A new enabling proteomics methodology to investigate membrane associated proteins from parasitic nematodes: case study using ivermectin resistant and ivermectin susceptible isolates of Caenorhabditis elegans and Haemonchus contortus.

The mechanisms involved in anthelmintic resistance (AR) are complex but a greater understanding of AR management is essential for effective and sustainable control of parasitic helminth worms in livestock. Current tests to measure AR are time consuming and can be technically problematic, gold standard diagnostics are therefore urgently required to assist in combatting the threat from drug resistant parasites. For anthelmintics such as ivermectin (IVM), target proteins may be present in the cellular membrane. As proteins usually act in complexes and not in isolation, AR may develop and be measurable in the target associated proteins present in the parasite membrane. The model nematode Caenorhabditis elegans was used to develop a sub-proteomic assay to measure protein expression differences, between IVM resistant and IVM susceptible isolates in the presence and absence of drug challenge. Evaluation of detergents including CHAPS, ASB-14, C7BzO, Triton ×100 and TBP (tributyl phosphine) determined optimal conditions for the resolution of membrane proteins in Two Dimensional Gel Electrophoresis (2DE). These sub-proteomic methodologies were then translated and evaluated using IVM-susceptible and IVM-resistant Haemonchus contortus; a pathogenic blood feeding parasitic nematode which is of global importance in livestock health, welfare and productivity. We have demonstrated the successful resolution of membrane associated proteins from both C. elegans and H. contortus isolates, using a combination of CHAPS and the zwitterionic amphiphilic surfactant ASB-14 to further support the detection of markers for AR.

The soluble proteome phenotypes of ivermectin resistant and ivermectin susceptible Haemonchus contortus females compared.

Anthelmintics in the absence of vaccines have underpinned a parasite control strategy for over 50 years. However, the continued development of anthelmintic resistance (AR) threatens this control. Measuring early AR is difficult as there many routes that resistance can arise from within multi-nematode populations operating complex metabolism capabilities coupled to different drug management pressures. There is an urgent need to identify and measure early resistance in the field situation. Proteomic profiling of expressed soluble proteins offers a new approach to reveal a drug resistant phenotype within a complex protein pattern. The hypothesis under test was that established differences in drug response phenotypes between nematode isolates can also be measured in their comparative proteomes. As a case study, proteomic differences were measured between an ivermectin resistant and susceptible adult female Haemonchus contortus. Adult H. contortus females were extracted from the abomasa of six lambs. The nematodes had been maintained in the lambs as monospecific isolates of either ivermectin susceptible or ivermectin resistant worms. Comparative analysis of the soluble proteome was completed along with immuno-proteomic analysis using pooled infection sera from the lambs. Following image analysis, spots of interest were excised and analysed by peptide mass fingerprinting and the proteins putatively identified using BLAST. Overall, a relative increase in the expression of proteins involved in the detoxification metabolic area was observed in the resistant isolate. In addition, Western blotting analysis also revealed differences in immuno-reactivity profiles between resistant and susceptible isolates. It can be concluded from this study that proteomic differences can be detectable between ivermectin susceptible and a resistant isolates of H. contortus, which could be further explored using other isolates to confirm if proteomic based fingerprinting offers molecular phenotyping or a new panel of resistance biomarkers.

Proteomic analysis of embryonic Fasciola hepatica: characterization and antigenic potential of a developmentally regulated heat shock protein.

Fasciola hepatica is responsible for human disease and economic livestock loss on a global scale. We report the first post-genomic investigation of cellular proteins expressed by embryonic F. hepatica via two-dimensional electrophoresis, image analysis and tandem mass spectrometry. Antioxidant proteins and protein chaperones are prominently expressed by embryonic F. hepatica. Molecular differences between the egg and other characterized F. hepatica lifecycle stages were noted. Furthermore, proteins expressed within liver fluke eggs differ to those isolated from the well-characterized eggs of the human blood flatworm Schistosoma mansoni were revealed. Plasticity in expression of major proteins, particularly a prominently expressed 65kDa protein cluster was seen between natural populations of embryonating F. hepatica eggs suggesting that liver fluke embryogenisis is a plastic process. Immunoblotting revealed that the abundant 65kDa protein cluster is recognised by infection sera from three F. hepatica challenged host species. Mass spectrometry and BLAST analyses demonstrated that the 65kDa antigen shows homology to egg antigens of other flatworm parasites, and is represented in a F. hepatica EST database constructed from adult fluke transcripts. EST clones encoding the egg antigen were re-sequenced, predicting two forms of the protein. Four clones predict a 312 aa polypeptide, three clones encode a putative 110 amino acid extension at the N-terminus which may be involved in protein secretion, although this extension was not expressed by natively extracted proteins. Consistent expression of alpha crystallin domains confirmed the protein to be a member of the alpha crystallin containing small heat shock protein (AC/sHSP) superfamily. AC/sHSPs are ubiquitous in nature, however, this is the first time a member of this protein superfamily has been described from F. hepatica. The antigenic AC/sHSP was named Fh-HSP35alpha based on predictions of molecular weight. Production of recombinant Fh-HSP35alpha reveals considerable mass discrepancy between native and recombinant proteins, although descriptions of other characterized flatworm AC/sHSPs, suggest that the native form is a dimer. Immunoblot analyses confirm that the recombinant protein is recognised by F. hepatica challenged hosts, but does not react with sera from non-infected animals. We discuss the potential of recombinant Fh-HSP35alpha as an egg-based diagnostic marker for liver fluke infection.

Plasticity demonstrated in the proteome of a parasitic nematode within the intestine of different host strains.

The soluble global proteome of adult nematode Heligmosomoides polygyrus (H. p.) bakeri, a hookworm laboratory model was compared for the first time in the intestines of a slow-responder mouse host strain (C57/BL10) that is known to support a primary parasite infection for many months, and rapid-responder mouse host (SWR) that is known to eliminate the nematode infection by week 6 postinfection. At week 4 postinfection, major adult nematode proteins selectively produced following establishment of infection in C57/BL10 hosts include several globin forms, calreticulin and a phosphatidylethanolamine-binding protein. The increased synthesis of forms of myosin, actin and troponin in the nematode living in the rapid-responder SWR host may relate to the attempted reorganisation or repair of the cytoskeleton and/or muscle layer in the host immune initiated, increased mucus production and smooth muscle activity within intestinal environment. Initial evidence suggests weakly antigenic forms of globins dominant in the cytosol of H. p. bakeri adults in the intestinal environment compared to their low production in a related free-living nematode. The demonstration of considerable plasticity within a parasitic nematode proteome provides a molecular basis for the previously observed phenotypic plasticity within different host environments. Proteome plasticity has relevance to the efficiency of future vaccine and drug therapy, and the continued failure of defined antigen vaccines in mammalian populations.

Epithelial innate immunity. A novel antimicrobial peptide with antiparasitic activity in the blood-sucking insect Stomoxys calcitrans.

The gut epithelium is an essential interface in insects that transmit parasites. We investigated the role that local innate immunity might have on vector competence, taking Stomoxys calcitrans as a model. S. calcitrans is sympatric with tsetse flies, feeds on many of the same vertebrate hosts, and is thus regularly exposed to the trypanosomes that cause African sleeping sickness and nagana. Despite this, S. calcitrans is not a cyclical vector of these trypanosomes. Trypanosomes develop exclusively in the lumen of digestive organs, and so epithelial immune mechanisms, and in particular antimicrobial peptides (AMPs), may be the prime determinants of the fate of an infection. To investigate why S. calcitrans is not a cyclical vector of trypanosomes, we have looked in its midgut for AMPs with trypanolytic activity. We have identified a new AMP of 42 amino acids, which we named stomoxyn, constitutively expressed and secreted exclusively in the anterior midgut of S. calcitrans. It displays an amphipathic helical structure and exhibits a broad activity spectrum affecting the growth of microorganisms. Interestingly, this AMP exhibits trypanolytic activity to Trypanosoma brucei rhodesiense. We argue that stomoxyn may help to explain why S. calcitrans is not a vector of trypanosomes causing African sleeping sickness and nagana.