Assessing the Putative Anticryptococcal Properties of Crude and Clarified Extracts from Mollusks

This protocol allows the isolation of compounds from mollusks with anti-cryptococcal properties. This technique offers an unbiased approach to identify and isolate compounds from mollusks with potential properties, including killing the human fungal pathogens. There are other compounds, such as HIV protease inhibitors, being used to treat HIV patients.

However, expansion to other pathogens, as well as investigating natural sources, represents new avenues of discovery. The last step is the most difficult step since the samples may not get filtered. As a result, samples may need to be diluted for filtering.

Begin by collecting mollusks like Cipangopaludina chinensis from a designated and approved natural area. Select native and invasive species to assess a broad range of potential antifungal effects. Gently break the shell of the C.chinensis using a pestle and mortar and remove the solid pieces with a pair of tweezers.

Generally, 10 mollusks are pooled for the protocol. Collect and weigh approximately 15 to 20 grams of the sample and use scissors to cut the organs into small pieces approximately 0.5 to one centimeter in size. Flash freeze the dissected and cut organ samples using liquid nitrogen before grinding the organ samples into a fine powder using a pestle and mortar.

Once done, add approximately 15 grams of the organ sample powder to 30 milliliters of cold distilled water to achieve a ratio of one to two. Pour two milliliters of the sample into high impact two milliliter tubes and add approximately 500 micrograms of three millimeters stainless steel beads to each tube. Homogenize for three minutes at 1, 200 RPM in four degrees Celsius using a bullet blender or bead beater.

Centrifuge the tube at 12, 000 G for 20 minutes at four degrees Celsius. Collect the supernatant in a fresh 50 milliliter tube and discard the pellet. Filter sterilize the supernatant using a 0.22 micrometer membrane and store the samples on ice before clarifying the extract.

Place the supernatant sample in a thermal bath at 60 degrees Celsius for 30 minutes and immediately cool it down by transferring the sample to a bucket filled with ice for 20 minutes. Centrifuge the cold samples at 15, 000 G for 45 minutes at four degrees Celsius. Collect the supernatant in a fresh 50 milliliter tube and discard the pellet.

Filter sterilize the samples using a 0.22 micron membrane filter and then store them on ice. Once done, determine the protein concentration in the crude and the clarified extracts using a quantification assay. The optimal protein concentration range is four to eight micrograms per milliliter.

Incubate the samples on ice for up to one hour or flash freeze in liquid nitrogen before storing them at minus 20 degrees Celsius until needed. In a 96-well flat bottom plate, incubate 10 microliters of four milligrams per milliliter crude or clarified mollusk protein extract, 10 microliters of subtilisin A, having a concentration within the measured linear range, and 220 microliters of 100 millimolar tris hydrochloride of 8.6 pH for 10 minutes at 25 degrees Celsius. Then add 10 microliters of N-succinyl-alanine, alanine, proline, phenolalanine, p-nitroaniline, the substrate for subtilisin A, at a final concentration of one KM for a final reaction volume of 250 microliters.

Once done, measure the enzymatic activity by monitoring the optical density at 405 nanometers every 15 seconds for three minutes. Ensure that the wells with no extracts produce a straight curve during the reading. To determine the residual enzymatic activity, calculate the ratio of enzymatic activity in the presence of mollusk extract to that in the absence of the extract.

If inhibitory activity is observed, measure the inhibitory concentration 50 or IC50 value using a range of concentrations of the extracts. Introduce a single colony of C.neoformans H99 wild type into five milliliters of yeast extract peptone dextrose or YEPD medium using a 10 microliter pipette tip and incubate for 16 to 18 hours at 30 degrees Celsius and 200 RPM. Collect 500 microliters of the culture in a cuvette and measure the growth by reading the optical density at 600 nanometers.

Dilute the culture with yeast nitrogen base or YNB medium to a final optical density of 0.02. Co-culture C.neoformans cells with different concentrations of crude and clarified extracts by mixing 10 microliters of the extracts with 190 microliters of the diluted C.neoformans culture in a 96-well plate. Measure the growth by reading the optical density at 600 nanometers using a plate reader every 15 minutes to one hour for 72 hours at 200 RPM and 37 degrees Celsius.

The extracts of C.chinensis were able to inhibit the proteolytic activity of subtilisin A, which is related to virulence in Cryptococcus neoformans with an IC50 value of 5.3 micrograms per milliliter in crude extracts and 4.53 micrograms per milliliter in clarified extracts. The assessment of the C.chinensis extracts against processes associated with C.Neoformans virulence factor production including fungal growth, showed that there was a significant reduction in fungal growth at 37 degrees Celsius in the presence of the crude and clarified C.chinensis extracts. The assessment of the extracts against capsule, melanin production, and the formation of biofilms showed that there were no changes in capsule or melanin production in the presence of crude or clarified C.chinensis extracts.

However, a significant reduction of 70 to 80%in biofilm formation was observed at high concentrations of the crude and clarified extracts relative to the untreated control. The protocol success depends on the proper extraction procedure including grinding, the addition of the appropriate amount of enzyme, and just reading after the addition of the substrate. The technique focuses on thermal tolerance as an important cryptococcal virulence factor.

However, other virulence factors can be assessed such as melanin and capsule production, as well as biofilm formation. These results open new pathways that constitute new approaches on antifungal strategies using natural compounds that offers more stability, specificity, and are less prone to resistance. The long-term goal is to find new methods against these fungal pathogens and minimize resistance mechanisms.