The authors thank members of the Geddes-McAlister Lab for their valuable support throughout this investigation and their manuscript feedback. The authors acknowledge the funding support from the Ontario Graduate Scholarship and International Graduate Research Award - University of Guelph to D. G.-G and from the Canadian Foundation of Innovation (JELF 38798) and Ontario Ministry of Colleges and Universities - Early Researcher Award for J. G.-M.

1. Protein extraction from mollusks
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Add the organ sample powder (approximately 15 g) into 30 mL of cold distilled water (4 &#176;C) to achieve a 1:2 ratio (w/v).</li>
	<li>Pour 2 mL of the sample into high-impact 2 mL tubes. Add a scoopful of 3 mm stainless-steel beads (approximately 500 &#181;g) to each tube, and homogenize using a blender (see Table of Materials) for 3 min at 1,200 rpm and 4 &#176;C.</li>
	<li>Centrifuge at 12,000 &#215; g for 20 min at 4 &#176;C. Collect all the supernatants with a pipette in a fresh 50 mL tube, discarding the pellet.</li>
	<li>Filter-sterilize the supernatant using a 0.22 &#181;m membrane. Store the samples on ice, and proceed to the clarification protocol (section 2), as applicable. 
	&#8203;NOTE: The samples can be flash-frozen in liquid nitrogen and stored at &#8722;20 &#176;C to be used as crude extracts.</li>
</ol>
2. Clarification of the mollusk extract
<ol>
	<li>Place the supernatant sample from step 1.8 in a thermal bath at 60 &#176;C for 30 min. Then, cool the samples quickly by transferring them to a bucket with ice for 20 min.</li>
	<li>Centrifuge the samples at 15,000 &#215; g for 45 min at 4 &#176;C. Collect the supernatant in a fresh 50 mL tube, and discard the pellet. Filter-sterilize the samples using a 0.22 &#181;m membrane filter, and then store them on ice.</li>
	<li>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.</li>
	<li>Store the samples on ice for up to 1 h or flash-freeze in liquid nitrogen, and store them at &#8722;20 &#176;C until needed.</li>
</ol>
3. Inhibitory activity assay
<ol>
	<li>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 virulence16,17,18; usually around 1 x 10&#8722;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.</li>
	<li>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.</li>
	<li>Incubate the crude (step 1.11) or clarified (step 2.5) mollusk protein extracts (e.g., 10 &#181;L containing 4 mg/mL protein) with 10 &#181;L of subtilisin A (concentration determined from step 3.2), and 220 &#181;L of 100 mM Tris-HCl at pH 8.6 (for the control, add 230 &#181;L of Tris buffer without adding the extract) for 10 min at 25 &#176;C in a 96-well flat-bottom plate.</li>
	<li>Add 10 &#181;L of N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, the substrate for subtilisin A, at a final concentration of 1 Km (Michaelis-Menten constant; 0.2 mM for this substrate with subtilisin A) for a final reaction volume of 250 &#181;L.</li>
	<li>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.</li>
	<li>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.</li>
	<li>If inhibitory activity is observed (i.e., residual activity below 1), measure the inhibitory concentration 50 (IC50)&#160;value using a range of concentrations of the extracts (e.g., a twofold dilution series from 200 &#181;g/mL to 1 &#181;g/mL) following standard methods19.</li>
</ol>
4. Effect of mollusk extracts on C. neoformans growth
<ol>
	<li>Use a 10 &#181;L 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 &#176;C and 200 rpm. Perform the experiment in biological triplicate and technical duplicate.</li>
	<li>Collect 500 &#181;L 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.</li>
	<li>Co-culture C. neoformans cells with different concentrations (e.g., a threefold dilution series from 440 &#181;g/mL to 15 &#181;g/mL protein) of the extracts (i.e., crude and clarified). To do this, mix 10 &#181;L of the extracts with 190 &#181;L of the diluted C. neoformans culture (step 4.2) in a 96-well plate.</li>
	<li>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 &#176;C.</li>
</ol>
5. Effect of mollusk extracts on C. neoformans melanin production
<ol>
	<li>Prepare L-3,4-dihydroxyphenylalanine (L-DOPA) plates.
	<ol>
		<li>Autoclave 230 mL of 14 g/L agar, and let it cool until it reaches 50-60 &#176;C.</li>
		<li>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 &#181;L of 10 mM thiamine, and 2.5 mL of 100 mM L-DOPA (filter-sterilized) to the liquid agar.</li>
		<li>Pour 15 mL of the mixture into Petri plates, let them dry for approximately 1 h in the dark, and store at 2-4 &#176;C for up to 1 week. Ensure the lids are partially lifted for the agar to properly dry.</li>
	</ol>
	</li>
	<li>Inoculate 5 mL of YNB medium with 50 &#181;L of C. neoformans culture from step 4.1. Incubate for 16-18 h at 30 &#176;C and 200 rpm.</li>
	<li>Spin down the cells at 1,000 &#215; g for 5 min at 4 &#730;C. Wash the cells 2x with sterile phosphate-buffered saline (PBS) at pH 7.4.</li>
	<li>Count the cells using a hemocytometer. Resuspend the cells in 1 mL of PBS to reach a final concentration of 106 cells/mL.</li>
	<li>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.</li>
	<li>Spread 200 &#181;L of the extracts onto Petri plates containing L-DOPA agar using a swab, and let them dry for 15 min.</li>
	<li>Spot 5 &#181;L of culture from each well onto the L-DOPA plate, leaving 1 cm between drops. Let it dry in the dark for 15 min.</li>
	<li>Incubate the plates at 30 &#176;C and 37 &#176;C in a static incubator for 3-5 days, capturing images every 24 h with a camera or plate imager (e.g., scanner). 
	&#8203;NOTE: Two temperature conditions are used here to explore the influence of temperature on the growth inhibitory effects of the mollusk extracts, with 30 &#176;C representing environmental and 37 &#730;C representing human physiological temperatures.</li>
</ol>
6. Effect of mollusk extracts on C. neoformans polysaccharide capsule production
<ol>
	<li>Prepare low iron medium (LIM) as described below.
	<ol>
		<li>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.</li>
		<li>Prepare low iron water (LI-water) by passing 2 L of sterile ultrapure water through the chelating column. Store the LI-water at 4 &#730;C for up to 3 months.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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 &#181;M.</li>
		<li>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 &#181;m membrane. Add 100 &#181;L of sterile thiamine (4 mg/mL) to the medium.</li>
	</ol>
	</li>
	<li>Inoculate 5 mL of YNB medium with 50 &#181;L of the C. neoformans culture from step 4.1. Incubate for 16-18 h at 30 &#176;C and 200 rpm. Use this culture to inoculate 5 mL of LIM to a final concentration of 105 cells/mL.</li>
	<li>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 &#176;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 &#215; g for 2 min at 4 &#730;C.</li>
	<li>Gently resuspend the cells in 1 mL of PBS. Mix the cells with India Ink at 1:1 ratio (e.g., 4 &#181;L of India Ink with 4 &#181;L of cell suspension) over a glass microscope slide. Place a coverslip on top of the slide.</li>
	<li>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.</li>
	<li>Using the ruler tool in ImageJ20, quantify the ratio of the total cell size (including the capsule) to the cell body size.</li>
</ol>
7. Effect of mollusk extracts on C. neoformans biofilm production
<ol>
	<li>Inoculate 5 mL of YNB medium with 50 &#181;L of C. neoformans culture from step 4.1. Incubate for 16-18 h at 30 &#176;C and 200 rpm.</li>
	<li>Harvest cells from the 5 mL culture. Wash the cells with sterile PBS, pH 7.4, at 1,500 &#215; g for 2 min at 4 &#730;C.</li>
	<li>Count the cells using a hemocytometer. Resuspend the cells in 3 mL of Dulbecco&#39;s Modified Eagle Medium (DMEM) to a final concentration of 107 cells/mL.</li>
	<li>Transfer 300 &#181;L 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.</li>
	<li>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 &#181;g/mL protein. Ensure that the extract volume is not greater than 10% of the total volume.</li>
	<li>Wrap the plates with aluminum foil (to avoid media evaporation), and incubate for 48 h using a static incubator at 30 &#176;C and 37 &#176;C.</li>
	<li>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 &#181;L of 0.3% crystal violet solution to each well (including the medium-only control wells), and incubate at room temperature for 10 min.</li>
	<li>Wash the wells thoroughly 3x with sterile water (the biofilms will not be disrupted during washing). Add 200 &#181;L of 100% ethanol, and incubate for 10 min at RT.</li>
	<li>Transfer 75 &#181;L to a new 96-well flat-bottom plate, and read the OD550. Quantify the biofilm formation as (OD550nm of sample - OD550nm of blank).</li>
</ol>

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The authors declare no conflicts of interest.

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 pigments that obstructed the filter membrane. To overcome this limitation, it is recommended to filter first through a 5 &#181;m membrane to exclude large compounds (e.g., disrupted cell membranes, genome DNA), thus allowing the proteins to pass through the filter, and then to filter again through a 0.22 &#181;m membrane. These steps are critical to the protocol as they restrict the presence of microbes that may contaminate the samples and interfere with downstream experiments.
During this investigation, peptidase inhibitors were detected against subtilisin A, a model enzyme for the S8 family of subtilisin. Members of this enzyme family are widely distributed among organisms and have varying roles, such as in protein processing, nutrition, and virulence mechanisms21,22, which support the phenotypic effects observed in this study. For example, a significant reduction in fungal growth was observed in the presence of both crude and clarified extracts and at relatively high and low concentrations, suggesting that the inhibitory activity was robust in the tested model. It is notable that when measuring the OD with a plate reader, the presence of the extracts caused clumping of the fungal cells at the bottom of the well, interfering with the growth measurement. This limitation can be overcome by using a high-speed shaking incubator (e.g., 900 rpm), which would avoid cell clumping.
Other technical limitations may exist that may influence the anticipated phenotypic observations. For instance, subtilisin-like peptidases are associated with melanin synthesis and quorum sensing in C. neoformans, but the crude or clarified extracts from any mollusks do not show significant effects on melanin production16,17,18. This is possibly due to the natural protective effect of melanin and capsule against external agents, which could prevent the mollusk extracts from impacting the intracellular components of the fungus. Important factors to consider when working with organic substrates include the need to dissolve them in dimethyl sulfoxide (DMSO), which may present solubility issues within the agar plate (as used in the melanin assays). To overcome this problem, the extracts may be spread along the surface of the agar plate and allowed to dry prior to spotting the fungal cells onto the plate.
Previous work demonstrated the susceptibility of C. neoformans biofilms to two antifungal agents in vitro, including amphotericin B and caspofungin; however, the fungus was resistant to fluconazole and voriconazole23. Given the importance of fungal biofilms in virulence and antimicrobial resistance, uncovering new strategies to interfere with or disrupt biofilm formation would be valuable. In the current study, crude and clarified extracts from C. chinensis impaired the formation of biofilms of cryptococcal cells with apparent dose-response behavior. These results support the impact and novelty of the approach. Notably, biofilm formation was inhibited to a greater extent on treatment with the crude compared to the clarified extract, which may be due to a loss of inhibitory compounds during the clarification process or a reduction in inhibitory function under the tested conditions. It is possible that the proteins responsible for these inhibitory effects are of high molecular weight and were lost during the clarification process or were susceptible to degradation during the thermal treatment. These results highlight the importance of using both crude and clarified extracts to detect changes in inhibitory functions, as differences related to the inhibitor source may influence the outcome.
Overall, this protocol enables the extraction of compounds from mollusks and the measurement of putative inhibitory activity against a selected peptidase with demonstrated roles in fungal virulence. In this protocol, the high yield and strong peptidase inhibitory activity of the extracts and their effect against C. neoformans growth and biofilm formation were evaluated. While further experiments are needed, these results stress the importance of mollusks as putative new sources of compounds against this dangerous fungal pathogen. Furthermore, while this protocol focuses on the extraction of inhibitors from mollusks, the methodology is adaptable to other invertebrates and can be used to assess the effect of inhibitors derived from different invertebrates on virulence factor production in a variety of microorganisms, including fungi and bacteria24,25,26. Ultimately, the extraction of compounds from natural sources can increase the repertoire of putative novel antimicrobial agents and, thus, improve our abilities to combat infectious diseases.

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 mechanisms3,4. 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,&#160;and extracellular enzymes) as potential therapeutic targets4,6. 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 uncharacterized10,12,13. 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.

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

assessing the putative anticryptococcal properties of crude and clarified extracts from mollusks

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.

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 representative results demonstrate the assessment of the enzymatic activity and antifungal properties of C. chinensis extracts.The crude and clarified extracts were able to inhibit the proteolytic activity of subtilisin A (related to virulence in C. neoformans) (Figure 3), with IC50 values of 5.3 &#181;g/mL and 4.53 &#181;g/mL, respectively. The activity of the C. chinensis extracts was further tested against processes associated with C. neoformans virulence factor production, including fungal growth, capsule and melanin production, and the formation of biofilms. There was a significant reduction in fungal growth at 37 &#176;C in the presence of the crude (Figure 4A) and clarified (Figure 4B) C. chinensis extracts. Notably, there were no changes in capsule or melanin production in the presence of crude or clarified C. chinensis extracts (Figure 4C,D). However, a significant reduction of 70%-80% in biofilm formation was observed at high concentrations of the crude (Figure 4E) and clarified (Figure 4F) extracts relative to the untreated control.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64540/64540fig01.jpg" /> 
Figure 1: Workflow for total protein extraction from mollusks.&#160;Created with BioRender.com.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64540/64540fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64540/64540fig02.jpg" /> 
Figure 2: General strategy used to assess the effects of mollusk extracts on proteolytic activity, growth, and virulence factor production in Cryptococcus neoformans. DIC = differential interference contrast microscopy. Optical density measurement indicated with the wavelength in nanometers (nm). Created with BioRender.com.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64540/64540fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64540/64540fig03.jpg" /> 
Figure 3: Representative results of the effects of mollusk protein extracts on the proteolytic activity of subtilisin A. (A) Cipangopaludina chinensis crude extracts. (B) C. chinensis clarified extracts. Each point represents the average of three replicates. Bars indicate standard deviation. IC50 = half-maximal inhibitory concentration. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64540/64540fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64540/64540fig04.jpg" /> 
Figure 4: Representative results of the effects of C. chinensis extracts on virulence factor production in C. neoformans. (A,B) Growth of C. neoformans at 37 &#176;C in the presence of crude and clarified&#160;C. chinensis extracts, respectively. (C) DIC microscopy images of C. neoformans showing capsule production in the presence of crude (50 &#181;g/mL) and clarified (40 &#181;g/mL) extracts. Scale bar = 10 &#181;m. (D) Melanin production of C. neoformans in the presence of crude (440 &#181;g/mL) and clarified (410 &#181;g/mL) extracts at 30 &#176;C and 37 &#176;C. (E,F) Relative biofilm formation of C. neoformans in the presence of crude and clarified extracts, respectively.&#160;For statistical analysis, a one-way ANOVA test and a Dunnett&#39;s multiple comparison test were performed. *p &#60; 0.05; **p &#60; 0.01; and ****p &#60; 0.0001. Each value corresponds to an average of at least five biological replicates and two technical replicates. Error bars indicate standard deviation. Melanin images shown are representative of three biological replicates and two technical replicates. Capsule images shown are representative of 40-50 cells per condition. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64540/64540fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Assessing the Putative Anticryptococcal Properties of Crude and Clarified Extracts from Mollusks at JoVE.com

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

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.

Cryptococcus neoformans is an encapsulated human fungal pathogen with a global distribution that primarily infects immunocompromised individuals. The ...

assessing-putative-anticryptococcal-properties-crude-clarified

Nanyang Technological University

Research

JoVE Journal

Biology

1.0K Views.  University of Guelph. 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.

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

Passive Administration of Monoclonal Antibodies Against H. capsulatum and Others Fungal Pathogens

The purpose of the use of this methodology is 1) to advance our capacity to protect individuals with antibody or vaccine for preventing or treating histoplasmosis caused by the fungus Histoplasma capsulatum and 2) to examine the role of virulence factors as target for therapy. To generate mAbs, mice are immunized, the immune responses are assessed using a solid phase ELISA system developed in our laboratory, and the best responder mice are selected for isolation of splenocytes for fusion with hybridoma cells. C57BL/6 mice have been extensively used to study H. capsulatum pathogenesis and provide the best model for obtaining the data required. In order to assess the role of the mAbs in infection, mice are intraperitoneally administered with either mAb to H. capsulatum or isotype matched control mAb and then infected by either intravenous (i.v.), intraperitoneal (i.p.), or intranasal (i.n.) routes. In the scientific literature, efficacy of mAbs for fungal infections in mice relies on mortality as an end point, in conjunction with colony formin units (CFU) assessments at earlier time points. Survival (time to death) studies are necessary as they best represent human disease. Thus, efficacy of our intervention would not adequately be established without survival curves. This is also true for establishing efficacy of vaccine or testing of mutants for virulence. With histoplasmosis, the mice often go from being energetic to dead over several hours. The capacity of an intervention such as the administration of a mAb may initially protect an animal from disease, but the disease can relapse which would not be realized in short CFU experiments. In addition to survival and fungal burden assays, we examine the inflammatory responses to infection (histology, cellular recruitment, cytokine responses). For survival/time to death experiments, the mice are infected and monitored at least twice daily for signs of morbidity. To assess fungal burden, histopathology, and cytokine responses, the mice are euthanized at various times after infection. Animal experiments are performed according to the guidelines of the Institute for Animal Studies of the Albert Einstein College of Medicine.

Passive Administration of Monoclonal Antibodies Against H. capsulatum and Others Fungal Pathogens

C57BL/6 mice have been used to study Hc pathogenesis and provide the best model. We are exploring the potential benefits of humoral immunity against this fungus and generated several mAbs [to histone H2B and a heat shock protein 60kDa] that we tested for their protective efficacy after intraperitoneal administration.

The purpose of the use of this methodology is 1) to advance our capacity to protect individuals with antibody or vaccine for preventing or treating ...

Immunology and Infection

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are able to replicate in the deep lung. Phagocytosis of Cryptococcus by macrophages is one of the ways that the disease is able to spread into the central nervous system to cause lethal meningoencephalitis. Therefore, study of the association between Cryptococcus and macrophages is important to understanding the progression of the infection. The present study describes a step-by-step protocol to study macrophage infectivity by C. neoformansin vitro. Using this protocol, the role of host sterols on host-pathogen interactions is studied. Different concentrations of methyl--cyclodextrin (MCD) were used to deplete cholesterol from murine reticulum sarcoma macrophage-like cell line J774A.1. Cholesterol depletion was confirmed and quantified using both a commercially available cholesterol quantification kit and thin layer chromatography. Cholesterol depleted cells were activated using Lipopolysacharide (LPS) and Interferon gamma (IFN&#947;) and infected with antibody-opsonized Cryptococcus neoformans wild-type H99 cells at an effector-to-target ratio of 1:1. Infected cells were monitored after 2 hr of incubation with C. neoformans and their phagocytic index was calculated. Cholesterol depletion resulted in a significant reduction in the phagocytic index. The presented protocols offer a convenient method to mimic the initiation of the infection process in a laboratory environment and study the role of host lipid composition on infectivity.

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

In this article, a protocol for infection of macrophages with Cryptococcus neoformans is described. Also, a method for sterol depletion from the macrophages is explained. These protocols provide a guide to study fungal infections in vitro and examine the role of sterols in such infections.

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are ...

Extraction of Plant-based Capsules for Microencapsulation Applications

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin exine capsules (SECs) are obtained when spores or pollen are processed so as to remove the internal sporoplasmic contents. The resulting hollow microcapsules exhibit a high degree of micromeritic uniformity and retain intricate microstructural features related to the particular plant species. Herein, we demonstrate a streamlined process for the production of SECs from Lycopodium clavatum spores and for the loading of hydrophilic compounds into these SECs. The current SEC isolation procedure has been recently optimized to significantly reduce the processing requirements which are conventionally used in SEC isolation, and to ensure the production of intact microcapsules. Natural L. clavatum spores are defatted with acetone, treated with phosphoric acid, and extensively washed to remove sporoplasmic contents. After acetone defatting, a single processing step using 85% phosphoric acid has been shown to remove all sporoplasmic contents. By limiting the acid processing time to 30 hr, it is possible to isolate clean SECs and avoid SEC fracturing, which has been shown to occur with prolonged processing time. Extensive washing with water, dilute acids, dilute bases, and solvents ensures that all sporoplasmic material and chemical residues are adequately removed. The vacuum loading technique is utilized to load a model protein (Bovine Serum Albumin) as a representative hydrophilic compound. Vacuum loading provides a simple technique to load various compounds without the need for harsh solvents or undesirable chemicals which are often required in other microencapsulation protocols. Based on these isolation and loading protocols, SECs provide a promising material for use in a diverse range of microencapsulation applications, such as, therapeutics, foods, cosmetics, and personal care products.

This protocol/manuscript describes a streamlined process for the production of sporopollenin exine capsules (SECs) from Lycopodium clavatum spores and for the loading of hydrophilic compounds into these SECs.

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin ...

Bioengineering

Identification of Hemolytic and Phospholipase Activity in Crude Extracts from Sea Anemones by Straightforward Bioassays

Sea anemone venom composition includes polypeptide and non-proteins molecules. Cytolytic components have a high biotechnological and biomedical potential for designing new molecular tools. Sea anemone venom locates in glandular cells from ectoderm and sub-cellular structures called nematocysts, both of which are distributed throughout the sea anemone body. This characteristic implies challenges because the cells and nematocyst must be lysed to release the venom components with other non-toxic molecules. Therefore, first, the venom is derived from a crude extract (mixture of different and diverse molecules and tissue debris). The next step is to detect polypeptides with specific bioactivities. Here, we describe an efficient strategy to obtain the sea anemone crude extract and bioassay to identify the presence of cytolysins. The first step involves inexpensive and straightforward techniques (stirred and freeze-thaw cycle) to release cytolysins. We obtained the highest cytolytic activity and protein (~500 mg of protein from 20 g of dry weight). Next, the polypeptide complexity of the extract was analyzed by SDS-PAGE gel detecting proteins with molecular weights between 10 kDa and 250 kDa. In the hemolytic assay, we used sheep red blood cells and determined HU50 (11.1 &plusmn; 0.3 &#181;g/mL). In contrast, the presence of phospholipases in the crude extract was determined using egg yolk as a substrate in a solid medium with agarose. Overall, this study uses an efficient and inexpensive protocol to prepare the crude extract and applies replicable bioassays to identify cytolysins, molecules with biotechnological and biomedical interests.

Here, we describe a protocol to obtain crude venom extract from sea anemone and detect its hemolytic and phospholipase activity.

Sea anemone venom composition includes polypeptide and non-proteins molecules. Cytolytic components have a high biotechnological and biomedical potential ...

Biochemistry

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in the caseous cores of cavities and lesions is especially crucial because they harbor subpopulations of drug-tolerant bacteria also commonly referred to as persisters. Existing methods for the measurement of drug penetration in tuberculosis lesions involve costly and time-consuming in vivo pharmacokinetic studies coupled to bioanalytical or imaging techniques. The in vitro measurement of drug binding to caseum macromolecules was proposed as an alternative to such techniques since this binding hinders the passive diffusion of drug molecules through caseum. Rapid equilibrium dialysis is a fast and reliable system for performing plasma protein and tissue binding studies. In this protocol, we used a rapid equilibrium dialysis (RED) device to measure drug binding to homogenates of caseum that is excised from the lesions and cavities of tuberculosis-infected rabbits. The protocol also describes how to generate a surrogate matrix from lipid loaded THP-1 macrophages to use in place of caseum. This caseum/surrogate binding assay is an important tool in tuberculosis drug discovery and can be adapted to help study drug distribution in lesions or abscesses caused by other diseases.

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

Here we describe a rapid equilibrium dialysis (RED) method to measure drug binding to caseum from pulmonary tuberculosis lesions and cavities. The protocol is also used with a foamy macrophage-derived matrix that is an effective surrogate to caseum.

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in ...

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for macrophages. This predatory organism, similar to macrophages, employs phagocytosis to kill internalized cells. With the aid of a confocal laser-scanning microscope, images depicting interactive moments between cryptococcal cells and amoeba are captured. The resolution power of the electron microscope also helps to reveal the ultrastructural detail of cryptococcal cells when trapped inside the amoeba food vacuole. Since phagocytosis is a continuous process, quantitative data is then integrated in the analysis to explain what happens at the timepoint when an image is captured. To be specific, relative fluorescence units are read in order to quantify the efficiency of amoeba in internalizing cryptococcal cells. For this purpose, cryptococcal cells are stained with a dye that makes them fluoresce once trapped inside the acidic environment of the food vacuole. When used together, information gathered through such techniques can provide critical information to help draw conclusions on the behavior and fate of cells when internalized by amoeba and, possibly, by other phagocytic cells.

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

This paper details a protocol for preparing a co-culture of cryptococcal cells and amoebae that is studied using still, fluorescent images and high-resolution transmission electron microscope images. Illustrated here is how quantitative data can complement such qualitative information.

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for ...

High-Throughput Screening Assay for Mycobacterium abscessus Using Dual Reporters

Assay Development for High-Throughput Drug Screening Against Mycobacteria

Mycobacterium abscessus (Mab) infections are challenging to treat due to high intrinsic drug resistance, comparable to multidrug-resistant tuberculosis. Treatments are extremely ineffective and based on a multi-drug regimen, resulting in low patient compliance. Consequently, the scientific community is urged to identify new and effective drugs to treat these infections. One of the strategies employed to this end is drug repurposing - the process of identifying new therapeutic opportunities for existing drugs in the market, circumventing the time required to establish pharmacokinetic and safety profiles of new drugs. With most studies on drug development against Mab relying on traditional and time-consuming methods, an assay for high-throughput drug screening was developed against mycobacteria using an in house developed double-reporter strain of Mab. Using liquid-handling robotics, automated microscopy, and analysis, alongside in house developed double reporter strains, bacterial viability can be rapidly measured using two different readouts, luminescence and fluorescence, without adding reagents or performing any extra steps. This reduces time and variability between assays, a major advantage for high-throughput screenings. The described protocol was validated by screening a library of 1280 compounds. The obtained results were corroborated by the literature, with efficient detection of active compounds. Thus, this work fulfilled the aim of supplying the field with a new tool to help fight this extremely drug-resistant bacteria.

Author Spotlight: Scalable Drug Screening Protocol for Efficient Discovery of M. abscessus Treatments

This study developed an assay for high-throughput screening against Mycobacterium abscessus with a&#160;recently created double reporter strain, using fluorescence and luminescence to assess bacterial viability quickly. This protocol will be relevant for researchers aiming to screen new drugs against this drug-resistant bacteria.

Mycobacterium abscessus (Mab) infections are challenging to treat due to high intrinsic drug resistance, comparable to multidrug-resistant tuberculosis. ...

Isolation and Purification of Fungal &#946;-Glucan as an Immunotherapy Strategy for Glioblastoma

One of the biggest challenges in developing effective therapies against glioblastoma is overcoming the strong immune suppression within the tumor microenvironment. Immunotherapy has emerged as an effective strategy to turn the immune system response against tumor cells. Glioma-associated macrophages and microglia (GAMs) are major drivers of such anti-inflammatory scenarios. Therefore, enhancing the anti-cancerous response in GAMs may represent a potential co-adjuvant therapy to treat glioblastoma patients. In that vein, fungal &#946;-glucan molecules have long been known as potent immune modulators. Their ability to stimulate the innate immune activity and improve treatment response has been described. Those modulating features are partly attributed to their ability to bind to pattern recognition receptors, which, interestingly, are greatly expressed in GAMs. Thus, this work is focused on the isolation, purification, and subsequent use of fungal &#946;-glucans to enhance the tumoricidal response of microglia against glioblastoma cells. The mouse glioblastoma (GL261) and microglia (BV-2) cell lines are used to test the immunomodulatory properties of four different fungal &#946;-glucans extracted from mushrooms heavily used in the current biopharmaceutical industry: Pleurotus ostreatus, Pleurotus djamor, Hericium erinaceus,&#160;and Ganoderma lucidum. To test these compounds, co-stimulation assays were performed to measure the effect of a pre-activated microglia-conditioned medium on the proliferation and apoptosis activation in glioblastoma cells.

The present protocol describes the purification steps and subsequent studies of four different fungal &#946;-glucans as potential immunomodulatory molecules that enhance the anti-tumoral properties of microglia against glioblastoma cells.

One of the biggest challenges in developing effective therapies against glioblastoma is overcoming the strong immune suppression within the tumor ...

Neuroscience

Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds

Fungal infections have become an important medical condition in the last decades, but the number of available antifungal drugs is limited. In this scenario, the search for new antifungal drugs is necessary. The protocol reported here details a method to screen peptides for their antifungal properties. It is based on the broth microdilution susceptibility test from the Clinical and Laboratory Standards Institute (CLSI) M27-A3 guidelines with modifications to suit the research of antimicrobial peptides as potential new antifungals. This protocol describes a functional assay to evaluate the activity of antifungal compounds and may be easily modified to suit any particular class of molecules under investigation. Since the assays are performed in 96-well plates using small volumes, a large-scale screening can be completed in a short amount of time, especially if carried out in an automation setting. This procedure illustrates how a standardized and adjustable clinical protocol can help the bench-work pursuit of new molecules to improve the therapy of fungal diseases.

Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds

An easy and adaptable broth microdilution method for screening antifungal compounds and extracts.

Fungal infections have become an important medical condition in the last decades, but the number of available antifungal drugs is limited. In this ...

Rapid Freezing using Sandwich Freezing Device for Good Ultrastructural Preservation of Biological Specimens in Electron Microscopy

Chemical fixation has been used for observing the ultrastructure of cells and tissues. However, this method does not adequately preserve the ultrastructure of cells; artifacts and extraction of cell contents are usually observed. Rapid freezing is a better alternative for the preservation of cell structure. Sandwich freezing of living yeast or bacteria followed by freeze-substitution has been used for observing the exquisite natural ultrastructure of cells. Recently, sandwich freezing of glutaraldehyde-fixed cultured cells or human tissues has also been used to reveal the ultrastructure of cells and tissues. 
These studies have thus far been carried out with a handmade sandwich freezing device, and applications to studies in other laboratories have been limited. A new sandwich freezing device has recently been fabricated and is now commercially available. The present paper shows how to use the sandwich freezing device for rapid freezing of biological specimens, including bacteria, yeast, cultured cells, isolated cells, animal and human tissues, and viruses. Also shown is the preparation of specimens for ultrathin sectioning after rapid freezing and procedures for freeze-substitution, resin embedding, trimming of blocks, cutting of ultrathin sections, recovering of sections, staining, and covering of grids with support films.

Here we show how to use the sandwich freezing device for rapid freezing of biological specimens, including bacteria, yeast, cultured cells, isolated cells, animal and human tissues, and viruses. We also show how to prepare specimens for ultrathin sectioning after rapid freezing.

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

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Rapid Freezing using Sandwich Freezing Device for Good Ultrastructural Preservation of Biological Specimens in Electron Microscopy