Victor S Njom1,2, Tim Winks1,3, Oumu Diallo1,3, Ann Lowe4, Jerzy Behnke4, Mark J Dickman5, Ian Duce4, Iain Johnstone6, David J Buttle7. 1. Department of Infection, Immunity and Cardiovascular Disease, The University of Sheffield Medical School, Beech Hill Road, Sheffield, S10 2RX, UK. 2. Department of Applied Biology and Biotechnology, Enugu State University of Science and Technology, Enugu, 1660, PMB, Nigeria. 3. Department of Biosciences and Chemistry, Sheffield Hallam University, Sheffield, S1 1WB, UK. 4. School of Life Sciences, University of Nottingham, University Park, Nottingham, NG7 2RD, UK. 5. Department of Chemical and Biological Engineering, ChELSI Institute, The University of Sheffield, Sheffield, S1 3JD, UK. 6. Department of Life Sciences and Biomolecular Sciences, University of Glasgow, Glasgow, UK. 7. Department of Infection, Immunity and Cardiovascular Disease, The University of Sheffield Medical School, Beech Hill Road, Sheffield, S10 2RX, UK. d.j.buttle@sheffield.ac.uk.
Abstract
BACKGROUND: Plant-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. METHODS: This 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. RESULTS: Immunohistochemistry 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. CONCLUSIONS: Scanning 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.
BACKGROUND: Plant-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. METHODS: This study followed the course of nematode cuticle disruption using immunohistochemistry, scanning electron microscopy and proteomics, using a free-living nematode, Caenorhabditis elegans and the murineGI nematode Heligmosomoides bakeri (H. polygyrus) as target organisms. RESULTS: Immunohistochemistry indicated that DPY-7collagen 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. CONCLUSIONS: Scanning 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.
Entities:
Keywords:
Anthelmintic; C. elegans; Cuticle; H. bakeri; Imaging; Immunohistochemistry; Papain; Papaya latex; Proteomics
Authors: Marco Albonico; Laura Rinaldi; Sonia Sciascia; Maria E Morgoglione; Monica Piemonte; Maria P Maurelli; Vincenzo Musella; Jürg Utzinger; Said M Ali; Shaali M Ame; Giuseppe Cringoli Journal: Trans R Soc Trop Med Hyg Date: 2013-08 Impact factor: 2.184
Authors: W A Edens; L Sharling; G Cheng; R Shapira; J M Kinkade; T Lee; H A Edens; X Tang; C Sullards; D B Flaherty; G M Benian; J D Lambeth Journal: J Cell Biol Date: 2001-08-20 Impact factor: 10.539