Use of In Vivo Imaging to Screen for Morphogenesis Phenotypes in Candida albicans Mutant Strains During Active Infection in a Mammalian Host

Candida albicans morphogenesis is triggered by a myriad of environmental signals. By studying morphogenesis in vivo, we're determining how the organism integrates and responds to all of these environmental signals. The main advantage of this technique is that it allows us to study morphogenesis in the absence of bias or artifacts that might be introduced using in vitro model systems.

Very few people have experience with intradermal injections. I strongly recommend that someone new to this technique contact their animal resource veterinarian for help learning it. Demonstrating the procedure will be Rohan Wakade, a post-doctoral fellow from our laboratory group.

Begin by preparing the animal for inoculation by feeding chlorophyll-free chow for at least seven days before inoculation. One day prior to injection, take a dab of Canada albicans cells from one colony using a toothpick. Inoculate 25 milliliters of YPD and repeat this process several times to obtain cells from several different colonies.

Incubate the culture overnight at 30 degrees Celsius in an orbital shaker incubator at 175 RPM. Centrifuge one milliliter of culture for two minutes at 500 x g, then wash the yeast three times with one milliliter of sterile DPBS. Resuspend the pellet in one milliliter of sterile DPBS and dilute the culture at 1 to 100.

Count the cells using a hemocytometer and accordingly adjust the cell density. Next, create the inoculum for injection by mixing equal volumes of the referenced and experimental strain. Using a cotton tipped swab, apply an over-the-counter depilatory cream liberally to the inner and outer surface of both ears of an anesthetized animal.

After two to three minutes, gently wipe the ear with a dry gauze followed by gauze pads saturated in sterile water to thoroughly remove depilatory cream residue. After sterilizing the ears'surface with 70%ethanol, mix the inoculum well by inverting the vial multiple times or vortexing it. Draw 20 to 30 microliters of inoculum into an insulin syringe.

Hold the needle in the upright position and tap the syringe gently to ensure that any air in the barrel is at the top. Carefully eject air and excess inoculum back into the inoculum tube or a waste tube so that the plunger is at the 10 microliter mark. Apply over the counter double-sided skin tape to a small conical or round-bottomed plastic tube and drape the ear across the tape, avoiding dislodging the anesthesia nose cone.

Keeping the syringe needle almost parallel to the skin and avoiding any large veins, insert the tip of the needle into the outermost layer of the skin until the bevel is just covered and slowly inject the inoculum intradermally. A good intradermal injection will raise a small bubble in the skin. Keep the needle in place for 15 to 20 seconds before removing it from the ear to minimize leakage.

Following institutional protocols, mark the cage with biohazard labels indicating that animals in the cage are infected with Candida albicans. Using the same washed cultures that were used to prepare the inoculum, create a 1 to 50 dilution of cells in RPMI 1640, supplemented with 10%heat and activated fetal bovine serum and incubate at 37 degrees Celsius with tumbling for four hours. Examine the sample using a microscope.

Count at least 100 cells, recording the number of yeast and filamentous cells for each strain. Rotate the long working distance objective into place and place a droplet of immersion fluid on the lens. Lower the lens to avoid possible damage when placing the cover slip.

Place number 1.5 cover slip over the stage opening and tape it in place. Raise the objective so that the immersion fluid is in contact with both the objective lens and the cover slip. Secure an anesthesia nose cone on the microscope stage in such a way that it will cover the animal's nose fully when it is in position for imaging.

Place the nose of an anesthetized mouse into the nose cone. Place a drop of sterile water on the cover slip above the objective lens. Adjust the anesthesia nose cone and position the mouse such that the mouse's ear is above the water droplet.

Take another cover slip and place the cover slip's edge parallel to the mouse's body with the edge touching the mouse where the ear meets the head. Using a hinge motion, lower the free edge of the cover slip to the microscope stage. As the cover slip comes against the microscope stage, it will flatten the ear.

Tape the top cover slip securely to hold just enough pressure to keep the ear flat and avoid catching the mouse's hair or whiskers in the tape. Unless a microscope with an environmental chamber is used, loosely cover the mouse's body with a sterile drape to maintain a normal thermic environment. Using white light, widefield imaging, adjust the focus plane into the ear tissue and focus on the red blood cells moving in the blood vessels.

To obtain a strong enough signal to noise ratio so that morphology can be determined for all cells in the field of view, adjust the laser power or imaging speed. To avoid tissue damage, use the lowest possible laser power. Choose a field of view with clear filament formation in the referenced strain and where most organisms are spread out enough that their morphology can be determined.

Set the top and bottom planes of focus for a Z-stack. Covering the entire depth of the infected area is not necessary, but bear in mind that organisms at the top or bottom of the imaged volume are typically excluded from the analysis. Acquire Z-stack images, pseudo color each channel to distinguish each strain and overlay the channels.

Save the images. Use imaging software to perform a maximum projection of the Z-stack into a two-dimensional image. Count each organism seen in the maximum projection images by strain type and morphology.

The referenced strain rapidly forms filaments in vitro. Whereas the EFG1 Null failed to form the filaments. The EFG1 Null exhibits a significant filamentation defect in vivo as well.

Approximately 9%of EFG1 Null cells grew as filaments in vivo. Similarly, the CYR1 Null mutant strain showed a filamentation defect in vitro compared to the referenced strain. In contrast, 53%of the CYR1 Null mutant cells grew as filaments in vivo.

The filaments formed by the CYR1 Null mutant were shorter than the referenced strain. It's critical to keep the needle parallel to the skin during inoculation. The goal is to place the inoculum just under the outer layer of the skin.

This technique can also be used with mouse strains that express a fluorescent protein in host cells of interest allowing us to study host pathogen interactions in vivo.