This study investigates the role of the proteolytic enzyme Sap2 secreted by Candida tropicalis in biofilm formation. The enzyme cleaves the inhibitory domain of Msb2, promoting fungal adhesion and growth into microcolonies.
Take a microplate containing the fungus Candida tropicalis.
The cells secrete Sap2, a proteolytic enzyme that cleaves a transmembrane glycoprotein called Msb2, removing its inhibitory domain.
This induces the fungus to adhere to the bottom and grow into a microcolony of yeast and filamentous forms. The cells secrete soluble components and biopolymers, initiating biofilm formation.
Add a serum sample containing Sap2-specific antibodies to selected wells, and incubate in the dark.
The antibodies bind to Sap2 and neutralize its activity, impeding cell viability and biofilm maturation.
In untreated wells, the biofilm matures into a dense network of cells and extracellular matrix.
Aspirate the liquid, wash the biofilm, and air-dry the plate. Add a chromogenic substrate, and incubate in the dark.
Viable cells, utilizing membrane-bound oxidoreductases, reduce the substrate to a colored product.
Transfer the colored supernatant and read the absorbance.
A lower absorbance in the serum-treated wells indicates antibody-mediated biofilm inhibition.
Prepare serial dilutions of heat-inactivated serum samples in sterile RPMI1640 MOPS medium. Add 100 microliters of the selected serum dilution to each well of the 96-well microtiter plate.
In column 10, add only RPMI1640-MOPS medium for the fungus-only positive control. Add a 1-to-50 dilution of serum to all wells in column 11 to serve as the no-fungus plus serum negative control. After covering the plate with a lid and aluminum foil, incubate the plate for 24 hours at 37 degrees Celsius.
The next day, after aspirating the serum carefully using a multichannel pipette, tap the inverted plate gently to remove any residual serum. Repeat the PBS wash three times, and air-dry the plate for 30 minutes at room temperature inside a biological safety cabinet to dry any excess PBS.
Then, dissolve 25 milligrams of XTT in 50 milliliters of filter-sterilized Ringer's lactate. Aliquot 10 milliliters in separate tubes. Cover it with aluminum foil, and store it at minus 80 degrees Celsius. Next, dissolve 8.6 milligrams of menadione in 5 milliliters of acetone, and after distributing 50 microliters in 100 separate microtubes, store the aliquots at minus 80 degrees Celsius.
Take 10 milliliters of XTT, and add 1 microliter of menadione to obtain a 1-micromolar working solution. Add 100 microliters of the XTT menadione solution per well of the 96-well microtiter plate. Next, after covering the plate with a lid and aluminum foil, incubate the plate for two hours at 37 degrees Celsius in the dark. Finally, transfer 80 microliters of the colored supernatant from each well into a fresh 96-well plate, and read the plate at 490 nanometers.
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