Abstract
BackgroundPlant-derived cysteine proteinases of the papain family (CPs) attack nematodes by digesting the cuticle, leading to rupture and death of the worm. The nematode cuticle is composed of collagens and cuticlins, but the specific molecular target(s) for the proteinases have yet to be identified.MethodsThis study followed the course of nematode cuticle disruption using immunohistochemistry, scanning electron microscopy and proteomics, using a free-living nematode, Caenorhabditis elegans and the murine GI nematode Heligmosomoides bakeri (H. polygyrus) as target organisms.ResultsImmunohistochemistry indicated that DPY-7 collagen is a target for CPs on the cuticle of C. elegans. The time course of loss of DPY-7 from the cuticle allowed us to use it to visualise the process of cuticle disruption. There was a marked difference in the time course of damage to the cuticles of the two species of nematode, with H. bakeri being more rapidly hydrolysed. In general, the CPs’ mode of attack on the nematode cuticle was by degrading the structural proteins, leading to loss of integrity of the cuticle, and finally death of the nematode. Proteomic analysis failed conclusively to identify structural targets for CPs, but preliminary data suggested that COL-87 and CUT-19 may be important targets for the CPs, the digestion of which may contribute to cuticle disruption and death of the worm. Cuticle globin was also identified as a cuticular target. The presence of more than one target protein may slow the development of resistance against this new class of anthelmintic.ConclusionsScanning electron microscopy and immunohistochemistry allowed the process of disruption of the cuticle to be followed with time. Cuticle collagens and cuticlins are molecular targets for plant cysteine proteinases. However, the presence of tyrosine cross-links in nematode cuticle proteins seriously impeded protein identification by proteomic analyses. Multiple cuticle targets exist, probably making resistance to this new anthelmintic slow to develop.Graphical
Highlights
Plant-derived cysteine proteinases of the papain family (CPs) attack nematodes by digesting the cuticle, leading to rupture and death of the worm
The C. elegans strains were cultured on plates of nematode growth medium (NGM) agar spread with an Escherichia coli (OP50) lawn
Effects of CPs on the DPY‐7 cuticle collagen of C. elegans Figure 1 illustrates the immunochemical staining of the cuticles of C. elegans after incubation with 1 μM papain
Summary
Plant-derived cysteine proteinases of the papain family (CPs) attack nematodes by digesting the cuticle, leading to rupture and death of the worm. The intensive use of drugs and the dependence of treatment of nematode infection on only a few drugs with similar modes of action have put pressure on the drug candidates with resulting loss of potency due to development of resistance by target nematodes [7,8,9]. In the GI tract they are not exposed to high concentrations of cysteine proteinases (CPs) and do not experience selective pressure to develop inhibitors to this class of proteinase in order to survive in the gut. Their protective cuticle may be sensitive to digestion by this class of proteinases. Tropical countries have relied to some extent on plant extracts for the treatment of nematode infections [19], including extracts that contain CPs [20,21,22]
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