloader pipette tips	Eppendorf	930001007
Glass culture tubes (16 x 150 mm)	VWR Scientific	60825-435
NaCl	VWR Scientific	BDH4534-500GP
KCl	VWR Scientific	BDH4532-500GP
MgSO4	VWR Scientific	BDH0246-500GP
HEPES (Corning)	VWR Scientific	BDH4520-500GP
Children clay (Play-Doh)	Hasbro
CaCl2	Fisher Scientific	C69-500
Methylene blue	VWR Scientific	VW6276-0
PTU	Sigma	P7629-10G
Petri dishes	Fisher Scientific	FB0875712
Hemocytometer (Hausser scientific)	VWR Scientific	15170-172
Type A immersion oil	Blue Marble Products	51935
Centrifuge	Eppendorf	5424
Vortex Genie	VWR Scientific	14216-184
Agarose (Lonza)	VWR Scientific	12001-870
Na2HPO4	Fisher Scientific	S374-500
KH2PO4	Fisher Scientific	P285-500
Fishing wire	Stren
96 well imaging plate (Sensoplate)	Greiner Bio-One	655892
High vacuum grease (Dow Corning)	VWR Scientific	59344-055
Microslide (25 x 75 mm)	VWR Scientific	48300-025
Cover slips (18 x 18 mm), No 1.5	VWR Scientific	48366-045
15 cm Petri dish (Olympus plastics)	Genesee Scientific	32-106
Glycerol (EMD chemicals)	VWR Scientific	EMGX0185-5
24-well culture dish (Olympus plastics)	Genesee Scientific	25-107
Weight boats (8.9 cm)	VWR Scientific	89106-766