title: A novel assay for the detection of anthelmintic activity mediated by cuticular damage to nematodes: validation on Caenorhabditis elegans exposed to cysteine proteinases.
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.
authors.affiliation: School of Life Sciences, University of Nottingham,Nottingham NG7 2RD,UK.
authors.forename: A M
authors.initials: AM
authors.lastname: Phiri
authors.affiliation: School of Life Sciences, University of Nottingham,Nottingham NG7 2RD,UK.
authors.forename: D I
authors.initials: DI
authors.lastname: DE Pomerai
authors.affiliation: Department of Infection, Immunity & Cardiovascular Disease,University of Sheffield Medical School,Sheffield S10 2RX,UK.
authors.forename: D J
authors.initials: DJ
authors.lastname: Buttle
authors.affiliation: School of Life Sciences, University of Nottingham,Nottingham NG7 2RD,UK.
authors.forename: J M
authors.initials: JM
authors.lastname: Behnke
mesh_terms: D000818:Animals; D000871:Anthelmintics / Q000302:isolation & purification / Q000494:pharmacology*; D017173:Caenorhabditis elegans / Q000187:drug effects* / Q000235:genetics / Q000648:ultrastructure; D029441:Carica / Q000201:enzymology*; D015891:Cystatins / Q000235:genetics; D057056:Cysteine Proteases / Q000302:isolation & purification / Q000494:pharmacology*; D015853:Cysteine Proteinase Inhibitors; D007202:Indicators and Reagents; D009154:Mutation; D009499:Neutral Red; D010940:Plant Proteins / Q000494:pharmacology*; D015800:Protozoan Proteins / Q000235:genetics
chemical_list: D000871:Anthelmintics; D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D007202:Indicators and Reagents; D010940:Plant Proteins; D015800:Protozoan Proteins; D009499:Neutral Red; D057056:Cysteine Proteases
keywords: 
                 Caenorhabditis elegans
              ; Neutral red; cuticular damage; cysteine proteinases