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

Summary

The human fungal pathogen Cryptococcus neoformans produces a variety of virulence factors (e.g., peptidases) to promote its survival within the host. Environmental niches represent a promising source of novel natural peptidase inhibitors. This protocol outlines the preparation of extracts from mollusks and the assessment of their effect on fungal virulence factor production.

Abstract

Cryptococcus neoformans is an encapsulated human fungal pathogen with a global distribution that primarily infects immunocompromised individuals. The widespread use of antifungals in clinical settings, their use in agriculture, and strain hybridization have led to increased evolution of resistance. This rising rate of resistance against antifungals is a growing concern among clinicians and scientists worldwide, and there is heightened urgency to develop novel antifungal therapies. For instance, C. neoformans produces several virulence factors, including intra- and extra-cellular enzymes (e.g., peptidases) with roles in tissue degradation, cellular regulation, and nutrient acquisition. The disruption of such peptidase activity by inhibitors perturbs fungal growth and proliferation, suggesting this may be an important strategy for combating the pathogen. Importantly, invertebrates such as mollusks produce peptidase inhibitors with biomedical applications and anti-microbial activity, but they are underexplored in terms of their usage against fungal pathogens. In this protocol, a global extraction from mollusks was performed to isolate potential peptidase inhibitors in crude and clarified extracts, and their effects against classical cryptococcal virulence factors were assessed. This method supports the prioritization of mollusks with antifungal properties and provides opportunities for the discovery of anti-virulence agents by harnessing the natural inhibitors found in mollusks.

Introduction

Cryptococcus neoformans is a human fungal pathogen that produces severe disease in immunocompromised hosts, such as individuals living with HIV/AIDS¹, and leads to approximately 19% of AIDS-related deaths². The fungus is susceptible to several classes of antifungals, including azoles, polyenes, and flucytosine, which exert fungicidal and fungistatic activity using distinct mechanisms^3,4. However, the extensive use of antifungals in clinical and agricultural settings combined with strain hybridization have amplified the evolution of resistance in multiple fungal species, including C. neoformans⁵.

To overcome the challenges of antifungal resistance and reduce the prevalence of fungal infections on a global scale, a promising approach is to use the virulence factors of Cryptococcus spp. (e.g., temperature adaptability, polysaccharide capsule, melanin, and extracellular enzymes) as potential therapeutic targets^4,6. This approach has several advantages, as these virulence factors are well-characterized in the literature, and targeting these factors could potentially reduce the rates of antifungal resistance by imposing a weaker selective pressure through impairing virulence rather than targeting cell growth⁶. In this context, numerous studies have assessed the possibility of targeting extracellular enzymes (e.g., proteases, peptidases) to reduce or inhibit the virulence of Cryptococcus spp.^7,8,9.

Organisms like invertebrates and plants do not possess an adaptive immune system to protect themselves from pathogens. However, they rely on a strong innate immune system with an immense array of chemical compounds to deal with microorganisms and predators¹⁰. These molecules include peptidase inhibitors, which play important roles in many biological systems, including the cellular processes of invertebrate immunity, such as the coagulation of hemolymph, the synthesis of cytokines and antimicrobial peptides, and the protection of hosts by directly inactivating the proteases of pathogens¹¹. Thus, peptidase inhibitors from invertebrates such as mollusks possess potential biomedical applications, but many remain uncharacterized^10,12,13. In this context, there are approximately 34 species of terrestrial mollusks in Ontario and 180 freshwater mollusks in Canada¹⁴. However, their in-depth profiling and characterization are still limited¹⁵. These organisms present an opportunity for the identification of new compounds with potential anti-fungal activity¹⁰.

In this protocol, methods to isolate and clarify extracts from invertebrates (e.g., mollusks) (Figure 1) followed by measuring the putative peptidase inhibitory activity are described. The antifungal properties of these extracts are then assessed by measuring their impact on C. neoformans virulence factor production using phenotypic assays (Figure 2). It is important to note that differences in antifungal properties between crude and clarified extracts may be indicative of microbial factors (e.g., secondary metabolites or toxins produced by the host microbiome) of the mollusk, which may influence experimental observations. Such findings support the need for this protocol to assess both crude and clarified extracts independently to unravel the modes of action. Additionally, the extraction process is unbiased and may enable the detection of antimicrobial properties against a plethora of fungal and bacterial pathogens. Therefore, this protocol provides an initiation point for the prioritization of mollusk species with antifungal properties against C. neoformans and an opportunity to evaluate the connections between enzymatic activity and virulence factor production through putative inhibitory mechanisms.

Protocol

1. Protein extraction from mollusks

1. Collect mollusks from a designated and approved natural area (e.g., Speed River, Guelph, Ontario). For this study, both native and invasive species were selected to assess a broad range of potential antifungal effects.
2. Gently break the shell of the mollusks (e.g., Cepaea nemoralis, Planorbella pilsbryi, and Cipangopaludina chinensis) using a pestle and mortar, and remove the solid pieces with a pair of tweezers. Generally, 10 mollusks are pooled for use throughout the protocol.
3. Collect and weigh the organs. Approximately 15-20 g of sample is optimal. Use scissors to cut the organs into small pieces approximately 0.5-1 cm in size.
4. Flash-freeze the dissected and cut organ samples with liquid nitrogen. Then, grind the organ samples into a fine powder using a pestle and mortar.
5. Add the organ sample powder (approximately 15 g) into 30 mL of cold distilled water (4 °C) to achieve a 1:2 ratio (w/v).
6. Pour 2 mL of the sample into high-impact 2 mL tubes. Add a scoopful of 3 mm stainless-steel beads (approximately 500 µg) to each tube, and homogenize using a blender (see Table of Materials) for 3 min at 1,200 rpm and 4 °C.
7. Centrifuge at 12,000 × g for 20 min at 4 °C. Collect all the supernatants with a pipette in a fresh 50 mL tube, discarding the pellet.
8. Filter-sterilize the supernatant using a 0.22 µm membrane. Store the samples on ice, and proceed to the clarification protocol (section 2), as applicable.
   ​NOTE: The samples can be flash-frozen in liquid nitrogen and stored at −20 °C to be used as crude extracts.

2. Clarification of the mollusk extract

1. Place the supernatant sample from step 1.8 in a thermal bath at 60 °C for 30 min. Then, cool the samples quickly by transferring them to a bucket with ice for 20 min.
2. Centrifuge the samples at 15,000 × g for 45 min at 4 °C. Collect the supernatant in a fresh 50 mL tube, and discard the pellet. Filter-sterilize the samples using a 0.22 µm membrane filter, and then store them on ice.
3. Determine the protein concentration in the samples from step 1.8 (i.e., the crude extract) and step 2.2 (i.e., the clarified extract) using a quantification assay (e.g., bicinchoninic acid assay¹⁷). The optimal protein concentration range is 4-8 mg/mL.
4. Store the samples on ice for up to 1 h or flash-freeze in liquid nitrogen, and store them at −20 °C until needed.

3. Inhibitory activity assay

1. Measure the inhibitory activity of the crude and clarified extracts using enzymatic assays in technical triplicate.
   NOTE: Each enzymatic assay consists of an enzyme (e.g., subtilisin A, which is involved in fungal virulence^16,17,18; usually around 1 x 10⁻⁹ mol/L for chromogenic-based assays), a buffer, and a substrate. The reaction starts when the substrate encounters the enzyme. The enzymatic activity is proportional to the rate of substrate conversion into the product.
2. Assess the effect of enzyme concentration (EC) over enzymatic activity (EA) until linear dependence is obtained between both parameters. For the next steps, use an enzyme concentration within the measured linear range.
   NOTE: The enzymatic assay for the peptidase subtilisin A is detailed in the following steps.
3. Incubate the crude (step 1.11) or clarified (step 2.5) mollusk protein extracts (e.g., 10 µL containing 4 mg/mL protein) with 10 µL of subtilisin A (concentration determined from step 3.2), and 220 µL of 100 mM Tris-HCl at pH 8.6 (for the control, add 230 µL of Tris buffer without adding the extract) for 10 min at 25 °C in a 96-well flat-bottom plate.
4. Add 10 µL of N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, the substrate for subtilisin A, at a final concentration of 1 K_m (Michaelis-Menten constant; 0.2 mM for this substrate with subtilisin A) for a final reaction volume of 250 µL.
5. Measure the enzymatic activity by monitoring the appearance of the product over time by reading the optical density (OD) at 405 nm every 15 s for 3 min.
6. Determine the residual enzymatic activity by calculating the ratio of the enzymatic activity in the presence of the mollusk extract to that in the absence of the extract.
7. If inhibitory activity is observed (i.e., residual activity below 1), measure the inhibitory concentration 50 (IC₅₀) value using a range of concentrations of the extracts (e.g., a twofold dilution series from 200 µg/mL to 1 µg/mL) following standard methods¹⁹.

4. Effect of mollusk extracts on C. neoformans growth

1. Use a 10 µL pipette tip to introduce a single colony of C. neoformans var. grubii H99 wild-type (WT) into 5 mL of yeast extract-peptone-dextrose (YPD) medium (10 g/L yeast extract, 20 g/L peptone, and 20 g/L dextrose, pH 6.5), and incubate for 16-18 h at 30 °C and 200 rpm. Perform the experiment in biological triplicate and technical duplicate.
2. Collect 500 µL of the culture in a cuvette, and measure the growth by reading the OD at 600 nm. Dilute the culture with yeast nitrogen base (YNB) medium to a final OD₆₀₀ of 0.02.
3. Co-culture C. neoformans cells with different concentrations (e.g., a threefold dilution series from 440 µg/mL to 15 µg/mL protein) of the extracts (i.e., crude and clarified). To do this, mix 10 µL of the extracts with 190 µL of the diluted C. neoformans culture (step 4.2) in a 96-well plate.
4. Measure the growth by reading the OD₆₀₀ using a plate reader every 15 min to 1 h for 72 h at 200 rpm and 37 °C.

5. Effect of mollusk extracts on C. neoformans melanin production

1. Prepare L-3,4-dihydroxyphenylalanine (L-DOPA) plates.
   1. Autoclave 230 mL of 14 g/L agar, and let it cool until it reaches 50-60 °C.
   2. Add 3.25 mL of 1 M glycine, 7.35 mL of 1 M KH₂PO₄, 2.5 mL of 1 M MgSO₄.7H₂O, 0.6 mL of 40% glucose, 70 µL of 10 mM thiamine, and 2.5 mL of 100 mM L-DOPA (filter-sterilized) to the liquid agar.
   3. Pour 15 mL of the mixture into Petri plates, let them dry for approximately 1 h in the dark, and store at 2-4 °C for up to 1 week. Ensure the lids are partially lifted for the agar to properly dry.
2. Inoculate 5 mL of YNB medium with 50 µL of C. neoformans culture from step 4.1. Incubate for 16-18 h at 30 °C and 200 rpm.
3. Spin down the cells at 1,000 × g for 5 min at 4 ˚C. Wash the cells 2x with sterile phosphate-buffered saline (PBS) at pH 7.4.
4. Count the cells using a hemocytometer. Resuspend the cells in 1 mL of PBS to reach a final concentration of 10⁶ cells/mL.
5. Prepare a dilution series (e.g., twofold) of the crude and clarified mollusk extracts. Similarly, prepare a 10-fold dilution series of C. neoformans cells (step 5.4) from 1 x 10⁶ cells/mL to 1 x 10¹ cells/mL using sterile PBS in 96-well plates.
6. Spread 200 µL of the extracts onto Petri plates containing L-DOPA agar using a swab, and let them dry for 15 min.
7. Spot 5 µL of culture from each well onto the L-DOPA plate, leaving 1 cm between drops. Let it dry in the dark for 15 min.
8. Incubate the plates at 30 °C and 37 °C in a static incubator for 3-5 days, capturing images every 24 h with a camera or plate imager (e.g., scanner).
   ​NOTE: Two temperature conditions are used here to explore the influence of temperature on the growth inhibitory effects of the mollusk extracts, with 30 °C representing environmental and 37 ˚C representing human physiological temperatures.

6. Effect of mollusk extracts on C. neoformans polysaccharide capsule production

1. Prepare low iron medium (LIM) as described below.
   1. Dissolve 5 g of chelating resin (see Table of Materials) in 60 mL of ultrapure water, and pack into a glass column (2 cm diameter x 25 cm length). Wash the column with 100 mL of water, and discard the collected water.
   2. Prepare low iron water (LI-water) by passing 2 L of sterile ultrapure water through the chelating column. Store the LI-water at 4 ˚C for up to 3 months.
   3. Dissolve 5 g of glucose in 100 mL of LI-water. Dissolve 5 g of L-asparagine in 200 mL of LI-water in a separate beaker.
   4. Add the following to 500 mL of LI-water in order: 4.78 g of HEPES, 0.4 g of K₂HPO₄, 0.08 g of MgSO₄.7H₂O, 1.85 g of NaHCO₃, and 0.25 g of CaCl₂.2H₂O.
   5. Combine the solutions from step 6.1.3 and step 6.1.4. Add 1 mL of 100 mM bathophenanthrolinedisulfonic acid sodium salt (BPS) to a final concentration of 100 µM.
   6. Adjust the pH to 7.2 with 1 M LI-MOPS. Add LI-water to a final volume of 1 L, and filter- sterilize the medium by passing it through a 0.22 µm membrane. Add 100 µL of sterile thiamine (4 mg/mL) to the medium.
2. Inoculate 5 mL of YNB medium with 50 µL of the C. neoformans culture from step 4.1. Incubate for 16-18 h at 30 °C and 200 rpm. Use this culture to inoculate 5 mL of LIM to a final concentration of 10⁵ cells/mL.
3. Add appropriate volumes of the mollusk extracts (e.g., a twofold dilution or the volume determined from previous virulence assays) to reach the desired concentrations. Incubate for 72 h at 37 °C and 200 rpm with loosened caps. After incubation, collect 1 mL of cells, and wash 2x with sterile PBS, pH 7.4, at 1,500 × g for 2 min at 4 ˚C.
4. Gently resuspend the cells in 1 mL of PBS. Mix the cells with India Ink at 1:1 ratio (e.g., 4 µL of India Ink with 4 µL of cell suspension) over a glass microscope slide. Place a coverslip on top of the slide.
5. Visualize the samples using a differential interference contrast (DIC) microscope with a 63x oil objective (see Table of Materials). Set the contrast to automatic, and focus manually using the microscope dial. Select all the pictures, and export them in TIFF format. No filters need to be applied.
6. Using the ruler tool in ImageJ²⁰, quantify the ratio of the total cell size (including the capsule) to the cell body size.

7. Effect of mollusk extracts on C. neoformans biofilm production

1. Inoculate 5 mL of YNB medium with 50 µL of C. neoformans culture from step 4.1. Incubate for 16-18 h at 30 °C and 200 rpm.
2. Harvest cells from the 5 mL culture. Wash the cells with sterile PBS, pH 7.4, at 1,500 × g for 2 min at 4 ˚C.
3. Count the cells using a hemocytometer. Resuspend the cells in 3 mL of Dulbecco's Modified Eagle Medium (DMEM) to a final concentration of 10⁷ cells/mL.
4. Transfer 300 µL of the cell suspension into the individual wells of a sterile, polystyrene, flat-bottom 24-well plate. Use wells with media only as a control.
5. Add appropriate volumes of the mollusk extracts (e.g., a twofold dilution or the volume determined from previous virulence assays) to reach a final concentration of 10-20 µg/mL protein. Ensure that the extract volume is not greater than 10% of the total volume.
6. Wrap the plates with aluminum foil (to avoid media evaporation), and incubate for 48 h using a static incubator at 30 °C and 37 °C.
7. Using a bottle or dispenser wash the wells 2x with sterile water, and let them air-dry for 10-15 min at room temperature. Then, add 100 µL of 0.3% crystal violet solution to each well (including the medium-only control wells), and incubate at room temperature for 10 min.
8. Wash the wells thoroughly 3x with sterile water (the biofilms will not be disrupted during washing). Add 200 µL of 100% ethanol, and incubate for 10 min at RT.
9. Transfer 75 µL to a new 96-well flat-bottom plate, and read the OD₅₅₀. Quantify the biofilm formation as (OD_550nm of sample - OD_550nm of blank).

Results

The workflow described herein enables the isolation of proteins and peptides from mollusks with potential anti-virulence properties against C. neoformans. Similarly, assessing different forms of extracts (i.e., crude and clarified) allows for the semi-purification of the potential active compounds and supports downstream assessment (e.g., mass spectrometry-based proteomics). Typically, the protein extraction workflow produces homogenized solutions with protein concentrations of 4-8 mg/mL. Here, the representativ...

Discussion

The extraction protocol described here outlines the isolation of compounds from mollusks collected from Ontario, Canada, and demonstrates a novel investigation of using mollusk extracts against the human fungal pathogen, C. neoformans. This protocol adds to a growing body of research investigating peptidase inhibitor activity from invertebrates¹³. During the extraction, some extract samples were difficult to filter-sterilize, possibly due to the presence of soluble polysaccharides and/or ...

Materials

Name	Company	Catalog Number	Comments
0.2 μm Filters	VWR	28145-477 (North America)
1.5 mL Tubes (Safe-Lock)	Eppendorf	0030120086
2 mL Tubes (Safe-Lock)	Eppendorf	0030120094
3,4-Dihydroxy-L-phenylalanine (L-DOPA)	Sigma-Aldrich	D9628-5G	CAS #: 59-92-7
96-well plates	Costar (Corning)	3370
Bullet Blender Storm 24	NEXT ADVANCE	BBY24M
Centrifuge 5430R	Eppendorf	5428000010
Chelex 100 Resin	BioRad	142-1253
CO2 Incubator (Static)	SANYO	Not available
Cryptococcus neoformans H99	ATCC	208821
DIC Microscope	Olympus
DIC Microscope software	Zeiss
DMEM	Corning	10-013-CV
Glucose (D-Glucose, Anhydrous, Reagent Grade)	BioShop	GLU501	CAS #: 50-99-7
Glycine	Fisher Chemical	G46-1	CAS #: 56-40-6
GraphPad Prism 9	Dotmatics
Hemocytometer	VWR	15170-208
HEPES	Sigma Aldrich	H3375
Magnesium sulfate heptahydrate (MgSO4.7 H2O)	Honeywell	M1880-500G	CAS #: 10034-99-8
Peptone	BioShop	PEP403
Phosohate buffer salt pH 7.4	BioShop	PBS408	SKU: PBS408.500
Plate reader (Synergy-H1)	BioTek (Agilent)	Not available
Potassium phosphate monobasic (KH2PO4)	Fisher Chemical	P285-500	CAS #: 7778-77-0
Subtilisin A	Sigma-Aldrich	P4860	CAS #: 9014-01-01
Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide	Sigma-Aldrich	573462	CAS #: 70967-97-4
Thermal bath	VWR	76308-834
Thiamine Hydrochloride	Fisher-Bioreagents	BP892-100	CAS #: 67-03-8
Yeast extract	BioShop	YEX401	CAS #: 8013-01-2
Yeast nitrogen base (with Amino Acids)	Sigma-Aldrich	Y1250-250G	YNB