Mechanism of attack and molecular target(s) for plant cysteine proteinases on cuticles of parasitic nematodes and C. elegans

• Main Author: Njom, Victor S.
• Other Authors: Buttle, David J. ; Dickman, Mark J. ; Duce, Ian
• Published: University of Sheffield 2016
• Subjects: 632
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.696040

Parasitic nematodes cause enormous public health, agricultural and economic problems worldwide, as pathogens of humans, livestock and crops. Their impact is increasing due to lack of full efficacy of current anthelmintics and development of resistance by the nematodes, therefore there is an urgent need for an alternative. Plant cysteine proteinases (CP) from papaya (“papain”), fig (ficin), and pineapple (bromelain) have been shown to be effective on gastrointestinal (GI) nematodes. The enzymes attack by digesting the cuticle leading to rupture and death of the nematode. The nematode cuticle is composed of collagens and cuticlins but the specific molecular target(s) of the proteinases have yet to be identified. There are about 160 collagen genes and 8 identified functional cuticlins genes in the C. elegans genome. This study identified some of the molecular targets and thereby began to define the mechanism of action of this new class of anthelmintics, through imaging, proteomic, immunohistochemical, and automated motility assay techniques using a free-living nematode, Caenorhabditis elegans and murine GI nematode Heligmosomoides bakeri as target organisms. Through proteomic approach, Col-87 and Cut-19 were identified as CP targets on C. elegans and H. bakeri cuticles respectively, cuticle globin and chemosensory protein that localise in the cuticle were also target for CPs. Immunohistochemical staining indicated that DPY-7 collagen is also a target for CPs on the cuticle of C. elegans. Imaging showed that there was a marked difference in degree of damage done to the two model worms used. H. bakeri was most affected by CPs as its entire cuticle was digested more rapidly when compared to the two strains of C. elegans. Motility of the three strains of C. elegans was affected by exposure to CPs, in a concentration-, time- and CP type-dependent manner. In papaya latex supernatant (PLS), there was no detectable statistically significant difference in susceptibility between wild-type and cystatin-null mutants. Papain affected the motility of worm types and was more effective than PLS. CP's mechanism of attack on the nematode cuticle is by degrading the structural proteins, leading to loss of integrity, motility and finally death of the nematode.