The protocol demonstrated in this video allows us to understand the role of gut metabolites on fungal hyphal morphogenesis. This ex vivo technique more or less the closest approximation of the gut for studying fungal hyphal morphogenesis in a way that no in vitro study can replicate. This method can be applied to study the role of gut metabolites on other enteric pathogens.
Dissect mice using autoclaved sterilized sharp-ended scissors and forceps. After euthanasia, secure the animal to a dissection surface by pinning all limbs to expose the abdomen. Spray the abdominal region with 70%ethanol to prevent fur from sticking to forceps, scissors, or gut sections.
Use forceps to pinch and lift a section of skin at the base of the abdomen and create a small incision through the skin and underlying fascia taking care to avoid puncturing the cecum or intestinal wall. Extend this cut to the ribcage partially exposing the peritoneal cavity, then make a cut starting at the point of the initial incision on either side and extending upward and laterally. Pull these flaps laterally and pin them to the dissecting surface to fully expose the peritoneal cavity.
Extract the GI tract with forceps while using scissors to make cuts superior to the stomach and at the distal region of the large intestine to ensure collection of the greatest amount of gut content. When removing the GI tract, take care to avoid rupturing the individual components. Separate the stomach, small intestine, cecum, and large intestine at their proximal and distal ends.
For collection of gut contents from each section, make a single incision at the distal end, then manually expel the gut content into a 1.5 milliliter microcentrifuge tube with forceps. Store the samples at minus 80 degrees Celsius for ex vivo assays. Streak a fresh culture of C.albicans SC5314 onto a YPD agar plate and incubate it overnight at 30 degrees Celsius.
On the next day, pick two or three medium-sized individual colonies and resuspend them in one milliliter of PBS. Retrieve frozen gut contents from the freezer and thaw them at 25 degrees Celsius. Transfer approximately 150 milligrams of gut contents into a new 1.5 milliliter tube and resuspend them with 150 microliters of PBS.
Vortex the sample at high speed for 30 seconds to homogenize the gut contents and allow it to sit at room temperature for about a minute. Centrifuge the homogenates at 1, 000 times G for three minutes, then transfer the supernatant to a new 1.5 milliliter tube taking care to not transfer any debris. After repeating the centrifugation, add 10 microliters of the C.albicans inoculum to the supernatant, mix well, and incubate the sample at 37 degrees Celsius for four to five hours.
To test the effect of exogenous metabolites on C.albicans, add the desired concentration of metabolites to the gut content and PBS mixture, then vortex the sample at high speed for 30 seconds, let it sit at room temperature for 10 minutes, and perform centrifugation and inoculation as previously described. Centrifuge the fungal cell culture at 1, 000 times G for two minutes and discard the supernatant. Fix the samples in 100 microliters of 2%PFA for 15 minutes, then repeat the centrifugation and discard the supernatant.
Wash the samples twice with one milliliter of PBS according to manuscript directions, then incubate the samples at room temperature in 100 microliters of PBS with polyclonal C.albicans antibody for 30 minutes. After the incubation, wash the sample three more times with PBS, working in dim light to avoid photobleaching. In a dark room, incubate the samples at room temperature for 15 minutes in 100 microliters of PBS containing anti-rabbit IgG Alexa Fluor 488 antibody at a one to 500 dilution.
After washing the sample three times with one milliliter of PBS, resuspend it in 100 microliters of PBS and transfer it to a 96-well plate for imaging. Image the fungal cells with a fluorescence imaging microscope using 20X and 40X objective lenses and a green fluorescent protein filter. When C.albicans is grown ex vivo in gut homogenate extracts taken from the stomach, small intestines and large intestines of untreated control and antibiotic-treated mice, it generally develops with a yeast morphology.
However, when grown in the cecal extract from antibiotic-treated mice, C.albicans readily undergoes morphogenesis resulting in yeast and hyphae forms. This indicates that antibiotic treatment causes changes in the cecal environment, which induce hyphal morphogenesis of C.Albicans. Exogenous addition of glucose to the cecal homogenate of antibiotic-treated mice showed a massive hyphal development ex vivo.
These results suggest that addition of gut metabolites back to the cecal homogenate of the antibiotic-treated mice differentially regulates the morphogenesis of C.albicans. When attempting this protocol, keep in mind that optimization of the dilution of the gut content ensures sufficient collection of gut metabolites without collection of debris. Do not exceed a two-to-one media-to-gut content dilution and aim for a lower dilution if possible.
This technique is being used now to explore how specific metabolites in the gut impact hyphal development, allowing researchers to better understand the role of gut metabolites on fungal pathogenesis.
