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Biology

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

Published: December 2nd, 2022

DOI:

10.3791/64540

1Molecular and Cellular Biology Department, University of Guelph, 2School of Environmental Sciences, 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.

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.

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 ....

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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. Generall.......

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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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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 .......

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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.

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NameCompanyCatalog NumberComments
0.2 μm FiltersVWR28145-477 (North America)
1.5 mL Tubes (Safe-Lock)Eppendorf0030120086
2 mL Tubes (Safe-Lock)Eppendorf0030120094
3,4-Dihydroxy-L-phenylalanine (L-DOPA)Sigma-AldrichD9628-5GCAS #: 59-92-7
96-well platesCostar (Corning)3370
Bullet Blender Storm 24NEXT ADVANCEBBY24M
Centrifuge 5430REppendorf5428000010
Chelex 100 ResinBioRad142-1253
CO2 Incubator (Static)SANYONot available
Cryptococcus neoformans H99ATCC208821
DIC MicroscopeOlympus
DIC Microscope softwareZeiss
DMEMCorning10-013-CV
Glucose (D-Glucose, Anhydrous, Reagent Grade)BioShopGLU501CAS #: 50-99-7
GlycineFisher ChemicalG46-1CAS #: 56-40-6
GraphPad Prism 9Dotmatics
HemocytometerVWR15170-208
HEPESSigma AldrichH3375
Magnesium sulfate heptahydrate (MgSO4.7 H2O)HoneywellM1880-500GCAS #: 10034-99-8 
PeptoneBioShopPEP403
Phosohate buffer salt pH 7.4BioShopPBS408SKU: PBS408.500
Plate reader (Synergy-H1)BioTek (Agilent)Not available
Potassium phosphate monobasic (KH2PO4)Fisher ChemicalP285-500CAS #: 7778-77-0
Subtilisin ASigma-AldrichP4860CAS #: 9014-01-01
Succinyl-Ala-Ala-Pro-Phe-p-nitroanilideSigma-Aldrich573462CAS #: 70967-97-4
Thermal bathVWR76308-834
Thiamine HydrochlorideFisher-BioreagentsBP892-100CAS #: 67-03-8
Yeast extractBioShopYEX401CAS #: 8013-01-2
Yeast nitrogen base (with Amino Acids)Sigma-AldrichY1250-250GYNB 

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