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Developing a rapid throughput screen for detection of nematicidal activity of plant cysteine proteinases: the role of Caenorhabditis elegans cystatins

Published online by Cambridge University Press:  04 September 2013

A. M. PHIRI ,
D. DE POMERAI ,
D. J. BUTTLE  and
J. M. B. BEHNKE
A. M. PHIRI
Affiliation:
School of Biology, University of Nottingham, Nottingham, NG7 2RD, UK School of Veterinary Medicine, University of Zambia, Lusaka, Zambia
D. DE POMERAI
Affiliation:
School of Biology, University of Nottingham, Nottingham, NG7 2RD, UK
D. J. BUTTLE
Affiliation:
Department of Infection and Immunity, University of Sheffield Medical School, Sheffield, S10 2RX, UK
J. M. B. BEHNKE*
Affiliation:
School of Biology, University of Nottingham, Nottingham, NG7 2RD, UK
*
* Corresponding author: School of Biology, University of Nottingham, Nottingham NG7 2RD, UK. E-mail: [email protected]
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Summary

Plant cysteine proteinases (CPs) from papaya (Carica papaya) are capable of killing parasitic nematode worms in vitro and have been shown to possess anthelmintic effects in vivo. The acute damage reported in gastrointestinal parasites has not been found in free-living nematodes such as Caenorhabditis elegans nor among the free-living stages of parasitic nematodes. This apparent difference in susceptibility might be the result of active production of cysteine proteinase inhibitors (such as cystatins) by the free-living stages or species. To test this possibility, a supernatant extract of refined papaya latex (PLS) with known active enzyme content was used. The effect on wild-type (Bristol N2) and cystatin null mutant (cpi-1−/− and cpi-2−/−) C. elegans was concentration-, temperature- and time-dependent. Cysteine proteinases digested the worm cuticle leading to release of internal structures and consequent death. Both cystatin null mutant strains were highly susceptible to PLS attack irrespective of the temperature and concentration of exposure, whereas wild-type N2 worms were generally resistant but far more susceptible to attack at low temperatures. PLS was able to induce elevated cpi-1 and cpi-2 cystatin expression. We conclude that wild-type C. elegans deploy cystatins CPI-1 and CPI-2 to resist CP attack. The results suggest that the cpi-1 or cpi-2 null mutants (or a double mutant combination of the two) could provide a cheap and effective rapid throughput C. elegans-based assay for screening plant CP extracts for anthelmintic activity.

Keywords

Plant cysteine proteinasespapaya latex supernatantanthelminticCaenorhabditis eleganscystatins
Type
Research Article
Information
Parasitology , Volume 141 , Issue 2 , February 2014 , pp. 164 - 180
Copyright
Copyright © Cambridge University Press 2013 

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