CLARK, D

Geldhof, Peter

KNOX, DP

MOLLOY, C

English

Ghent University Academic Bibliography

KamIoHhiHioagneapoepbiofltgc0dMolecular & Biochemical Parasitology 152 (2007) 220–223Short communicationAssessment of Caenorhabditis elegans as a model inHaemonchus contortus vaccine researchP. Geldhof a,b, D. Clark a, C. Molloy a, D.P. Knox a,∗a Moredun Research Institute, Penicuik EH26 0PZ, UKb Laboratory for Parasitology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, BelgiumReceived 20 November 2006; received in revised form 12 December 2006; accepted 13 December 2006Available online 20 December 2006[sefdtqpl1tSpCt[etasatRmeywords: Caenorhabditis elegans; Parasite; Model; Vaccine; RNAi; AntibodyOver the past decade Caenorhabditis elegans has becomepopular model organism for parasitic nematode research andany examples have been published to illustrate its use [1].t has especially been useful for the research on mechanismsf action of anthelmintics and on anthelmintic resistance [2].owever, its validity in parasite vaccine research is less clear. Itas been suggested that studying genes in C. elegans by RNAnterference might help to identify new vaccine targets [3,4].owever, as far as we know, this approach has not yet resultedn the identification of new protective antigens. In contrast, theutcome of a recent study actually questions the utility of thispproach in vaccine research. C. elegans was used to investi-ate the function of intestinal cathepsin B-like proteinases inematodes [5]. These gut expressed enzymes have proven to beffective targets for vaccination against Haemonchus contortusnd Ancylostoma caninum, resulting in lower faecal egg out-ut and worm burdens [6,7]. Surprisingly, simultaneous RNAif the predominant intestinal cathepsin B enzymes in adult C.legans did not result in any visible phenotype despite the com-lete abolishment of all cysteine proteinase activity, detectabley substrate gel analysis of worm extracts. [5]. One possibil-ty is that the expected phenotype was masked by the activity ofther proteinases which could compensate for a loss of digestiveunction.Here, in the first instance, homologues of the most estab-ished H. contortus digestive enzyme vaccine candidates wereargeted simultaneously in C. elegans by RNAi. These anti-ens are the metallopeptidase III and pepsin of the H-gal-GPomplex, the H11 aminopeptidase and the cysteine proteinases∗ Corresponding author. Tel.: +44 131 445 5111; fax: +44 131 445 6111.E-mail address: david.knox@moredun.ac.uk (D.P. Knox).uasowod166-6851/$ – see front matter Published by Elsevier B.V.oi:10.1016/j.molbiopara.2006.12.0068]. These intestinal antigens induce high levels of protection inheep against a H. contortus challenge infection, reducing faecalgg counts by 77–90% and worm burdens by 47–78%.All members in C. elegans of the metallopeptidase (peptidaseamily M13), pepsin (peptidase family A1) and aminopepti-ase (peptidase family M1) protein families were identified inhe Wormbase database (http://www.wormbase.org) and subse-uently used in a phylogenetic analysis with the H. contortusroteins using ClustalX [9] and Treeview [10]. Two metal-opeptidases (CE14174, CE11095), 1 pepsin (CE16843) andaminopeptidase (CE21162) were identified which clusteredogether with the H. contortus proteins (results not shown).equence similarity between the C. elegans and H. contortusroteins were 20 and 42% for CE14174 and CE11095, 40% forE16843 and 39% for CE21162. The homologues of the H. con-ortus intestinal cysteine proteinases were previously identified5].RNA interference experiments were set-up to knock down thexpression of the metallopeptidases, the pepsin, the aminopep-idase and the CPR 4 cysteine proteinase both individuallynd simultaneously. The CPR4 cysteine proteinase was cho-en because it was shown previously [5] that RNAi directedt this transcript resulted in the complete abolishment of all cys-eine proteinase activity in the worm [5]. The double-strandedNA for each gene was produced and RNAi conducted usingethods described previously [5,11]. The primer sequencessed to amplify and clone each gene can be obtained from theuthors. Larvae soaked in PBS or dsRNA for a H. contortuspecific superoxide dismutase [5] served as controls. After 24 hf soaking, the larvae were transferred to NGM plates seededith OP50 Escherichia coli. Worms were visually examinedver a period of 5 days for phenotypic effects such as growth,evelopment and movement. A possible effect on reproductionemicwNpbffwmefqsp(tt[lcPpfcdosCstRapetAhdTa%wtdFswcdTiawgtfFatdsortP. Geldhof et al. / Molecular & Biochas checked by transferring 10 adult hermaphrodites to freshGM plates (2 worms per plate). Eggs were counted on eachlate after 24 h. An in vivo feeding assay with FITC-labelledacteria [5] was used to visualize possible effects on wormeeding and Nile Red staining was used as a measure ofood digestion and nutrition in the worms [12]. RNAi treatedorms, fed on Nile Red, were examined under a fluorescenticroscope with an excitation filter of 515–560 nm and anmission filter of 590 nm. The absence of the target transcriptsollowing RNAi treatment was confirmed by gene-specific semi-uantitative RT-PCRs using the Superscript One-Step RT-PCRystem (Invitrogen) on RNA from 15 adult hermaphrodites 24 host-RNAi treatment [11]. Transcript levels of a non-target genef54d5.3) were measured as a control for the specificity ofhe treatment [5]. The transcript levels of a heat shock pro-ein (hsp16–1) were measured to monitor stress in the worms13].Worms treated with individual dsRNA for the two metal-opeptidases and the CPR-4 cysteine proteinase showed almostomplete gene silencing for the target genes, as judged by RT-CR (results not shown). However, there were no detectablehenotypes (results not shown). RNA interference experimentsor the aminopeptidase, on the other hand, did not result in spe-ific knock down in the expression of this gene despite differentsRNA constructs being tested in repeated experiments, with-ut any success. RT-PCRs on RNA samples from different lifetages of C. elegans showed that the pepsin homologue (no.E16843) was not expressed in the adult hermaphrodite lifetage (results not shown). Therefore, the aminopeptidase andhe pepsin homologue were not included in the combinatorialNAi experiment.tiCpig. 1. Adult hermaphrodite C. elegans worms were treated with a dsRNA mixture tond a cysteine proteinase (cpr4) and were subsequently checked for effects on feediranscripts following RNAi. Semi-quantitative RT-PCRs were carried out on total RNsRNA (Hc-sodc) and 15 worms treated the dsRNA mixture (mix). RT-PCRs for twoamples. (B) Effect of the RNAi treatment on worm fecundity. The results are shownutput was increased by 21% (P < 0.02) in the RNAi treated worms compared to the ded is a lipophilic stain and when mixed with E. coli (0.1g/ml) the worms take up thhem red under a fluorescent microscope. The RNAi treated worms (mix) showed simal Parasitology 152 (2007) 220–223 221Worms treated with a mixture of dsRNA to the two met-llopeptidases (CE11095, CE14174) and the CPR4 cysteineroteinase showed a significant knock down in the expression ofach target gene, compared to expression levels in negative con-rol worms and worms treated with control dsRNA (Fig. 1, panel). The expression levels of the control gene (f54d5.3) and thesp16–1 gene were not affected, indicating that the gene knockown was specific and did not induce a major stress response.he RNAi treated worms did not show any obvious phenotypicbnormalities. Egg output (Fig. 1, panel B) was increased by 21in the RNAi treated worms compared to the dsRNA controlorms. The in vivo feeding and nutrition assays indicated thathere was no effect in the RNAi treated worms on food uptake andigestion. Typical results for the Nile Red staining are shown inig. 1 panel C. The RNAi treated worms (MEP + CPR4) showedimilar staining throughout the intestine compared to controlorms.Nowell et al. [13] used C. elegans to monitor immunologi-al stress in nematodes by incubating C. elegans in antibodiesirected against the worm cuticle to mimic an immune attack.he results indicated that the physical binding of antibodynduced a stress response with a significant reduction in growthnd fecundity of the adult worms. At the moment, the bestorking native vaccines against H. contortus are all based onut expressed proteins [8] and, more importantly, antibody ishought to mediate this protective immunity by blocking or inter-ering with the digestion within the worm. One possibility is thathis “physical” effect acts synergistically with specific enzymenhibition to impair worm digestion and that RNAi targeting the. elegans homologues does not induce a phenotype because thehysical effect is absent. To test this hypothesis, we targeted theknock down the expression of two metallopeptidases (CE11095 and CE14174)ng, development and reproduction. (A) Loss of CE11095, CE14174 and cpr4A isolated from 15 wild-type worms (control), 15 worms treated with controlcontrol genes (f54d5.3 and hsp16–1) were run to check the quality of the RNAas mean egg output for two worms after 24 h on the plate (five replicates). EggsRNA control worms. (C) In vivo nutrition assay with Nile Red staining. Nilee Nile Red and incorporate it into lipid droplets of the intestinal cells, stainingilar staining throughout the intestine compared to control worms.2 emicggeiowmUs(TwwpasaibLtafiicastcbsopsNwomapiisewapoaasptcltitbodttgFgswp1a22 P. Geldhof et al. / Molecular & Biochut expressed proteins in C. elegans with antibodies directed atlycoproteins expressed on the intestine surface [14] and soughtffects on feeding, development and reproduction. The antibod-es we used in this experiment were purified from pooled sera,btained either pre- or post-immunisation from sheep vaccinatedith C. elegans ConA-binding proteins [14], by affinity chro-atography over a HiTrap Protein G column (GE Healthcare,K). This ConA-binding protein fraction contains a complexet of glycoproteins, including cysteine and metallo proteinasesresults not shown), purified from the intestine of C. elegans.o determine any affect of IgG binding on worm fecundity, aell-based IgG assay was set-up. Two L3/L4 stage C. elegansere transferred to wells containing 90l PBS (pH 7.4) plusre- or post-immunisation ConA-binding fraction reactive IgGt a final dilution of 1:10. Ten replicates of each treatment wereet-up. To allow worms to feed, 10l OP50 strain E. coli fromn overnight bacterial culture was added to each well. Followingncubation at room temperature for 2 days, worms were killedy addition of 2l helminthological iodine and the number of1/L2 stage larvae in each well enumerated. FITC-labelled bac-eria and the Nile red staining were used to monitor food uptakend nutrition. Binding of antibody to the intestine was con-rmed by transferring 15 worms to NGM agar plates. Followingncubation for 1 h, worms were picked and incubated in PBSontaining FITC-labelled mouse anti-sheep IgG (Sigma, UK) atfinal dilution of 1:50 for 2 h at room temperature. Worms wereubsequently transferred to NGM agar for an hour after whichhey were transferred into 50l PBS for analysis under a fluores-ent microscope. Stress induced by the treatment was monitoredy measuring changes in transcript levels of the C. elegans heathock protein hsp16–1. This was done by a semi-quantitativene-step RT-PCR on total RNA as described above.The results from the immunostaining are shown in Fig. 2anel A. Worms incubated in antibodies purified from immuneerum clearly had antibodies bound to their intestinal surface.slwiig. 2. (A) Detection of antibodies bound to the intestinal surface of hermaphrodiut expressed proteins for 2 days, following this incubation they were transferred tubsequently transferred to NGM agar for an hour after which they were transferred ias used as a negative control. (B) Effect of IgG binding on worm fecundity. Two Llus pre- or post-immunisation ConA-binding fraction reactive IgG at a final dilution0l OP50 strain E. coli from an overnight bacterial culture was added to each welddition of 2l helminthological iodine and the number of L1/L2 stage larvae in eacal Parasitology 152 (2007) 220–223evertheless, there was no phenotypic difference betweenorms incubated with and without antibody, in the presencef pre-immune or immune antibodies. Growth, development,ovement, reproduction, food uptake and nutrition were notffected. The results for worm fecundity are shown in Fig. 2anel B. In addition, hsp16–1 transcript levels were not affectedn untreated worm and worms incubated in antibodies, both pre-mmune and immune (results not shown). To assess a potentiallyynergistic effect of combined RNAi and antibody treatment,xperiments were carried out in which worms were pre-treatedith a mixture of metallopeptidase and CPR4 or control dsRNAs before. Following RNAi, worms were incubated with eitherre- or post-immune IgG as described above. Five replicatesf each treatment were set-up. The results for worm fecundityre shown in Fig. 3. No treatment combination had a discern-ble effect on phenotype (assessed as before) and there was notatistical difference in fecundity between treatments.The aim of this study was to assess and compare the use of tworoposed C. elegans-based methods to identify parasite vaccineargets involved in digestion. Neither approach, either alone or inombination, induced any visible phenotypes in the worms. Theack of phenotype might be explained by the fact that the genesargeted in this study are all members of multigene families andt is possible that genes within such families have some func-ional redundancy. Alternatively, phenotypes might have beenecome visible if worms were kept in the presence of antibodyr dsRNA for longer periods. However, worms treated withsRNA were already observed for 5 days. During this periodhere was no sign that the development of the worms stopped orhat they were slowly starving, in contrast, the worms were stillrowing. Finally, the target genes were identified based on theirequence homology and although they are the closest homo-ogues, there is no guarantee that their function is conservedith the H. contortus genes. In conclusion, the data presentedn this paper clearly infers that both these screening methodste C. elegans worms. Worms were soaked in sheep antibodies raised againsto a 1:50 dilution of FITC labelled anti-sheep conjugate for 2 h. Worms werento 50l PBS for analysis under a fluorescent microscope. Pre-immune serum3/L4 stage C. elegans were transferred to wells containing 90l PBS (pH 7.4)of 1:10. Ten replicates of each treatment were set-up. To allow worms to feed,l. Following incubation at room temperature for 2 days, worms were killed byh well enumerated.P. Geldhof et al. / Molecular & BiochemicFig. 3. Effect of combined RNAi and antibody treatment on worm fecundity.Worms underwent RNAi by soaking for 12 h in the dsRNA mixture for the met-allopeptidases and CPR4. These worms were subsequently transferred to wells(2 worms/well) containing 90l PBS (pH 7.4) plus pre- or post-immunisationConA-binding fraction reactive IgG at a final dilution of 1:10. Five replicates ofeach treatment were set-up. To allow worms to feed, 10l OP50 strain E. colifrom an overnight bacterial culture was added to each well. Following incubationalFwaepA(R[[[[t room temperature for 2 days, worms were killed by addition of 2l helmintho-ogical iodine and the number of L1/L2 stage larvae in each well enumerated.ecundity was compared with worms which did not undergo the RNAi treatment.ould not identify gut expressed proteins as vaccine candidatesgainst H. contortus. This, in turn, suggests the need for consid-rable caution in interpreting C. elegans-based data to identifyotential parasite vaccine targets.cknowledgementPG is the recipient of a Marie Curie reintegration grantno. 028870).[al Parasitology 152 (2007) 220–223 223eferences[1] Britton C, Murray L. Using Caenorhabditis elegans for functional analysisof genes of parasitic nematodes. Int J Parasitol 2006;36:651–9.[2] Geary T, Thompson DP. Caenorhabditis elegans: how good a model forveterinary parasites? Vet Parasitol 2001;101:371–86.[3] Hashmi S, Tawe W, Lustigman S. Caenorhabditis elegans and the study ofgene function in parasites. Trends Parasitol 2001;17:387–93.[4] Kuwabara PE, Coulson A. RNAi—prospects for a general technique fordetermining gene function. Parasitol Today 2000;16:347–9.[5] Geldhof P, Molloy C, Knox DP. Combinatorial RNAi on intestinal cathepsinB-like proteinases in Caenorhabditis elegans questions the perception oftheir role in nematode biology. Mol Biochem Parasitol 2006;145:128–32.[6] Redmond D, Knox DP. Protection studies in sheep using affinity-purifiedand recombinant cysteine proteinases of adult Haemonchus contortus. Vac-cine 2004;22:4252–61.[7] Loukas A, Bethony JM, Williamson AL, et al. Vaccination of dogs withrecombinant cysteine protease from the intestine of canine hookwormsdiminishes the fecundity and growth of worms. J Infect Dis 2004:1952–61.[8] Knox DP, Redmond DL, Newlands GF, Skuce PJ, Pettit D, Smith D. Thenature and prospects for gut membrane proteins as vaccine candidatesfor Haemonchus contortus and other ruminant trichostrongyloids. Int JParasitol 2003;33:1129–37.[9] Thompson JD, Gibson TJ, Plewnaik F, Jeanmougin F, Higgins DG. TheClustalX windows interface: flexible strategies for multiple sequence align-ment aided by quality analysis tools. Nucl Acids Res 1997;25:4876–82.10] Page RDM. Treeview: an application to display phylogenetic trees onpersonal computers. Comput Appl Biosci 1996;12:357–8.11] Geldhof P, Whitton C, Gregory WF, Blaxter M, Knox DP. Characterisationof the two most abundant genes in the Haemonchus contortus expressedsequence tag dataset. Int J Parasitol 2005;35:513–22.12] Ashrafi K, Chang FY, Watts JL, et al. Genome-wide RNAi analysis ofCaenorhabditis elegans fat regulatory genes. Nature 2003;421:268–72.13] Nowell MA, Pomerai DIDE, Pritchard DI. Caenorhabditis elegans asa biomonitor for immunological stress in nematodes. Parasite Immunol1999;21:495–505.14] Redmond DL, Geldhof P, Knox DP. Evaluation of Caenorhabditis elegansglycoproteins as protective immunogens against Haemonchus contortuschallenge in sheep. Int J Parasitol 2004;34:1347–53.

Assessment of Caenorhabditis elegans as a model in Haemonchus contortus vaccine research

https://biblio.ugent.be/publication/413802/file/1130339.pdf

Assessment of Caenorhabditis elegans as a model in Haemonchus contortus vaccine research

Abstract

Similar works

Full text

Available Versions

Ghent University Academic Bibliography