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. Generall...

<ol>
	<li>Derek, J., Sloan, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryptococcal+meningitis:+Epidemiology+and+therapeutic+options.">Cryptococcal meningitis: Epidemiology and therapeutic options.</a> Clinical Epidemiology. 6, 169-182 (2014).</li><li>Rajasingham, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+global+burden+of+HIV-associated+cryptococcal+infection+in+adults+in+2020:+a+modelling+analysis.">The global burden of HIV-associated cryptococcal infection in adults in 2020: a modelling analysis.</a> The Lancet Infectious Diseases. , (2022).</li><li>Mourad, A., Perfect, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Present+and+future+therapy+of+Cryptococcus+infections.">Present and future therapy of Cryptococcus infections.</a> Journal of Fungi. 4 (3), 79 (2018).</li><li>Bermas, A., Geddes-McAlister, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combatting+the+evolution+of+antifungal+resistance+in+Cryptococcus+neoformans.">Combatting the evolution of antifungal resistance in Cryptococcus neoformans.</a> Molecular Microbiology. 114 (5), 721-734 (2020).</li><li>Geddes-McAlister, J., Shapiro, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+pathogens,+new+tricks:+Emerging,+drug-resistant+fungal+pathogens+and+future+prospects+for+antifungal+therapeutics.">New pathogens, new tricks: Emerging, drug-resistant fungal pathogens and future prospects for antifungal therapeutics.</a> Annals of the New York Academy of Sciences. 1435 (1), 57-78 (2019).</li><li>Kronstad, J. W., Hu, G., Choi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cAMP/protein+kinase+A+pathway+and+virulence+in+Cryptococcus+neoformans.">The cAMP/protein kinase A pathway and virulence in Cryptococcus neoformans.</a> Mycobiology. 39 (3), 143-150 (2018).</li><li>Olszewski, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urease+expression+by+Cryptococcus+neoformans+promotes+microvascular+sequestration,+thereby+enhancing+central+nervous+system+invasion.">Urease expression by Cryptococcus neoformans promotes microvascular sequestration, thereby enhancing central nervous system invasion.</a> The American Journal of Pathology. 164 (5), 1761-1771 (2004).</li><li>Shi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+imaging+of+trapping+and+urease-dependent+transmigration+of+Cryptococcus+neoformans+in+mouse+brain.">Real-time imaging of trapping and urease-dependent transmigration of Cryptococcus neoformans in mouse brain.</a> The Journal of Clinical Investigation. 120 (5), 1683-1693 (2010).</li><li>Vu, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasion+of+the+central+nervous+system+by+Cryptococcus+neoformans+requires+a+secreted+fungal+metalloprotease.">Invasion of the central nervous system by Cryptococcus neoformans requires a secreted fungal metalloprotease.</a> mBio. 5 (3), 01101-01114 (2014).</li><li>Gutierrez-Gongora, D., Geddes-McAlister, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+naturally-sourced+protease+inhibitors+to+new+treatments+for+fungal+infections.">From naturally-sourced protease inhibitors to new treatments for fungal infections.</a> Journal of Fungi. 7 (12), 1016 (2021).</li><li>Nakao, Y., Fusetani, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzyme+inhibitors+from+marine+invertebrates.">Enzyme inhibitors from marine invertebrates.</a> Journal of Natural Products. 70 (4), 689-710 (2007).</li><li>Reytor, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+of+protease+inhibitory+activity+in+extracts+of+five+Ascidian+species+from+Cuban+coasts.">Screening of protease inhibitory activity in extracts of five Ascidian species from Cuban coasts.</a> Biotecnologia Aplicada. 28 (2), 77-82 (2011).</li><li>Gonz&#225;lez, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+of+protease+inhibitory+activity+in+aqueous+extracts+of+marine+invertebrates+from+Cuban+coast.">Screening of protease inhibitory activity in aqueous extracts of marine invertebrates from Cuban coast.</a> American Journal of Analytical Chemistry. 7 (4), 319-331 (2016).</li><li>Brown, D. S., Werger, M. J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Freshwater+molluscs.">Freshwater molluscs.</a> Biogeography and Ecology of Southern Africa. , 1153-1180 (1978).</li><li>Forsyth, R. G., Oldham, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Terrestrial+molluscs+from+the+Ontario+Far+North.">Terrestrial molluscs from the Ontario Far North.</a> Check List. 12 (3), 1-51 (2016).</li><li>Eigenheer, R. A., Lee, Y. J., Blumwald, E., Phinney, B. S., Gelli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+glycosylphosphatidylinositol-anchored+mannoproteins+and+proteases+of+Cryptococcus+neoformans.">Extracellular glycosylphosphatidylinositol-anchored mannoproteins and proteases of Cryptococcus neoformans.</a> FEMS Yeast Research. 7 (4), 499-510 (2007).</li><li>Homer, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+action+of+a+secreted+peptide+required+for+fungal+virulence.">Intracellular action of a secreted peptide required for fungal virulence.</a> Cell Host &amp; Microbe. 19 (6), 849-864 (2016).</li><li>Clarke, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+activity+and+genetic+profiling+of+secreted+peptidases+in+Cryptococcus+neoformans+reveals+an+aspartyl+peptidase+required+for+low+pH+survival+and+virulence.">Integrated activity and genetic profiling of secreted peptidases in Cryptococcus neoformans reveals an aspartyl peptidase required for low pH survival and virulence.</a> PLoS Pathogens. 12 (12), 1006051 (2016).</li><li>Copeland, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Evaluation of Enzyme Inhibitors in Drug Discovery: A Guide for Medicinal Chemists and Pharmacologists. , (2013).</li><li>Collins, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ImageJ+for+microscopy.">ImageJ for microscopy.</a> Biotechniques. 43, 25-30 (2007).</li><li>Rawlings, N. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MEROPS+database+of+proteolytic+enzymes,+their+substrates+and+inhibitors+in+2017+and+a+comparison+with+peptidases+in+the+PANTHER+database.">The MEROPS database of proteolytic enzymes, their substrates and inhibitors in 2017 and a comparison with peptidases in the PANTHER database.</a> Nucleic Acids Research. 46, 624-632 (2018).</li><li>Gutierrez-Gongora, D., Geddes-McAlister, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptidases:+Promising+antifungal+targets+of+the+human+fungal+pathogen,+Cryptococcus+neoformans.">Peptidases: Promising antifungal targets of the human fungal pathogen, Cryptococcus neoformans.</a> Facets. 7 (1), 319-342 (2022).</li><li>Martinez, L. R., Casadevall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Susceptibility+of+Cryptococcus+neoformans+biofilms+to+antifungal+agents+in+vitro.">Susceptibility of Cryptococcus neoformans biofilms to antifungal agents in vitro.</a> Antimicrobial Agents and Chemotherapy. 50 (3), 1021-1033 (2006).</li><li>Culp, E., Wright, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+proteases,+untapped+antimicrobial+drug+targets.">Bacterial proteases, untapped antimicrobial drug targets.</a> Journal of Antibiotics. 70 (4), 366-377 (2017).</li><li>Ruocco, N., Costantini, S., Palumbo, F., Costantini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marine+sponges+and+bacteria+as+challenging+sources+of+enzyme+inhibitors+for+pharmacological+applications.">Marine sponges and bacteria as challenging sources of enzyme inhibitors for pharmacological applications.</a> Mar Drugs. 15 (6), 173 (2017).</li><li>Costa, H. P. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=JcTI-I:+A+novel+trypsin+inhibitor+from+Jatropha+curcas+seed+cake+with+potential+for+bacterial+infection+treatment.">JcTI-I: A novel trypsin inhibitor from Jatropha curcas seed cake with potential for bacterial infection treatment.</a> Frontiers in Microbiology. 5, 5 (2014).</li></ol>

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 ...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.2 &#956;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>CO2 Incubator (Static)</td>
			<td>SANYO</td>
			<td>Not available</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryptococcus neoformans 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 (MgSO4.7 H2O)</td>
			<td>Honeywell</td>
			<td>M1880-500G</td>
			<td>CAS #: 10034-99-8&#160;</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 (KH2PO4)</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&#160;</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 representativ...

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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 ...

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.

assessing-putative-anticryptococcal-properties-crude-clarified

Research

JoVE Journal

Biology

1.0K visualizações.  University of Guelph. Este protocolo permite o isolamento de compostos de moluscos com propriedades anti-criptocócicas. Esta técnica oferece uma abordagem imparcial para identificar e isolar compostos de moluscos com propriedades potenciais, incluindo matar os patógenos fúngicos humanos. Existem outros compostos, como os inibidores da protease do HIV, sendo usados para tratar pacientes com HIV.No entanto, a expansão para outros patógenos, bem como a investigação de fontes naturais, representam novos...