Phiri, A.M., De Pomerai, D.I., Buttle, D.J. orcid.org/0000-0003-2767-0082 et al. (1 more author) (2017) A novel assay for the detection of anthelmintic activity mediated by cuticular damage to nematodes: validation on Caenorhabditis elegans exposed to cysteine proteinases. Parasitology, 144 (5). pp. 583-593. ISSN 0031-1820
Abstract
Plant cysteine proteinases (CPs) from Carica papaya kill parasitic and free-living nematodes in vitro by hydrolysis of the worm cuticle, a mechanism that is different to all commercially available synthetic anthelmintics. We have developed a cheap and effective, rapid-throughput Caenorhabditis elegans-based assay for screening plant CP extracts for anthelmintic activity targeting cuticular integrity. The assay exploits colorimetric methodology for assessment of cuticular damage, and is based on the ability of viable cells to incorporate and bind Neutral red dye within lysosomes and to release the dye when damaged. Living worms are pre-stained with the dye, exposed to CPs and then leakage of the dye through the damaged cuticle is quantified by spectrophotometry. In contrast to motility assays and semi-subjective interpretation of microscopical images, this colorimetric assay is independent of observer bias. Our assay was applied to a series of C. elegans bus mutant strains with leaky cuticles and to cystatin knockout mutants. At ambient temperature and over 0.5–24 h, both bus mutants and the cystatin knockouts were highly susceptible to CPs, whereas wild-type Bristol N2 worms were essentially unstained by Neutral red and unaffected by CPs, providing validation for the utility of this assay.
Metadata
Item Type: | Article |
---|---|
Authors/Creators: |
|
Copyright, Publisher and Additional Information: | © 2017 Cambridge University Press. |
Keywords: | Caenorhabditis elegans; Neutral red; cuticular damage; cysteine proteinases |
Dates: |
|
Institution: | The University of Sheffield |
Academic Units: | The University of Sheffield > Faculty of Medicine, Dentistry and Health (Sheffield) > Department of Infection, Immunity and Cardiovascular Disease |
Depositing User: | Symplectic Sheffield |
Date Deposited: | 31 Aug 2017 09:37 |
Last Modified: | 31 Aug 2017 09:38 |
Published Version: | https://doi.org/10.1017/S0031182016002353 |
Status: | Published |
Publisher: | Cambridge University Press |
Refereed: | Yes |
Identification Number: | 10.1017/S0031182016002353 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:115264 |