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Volume 3 † Number 1 † March 2010 10.1093/biohorizons/hzq005 Advance Access publication 13 February 2010 ......................................................................................................................................................................................................................................... Research article Functional analysis of nematode nicotinic receptors James Nicholas Sleigh* Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, UK. * Corresponding author: MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Le Gros Clark Building, Oxford OX1 3QX, UK. Tel: þ44 1865 282273. Email: james.sleigh@hertford.ox.ac.uk Supervisor: Dr Adrian J. Wolstenholme, Department of Infectious Diseases, University of Georgia, Athens, GA 30602, USA. ........................................................................................................................................................................................................................................ Parasitic nematodes pose a significant threat to the health and economic welfare of communities worldwide, both directly through human disease and indirectly through infection of livestock and crop. At present, anthelmintic drugs such as the nicotinic agonists, which target nematode nicotinic acetylcholine receptors (nAChRs), offer a facile and cost-effective method of controlling both human and animal infection. Unfortunately, the continued heavy reliance on such compounds has led to the inevitable emergence of resistance in many different Nematoda species, thus making the subject of nematode nicotinic receptors of great importance. Using various levamisole-resistant, nAChR subunit mutants of the soil-dwelling nematode Caenorhabditis elegans (viz. unc-29, unc-38 and unc-63) and well-established resistance-monitoring assays, the responses of the different strains to the nicotinic agonists levamisole, morantel, oxantel and pyrantel have begun to be characterized. Wild-type adult C. elegans are relatively unaffected by 1 mM oxantel, contrasting with a developmental retardation of larvae. Also, unc-38 mutants appear to be less refractory to the anthelmintics than the unc-29 and unc-63 animals. In preparation for the use of C. elegans as an ectopic transformation system for parasitic receptor DNA, preliminary biolistic transformation experiments were performed using microparticle bombardment. It was found that transform- ation of unc-38 animals with myo-3::gfp construct has no significant effect on their ability to thrash in suspension. The mutant charac- terization data may possibly reflect differential expression of nAChRs at various stages of development. The transformation data suggest that microparticle bombardment has little or no effect on C. elegans, which is essential for future transgenic experiments. In summary, important baseline data on C. elegans nicotinic receptors have been generated and a fundamental transgenic control line in unc-38;myo- 3::gfp has been established. This will allow for exciting rescue experiments using parasitic nematode nAChR DNA. Key words: parasite resistance, nicotinic acetylcholine receptor (nAChR), Caenorhabditis elegans, parasitic nematode, anthelmintic drug, nicotinic agonist. Submitted on 28 September 2009; accepted on 22 January 2010 ........................................................................................................................................................................................................................................ There are three major classes of broad-spectrum anthel- Introduction mintic in widespread use: the benzimidazoles, the macrocyc- Parasitic nematode infections are of major global concern as lic lactones and the nicotinic agonists. A large quantity of they pose a considerable threat not only to human life, but these specifically target and bind to regions of the nematode 1 – 3 that of livestock and crops. In excess of 1 billion people nervous system, highlighting the importance of its study for 7, 8 are parasitized by intestinal nematodes in the developing medical and veterinary purposes, among others. The nic- world alone. Consequently, they have an impact on both otinic agonists, such as levamisole, morantel, oxantel and the health and economic welfare of communities and are pyrantel, specifically target nicotinic acetylcholine receptors 9, now recognized as one of the principal causes of human mor- (nAChRs) found at the nematode neuromuscular junction. 1, 5 bidity. Fortunately, anthelmintic chemotherapeutic agents Of particular significance, the identification of many offer a simple and inexpensive method of controlling both nicotinic agonist targets and potential resistance genes human and animal infections. was first achieved using the well-defined, free-living ......................................................................................................................................................................................................................................... The Author 2010. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distri- bution, and reproduction in any medium, provided the original work is properly cited. 29 Research article Bioscience Horizons † Volume 3 † Number 1 † March 2010 ......................................................................................................................................................................................................................................... nematode Caenorhabditis elegans rather than parasitic expression of the nicotine-insensitive L-type receptor in 11 – 15 19 Nematoda. Xenopus laevis oocytes. unc-29, unc-38 and unc-63 null The nicotinic agonists open ligand-gated receptors found mutants are all highly resistant to levamisole and electro- on muscle cell membranes, causing depolarization and physiological responses to levamisole, but not nicotine, are 9, 10, 12 28 hypercontraction. Levamisole is more potent than completely abolished. They also share a similar behaviour 16, 17 acetylcholine at nematode muscle nAChRs. These of uncoordinated locomotion, especially in early larval anthelmintics are not rapidly degraded, leading to prolonged stages. Surprisingly, the response of C. elegans to morantel, 2þ channel opening and Ca entry, causing spastic paralysis oxantel and pyrantel is at present ill-defined. It has often and ultimately death. In parasitic nematodes, this paralysis been assumed that the response to levamisole reflects that allows expulsion from the host. Anthelmintics can be used of the other three anthelmintics. Oxantel is an N-type to treat infections in animals because they have a selective agonist of Ascaris suum receptors, however, and not an therapeutic effect on nematode muscular nAChRs, and do L-type, suggesting that the effects of oxantel could not activate host receptors. The molecular basis for this perhaps be different to levamisole at least. specificity is largely unidentified. Transformation techniques such as microparticle bom- Unfortunately, the continued heavy reliance on and wide- bardment of DNA constructs (aka biolistic transformation) spread application of such compounds have led to the inevi- are integral to the use of C. elegans as a heterologous trans- table emergence of resistance to all three major classes of formation system to study parasitic receptors in vivo. The broad-spectrum anthelmintic in many nematode popu- generation of transgenic animals is important for the identi- 20 – 22 lations. This rise in drug resistance has predictably led fication of genes involved in anthelmintic resistance and their to a concomitant decrease in our ability to control infections. subsequent functional analysis. Microparticle bombard- Resistance to one anthelmintic usually confers resistance to ment involves accelerating DNA-coated beads (such as gold all other drugs in that chemical class and reversion to an particles) to high speeds, allowing the penetration of target 31, 32 anthelmintic-susceptible state once resistance has been estab- organism cells. Successfully transformed individuals lished has never been observed in a population. from subsequent generations are identified using a positive Consequently, it is imperative that the underlying mechan- marker, such as GFP. Combining both the ease and isms of resistance are better elucidated in order to both speed of using C. elegans with the efficiency and low cost prolong the lifetime of currently available anthelmintics of microparticle bombardment, much can be learned about and limit the future impact of resistance. anthelmintic drug action and mechanisms of parasitic nema- Caenorhabditis elegans has been important to the study of tode resistance. nematode nAChRs; a good proportion of what is known The main objective of the work presented here was to elu- about their function, genetics and pharmacology having cidate nematode nicotinic receptor function by analysing the been deduced using the small non-parasitic species. More effects of the nicotinic agonists levamisole, morantel, oxantel than 29 genes encode nAChR subunits in C. elegans, and pyrantel on various C. elegans strains. Surprisingly, little which is the most extensive and diverse collection of any is currently known about the effects of these anthelmintics, single organism characterized to date. Caenorhabditis so if C. elegans is to be used as a parasite model, there is elegans was first used in the study of parasitology over a urgent need to fully characterize the consequences of decade ago and its popularity as a model organism to their use. study parasitic nematodes has been on the rise ever since. This is due predominantly to the genetic and morphological similarities it shares with other nematodes and the major dif- Materials and methods ficulties associated with cultivating parasitic species in the Nematode strains and general methods lab. At least two distinct nAChR subtypes have been identified Caenorhabditis elegans strains were maintained using stan- in C. elegans muscle; N-type receptors, which are composed dard conditions. The following strains were used: N2 of five ACR-16 subunits and are most sensitive to nicotine, Bristol wild-type, CB904 unc-38(e264) I, ZZ13 and L-type receptors, which are composed of three unc-63(x13) I and CB193 unc-29(e193) I. All strains were a-subunits (LEV-8, UNC-38 and UNC-63) and two maintained on Nematode Growth Medium (NGM) (3 g/l non-a-subunits (LEV-1 and UNC-29) and are most sensitive NaCl, 17 g/l Bactoagar, 2.5 g/l Bactopeptone, 1 ml/l 11, 13, 15 to levamisole. Disturbance of both receptor subtypes 5mg/ml cholesterol, 1 ml/l 1 M CaCl ,1ml/l1M causes near complete debilitation of C. elegans, but disrup- MgSO ,25 ml/l potassium phosphate buffer [ pH6]) at tion of either one only causes slight locomotory deficiency, 198C and sustained on freshly seeded OP50 Escherichia 14, 27 suggestive of partial redundancy. In addition to the coli. Where possible, assays were blind and conducted at various nAChR subunits, three ancillary proteins (RIC-3, room temperature (18 – 228C). Reagents were obtained UNC-50 and UNC-74) are essential for heterologous from Sigma if not stated otherwise. ......................................................................................................................................................................................................................................... 30 Bioscience Horizons † Volume 3 † Number 1 † March 2010 Research article ......................................................................................................................................................................................................................................... Age synchronization bending at the mid-body. Animals that moved on to the well sides were discarded from the analysis. L4 stage animals were identified by the characteristic mor- phology of the developing vulva, which appears as a translu- Body bend assays cent, semi-circle region located in the middle of the body. Age-synchronized young adults were placed on unseeded Animals aged using this distinctive trait were picked to 1 mM NGM plates and allowed to acclimatize for 5 – NGM plates and placed at 188C for 24+ 1 h before 10 min. This short period is essential because well-fed wild- assaying. type C. elegans tend to show frequent directional switching 38, 39 immediately post-transferral. The number of body Dose–response assay bends was counted for 30 s and multiplied by 2 to obtain Adapted from the larval paralysis test, gravid adults were an estimate per minute. A complete body bend was defined placed in wells of a 96-well microtitre plate containing M9 as the bending of the head region across the central-line of solution (3 g/lKH PO ,6g/lNa HPO ,5g/l NaCl, 2 4 2 4 14, 40 the animal, in which a full sinusoid was completed. It 0.25 g/l MgSO 7H O) with varying concentrations of 4 2 has previously been observed that C. elegans reduce body anthelmintic or vehicle (99.5% dimethyl sulphoxide bend frequency when encountering bacteria; therefore, [DMSO]). Animals were then left for 10 – 12 min before animals found within 1 cm of unintentional food deposition being scored for immobilization. As in all experiments, during an assay were discarded from the analysis. anthelmintics were freshly prepared as 100 mM stocks in 99.5% DMSO. Suitable quantities of the stock were added Microparticle bombardment to the M9 to produce a final concentration of 1 mM– Biolistic transformation was performed using a BioRad 1 mM. Equivalent amounts of DMSO were used as a Biolistic PDS-1000/He particle delivery system and was control; the final concentration never exceeding 1%. scaled-up using a Hepta adapter gas splitter. Animals were categorized as immobilized following 10 s of ApaI-linearized Addgene plasmid pPD118.20 containing a inactivity. myo-3::gfp marker was used in the transformation exper- iments (courtesy of A. Fire). The following parameters Anthelmintic drug plates were implemented: in. gap distance, 9 mm macrocarrier NGM plates were made as described above with the addition to screen distance, 21 in. of Hg vacuum and 1550 p.s.i. of levamisole, morantel, oxantel, pyrantel or DMSO after rupture disc. Gold particles (0.3 – 3 mm) were prepared as autoclaving. A final concentration of 1 mM was used. described in the PDS-1000/He user manual and stored at Plates were foil-sealed and kept at room temperature for no 48C. Plasmid DNA was coated onto the gold microparti- longer than 4 weeks. For drug sensitivity assays, plates cles as outlined in Berezikov et al. were seeded with 150 mlof E. coli within 3 h of being Animals were washed from 4 to 6 crowded 9 cm plates poured and then incubated at room temperature for 6 days using M9 into a single Falcon tube and allowed to sediment. to allow growth of a robust bacterial lawn. Plates used in Following supernatant removal, they were spread onto a body bend assays remained unseeded. single unseeded 9 cm NGM plate and allowed to dry. Animals were cooled in order to reduce movement, improv- Growth sensitivity assays ing transformation efficiency and then subjected to Similar to the larval development test, C. elegans embryos bombardment. were transferred to plates with an E. coli lawn at time 0 h Post-bombardment, animals were allowed to recover for and their development was visually assessed at 48 and 1–2 h at 198C before being washed with M9 onto seeded 72 h. Animals were classified as being adult, L4, L1 – L3 or NGM plates. These were allowed to dry for 2 h before dead based on body size, vulval development and motility. being incubated at 198C for 3 – 5 days. Early-stage transfor- Animals were presumed dead following 10 s of immobility mants were identified by their positive GFP marker, picked in combination with a complete lack of response to prodding to new plates and allowed to reach the egg-laying stage. with a pick. Thrashing assays were subsequently performed vs. non- bombarded controls, as described above. Thrashing assays Statistical analysis Age-synchronized young adults were placed in individual wells containing M9 with or without drug at 1 mM. Each Data sets were statistically analysed using either an unpaired well contained 55 ml of solution in total; a volume chosen two-sample t-test or a Mann – Whitney U-test according to following preliminary experiments. After 5 – 10 min of parameters outlined in Townend. Normality of the data acclimatization, thrashes were counted for 30 s and multi- was assessed using the Kolmogorov – Smirnov test and plied by 2 to obtain an estimate per minute. A single both Levene’s and Bartlett’s tests were used to evaluate thrash was defined as a complete change in direction of homogeneity of variance. Minitab 15 Statistical Software ......................................................................................................................................................................................................................................... 31 Research article Bioscience Horizons † Volume 3 † Number 1 † March 2010 ......................................................................................................................................................................................................................................... was used for all statistical calculations. Some graphs lack Levamisole has the greatest effect on C. elegans statistical analyses; this is due to the data being presented development as percentages. To investigate the effect of the anthelmintics on develop- ment, growth sensitivity assays were performed (Fig. 2). Embryos of the various strains were passaged to 1 mM Results drug plates and observed 48 and 72 h post-transferral. At 48 h, there was little difference between the control and Mutant characterization drug plates for all four strains; no adult animals were present High concentrations of oxantel have no visible effect on and very few L1 – L3s were unresponsive to prodding with a wild-type mobility pick (data not shown). After 72 h, development of wild-type C. elegans was retarded when grown on plates containing To assess short-term, visible consequences of anthelmintic levamisole (Fig. 2A); the majority of animals were found at exposure, a dose – response assay was performed (Fig. 1). the L1 – L3 stage and no adults were present. This contrasts Gravid adults were passaged to wells containing M9 solution with control plates, which harboured a proportion of with varying drug concentrations and observed after 10 – adults well over 90%. Morantel and pyrantel also altered 12 min. the timing of animal development; only small percentages All mutants were visibly unaffected by the four drugs; no reached adulthood, but retardation was less severe than on single animal was paralysed even at the high concentration of levamisole. Conversely, a larger percentage of animals on 1 mM (data not shown). Some wild-type animals, however, oxantel plates reached maturity (.60%), with many more displayed inactivity in levamisole, morantel and pyrantel at reaching the L4 stage. 100 mM. All three drugs had similar effects; there was a In the previous assay, all four anthelmintics had no concomitant rise in the percentage of animals immobilized obvious effect on the mutants. This remains true for moran- with increasing drug concentration, such that at 1 mM, the tel, oxantel and pyrantel, since the proportions of animals at majority of animals were paralysed. Using an unpaired two- the different stages were comparable to the control for all sample t-test, 1 mM levamisole, morantel and pyrantel were three strains. unc-29 and unc-38 mutants raised on levami- all significantly different from the control. Oxantel had no sole, however, appeared to have slight developmental retar- visible effect on wild-type animals; movement was compar- dation; a higher percentage of animals remained at the able to the control at all concentrations. A 1 mM drug con- L1 – L3 stage (Fig. 2B and C). unc-63 development appeared centration was used in subsequent assays because it had the unaffected by all four drugs (Fig. 2D). greatest effect on wild-type with no apparent effect on mutants. Oxantel has no effect on C. elegans thrashing rate To measure the acute impact of the anthelmintics on C. elegans locomotion, thrashing assays were performed in M9 solution containing 1 mM drugs (Fig. 3). Thrashing rates of wild-type and unc-38 animals were sig- nificantly reduced by levamisole, morantel and pyrantel, whereas oxantel had no significant effect (Fig. 3A and C). The reduction was much more marked for wild-type. Only levamisole caused a reduction in unc-29 thrashing rate (Fig. 3B) and unc-63 locomotory activity was not signifi- cantly affected by any drug (Fig. 3D). The results for wild- type, unc-29 and unc-63, were similar to those of the growth sensitivity assay, in that the same drugs caused the same negative trends (Fig. 2). Conversely, unc-38 locomo- tory activity appeared to be more affected than its develop- ment by morantel and pyrantel. Plotting thrashing rates as a percentage of control data highlights the insensitivity of the wild-type to oxantel in comparison with the other drugs (Fig. 3E). It should also be noted that control wild-type Figure 1. Percentage of gravid wild-type animals immobilized following thrashing rates were about 4-fold faster than those of the 10–12 min in M9 solution with varying concentrations of anthelmintics. mutants (t-test, P, 0.001), and that control unc-63 Values are mean percentages +SE derived from three trials performed animals thrashed significantly less than the other unc on separate days. For each treatment, 50 animals of each strain were included in the analysis. mutants. ......................................................................................................................................................................................................................................... 32 Bioscience Horizons † Volume 3 † Number 1 † March 2010 Research article ......................................................................................................................................................................................................................................... Figure 2. Growth sensitivity data showing the percentage of adult, L4, L1–L3 and dead animals 3 days post-transferral of embryos to different 1 mM anthelmintic plates. (A) Wild-type, (B) unc-29,(C) unc-38 and (D) unc-63. Values are mean percentages +SE derived from three trials performed on separate days. One hundred animals of each strain were assayed on each drug. Oxantel has no effect on C. elegans body bend rate 2 – 3 min, but the body bend frequency was relatively unaffected. Thrashing assays indicated that the anthelmintics could Levamisole, morantel and pyrantel were all found to signifi- disrupt high-frequency C. elegans locomotion (Fig. 3). A cantly reduce body bend generation in gravid wild-type adults. body bend assay was subsequently performed to analyse Once again, oxantel had no significant effect. No anthelmintic less strenuous, non-maximal locomotion. The number of significantly altered the body bend rate of the mutants. The fre- body bends was counted in a 30 s period, 5 – 10 min post- quency of wild-type body bends on control plates was signifi- transferral and multiplied by 2 to get an estimate per cantly greater than all three mutants (t-test, P, 0.05), so rates minute (Fig. 4). of body bends were also measured as a percentage of control Upon passage to plates containing morantel and pyrantel (Fig. 4E). This emphasizes the insensitivity of the wild-type at 1 mM, wild-type animals were immediately incapacitated to oxantel in comparison with the other three drugs. It and did not regain the ability to move within the following should be noted that wild-type animals were instantaneously hour. When transferred to plates with levamisole at 1 mM, incapacitated on levamisole, morantel and pyrantel, but the majority of wild-type animals moved around the plate were able to survive and reach at least the L4 stage in the for 1 – 2 min then became rigid and paralysed. A small per- growth sensitivity assay (Fig. 2A). centage moved in the characteristic sinusoidal motion for up to 15 min; however, after 20 min, all animals were unable to Mutant characterization summary move. Control and oxantel had no obvious effect up to 1 h post-transferral. Anthelmintics caused the mutants to begin As expected, 1 mM levamisole, morantel and pyrantel signifi- to pirouette (i.e. move around in tight circles) after about cantly altered wild-type behaviour without fail (Table 1). In ......................................................................................................................................................................................................................................... 33 Research article Bioscience Horizons † Volume 3 † Number 1 † March 2010 ......................................................................................................................................................................................................................................... Figure 3. Thrashing rates after 5–10 min in M9 solution with various anthelmintic drugs at 1 mM. (A) Wild-type, (B) unc-29,(C) unc-38 and (D) unc-63. Values are composite means+ SE. Assays were performed on three separate days. For each drug, 18 animals of each strain were assayed. *P, 0.05; **P, 0.01; ***P, 0.001 Mann–Whitney U-test or unpaired two-sample t-test. NB: (A) has a larger scale than (B–D). (E) Thrashing rates plotted as a per- centage of the control. Values are mean percentages +SE derived from three trials performed on separate days. the dose – response assay (Fig. 1), a concentration of 1 mM (Fig. 2). Finally, in the thrashing (Fig. 3) and body bend caused immobilization of between 70% and 80% of gravid (Fig. 4) assays, they all caused a dramatic decrease. In stark adults. In the growth sensitivity assay, all three caused retar- contrast to this, oxantel had no significant effect on any of dation of development, levamisole being the most potent the C. elegans behaviours in any strain. ......................................................................................................................................................................................................................................... 34 Bioscience Horizons † Volume 3 † Number 1 † March 2010 Research article ......................................................................................................................................................................................................................................... Figure 4. Body bends per minute on NGM plates containing various anthelmintic drugs at 1 mM. (A) Wild-type, (B) unc-29,(C) unc-38 and (D) unc-63. Values are composite means+ SE. Assays were performed on three separate days. For each drug, at least nine animals of each strain were assayed. *P, 0.05; **P, 0.01; ***P, 0.001 unpaired two-sample t-test. (E) Body bend rates plotted as a percentage of the control. Values are mean percentages +SE derived from three trials performed on separate days. In contrast to wild-type, the mutant animals were rela- levamisole, morantel, and pyrantel causing a reduction in tively unaffected by the anthelmintic drugs. The behavioural thrashing rate (Table 1). unc-63 animals appeared to be phenotypes of unc-29 animals were completely unaffected the most refractory to the anthelmintics; not a single behav- except thrashing rate in levamisole (Table 1). unc-38 ioural phenotype was significantly altered by the anthelmin- animals were the most susceptible mutants assayed; tics tested (Table 1). ......................................................................................................................................................................................................................................... 35 Research article Bioscience Horizons † Volume 3 † Number 1 † March 2010 ......................................................................................................................................................................................................................................... Table 1. Mutant characterization summary Wild-type unc-29 1 231 2 3 ................................................................................................................ Levamisole ** *** *** NS ** NS Morantel *** *** *** NS NS NS Oxantel NS NS NS NS NS NS Pyrantel ** *** *** NS NS NS unc-38 unc-63 1 231 2 3 ................................................................................................................ Levamisole NS ** NS NS NS NS Morantel NS * NS NS NS NS Oxantel NS NS NS NS NS NS Pyrantel NS ** NS NS NS NS 1, dose–response assay; 2, thrashing assay; 3, body bend assay; NS, not Figure 5. Thrashing rates of transformant and control animals after 5– significant; *P, 0.05; **P , 0.01; ***P, 0.001. 10 min in M9 solution with various 1 mM anthelmintic drugs. Values are means +SE. For each drug, four animals of each strain were assayed. All Microparticle bombardment data were analysed using unpaired two-sample t-tests. To establish a transgenic control strain, biolistic transform- phenotypes analysed by the other assays. This could perhaps ation using a BioRad Biolistic particle delivery system was be due to differing nAChR expression profiles of larval and performed. unc-38 mutants were bombarded with adult C. elegans. In the growth assay, larvae were exposed ApaI-linearized plasmid pPD118.20 containing myo-3 pro- to anthelmintics from hatching; whereas adults were moter fused to gfp. myo-3 encodes a muscle-type specific assayed in the other experiments. It is possible that larval myosin heavy-chain isoform and is expressed in all 45 – 47 stage animals have differing proportions and/or distributions C. elegans muscles outside the pharynx. Transformed of nAChR subunits and receptor subtypes in order to better animals expressing the myo-3::gfp construct were identified exploit their surroundings and thus enhance survival. This 3 – 5 days post-bombardment via green fluorescence observed dynamic modulation may be needed in order to rapidly in body wall and vulval muscles. adapt to environmental changes. This is more likely for para- To observe whether microparticle bombardment had any sitic nematodes as the external environment experienced by significant effect on C. elegans locomotion, transformant larvae is often very different from that encountered by animals were allowed to reach the egg-laying stage and then adults; for instance, the sheep nematode Haemonchus assayed for thrashing rate (Fig. 5). Non-bombarded unc-38 contortus has both free-living and parasitic stages. mutants were used as a control. A thrashing assay was used Sangster and Bjorn have shown that the level of levamisole because thrashing of unc-38 animals appeared to be the most resistance of H. contortus varies throughout its life-cycle; anthelmintic-susceptible behaviour/phenotype (Table 1). immature parasites being more susceptible than adults both There was no significant difference between the thrashing fre- in vitro and in vivo. This variation in susceptibility could be quencies of unc-38;myo-3::gfp and control animals under all explained by differential nAChR expression profiles, for five conditions. Biolistic transformation also had no effect on example, in the muscles and neurons. Since H. contortus unc-38 resistance to the anthelmintics, as there was no differ- and C. elegans both belong to Nematoda Clade V, it is ence between control thrashing rates and those in the drugs. quite possible that this could also manifest in the latter. Similarly, there was no difference between unc-38 control Oxantel is a derivative of pyrantel developed to treat chil- thrashing rates and rates in the different drugs. Ultimately, an 30, 52 dren with whipworm infection. It has since been charac- unc-38;myo-3::gfp transformant control line was established terized as an N-type agonist of the Clade III parasite by the microparticle bombardment technique. 30, 51 A. suum. Contrastingly, oxantel is an antagonist of C. elegans N-type receptors, binding to nAChRs composed solely of ACR-16 subunits. The early developmental retar- Discussion dation of wild-type C. elegans, in combination with the Mutant characterization resistance at adult stages, could possibly be due to differen- tial expression of N-type receptors. If ACR-16 expression is Oxantel caused retardation of wild-type C. elegans develop- up-regulated in larvae, it is conceivable that larvae would be ment (Fig. 2A); whereas it had no significant effect on ......................................................................................................................................................................................................................................... 36 Bioscience Horizons † Volume 3 † Number 1 † March 2010 Research article ......................................................................................................................................................................................................................................... more susceptible to oxantel than adults. In contrast, wild- requirements of morantel and pyrantel may also explain type animals appeared to be relatively more susceptible to the discrepancy. Pyrantel requires a glutamate at a specific levamisole, an L-type agonist, at later stages of develop- position in loop D of UNC-63 for receptor binding, ment. Unlike oxantel, when passaging adult animals to whereas levamisole does not. Leading on from this, due plates containing 1 mM levamisole, the vast majority were to the apparent resistance of unc-63 to all anthelmintics completely paralysed within 5 min. In the growth sensitivity tested, it is possible that the most important residues for effi- assay, despite development being severely retarded compared cient binding of levamisole, morantel and pyrantel reside with the control and more so than oxantel, wild-type somewhere within the UNC-63 subunit. C. elegans larvae were still alive and moving about the When interpreting these C. elegans data, it is important to plate. These results are corroborated by the similar data for be wary of the drug molarities used. The high concen- pyrantel, which is also an L-type agonist. tration of 1 mM used in these resistance assays is likely to Altogether, these results are perhaps concordant with be toxic to a mammalian host and so cannot be used to greater expression of N-type nAChRs in larvae than adults. therapeutically treat nematode infections. If a concentration They also suggest that L-type receptor expression is possibly of 100 mM was administered to treat parasitic infection, up-regulated as C. elegans ages. The greater retardation of this would constitute a 10-fold difference when comparing wild-type development by levamisole over oxantel suggests with the C. elegans data generated here. that L-type receptors are more prevalent throughout the life- Microparticle bombardment cycle. Phosphorylation levels of nAChRs have been shown to have an effect on agonist potency, which may also explain The GFP expression pattern of unc-38 animals bombarded the differential susceptibilities of the various C. elegans with myo-3::gfp DNA construct is suggestive of successful stages. Oxantel has no such effect on the development of transformation because they resemble the endogenous 45 – 47 mutant animals, which all have fully functional N-type pattern of myo-3 expression. myo-3 is usually expressed receptors. This could be explained by the slower growth in body wall and vulval muscles, among others, which can be rate of unc-29 and unc-38 animals, which may mask the seen to fluoresce in the unc-38;myo-3::gfp animal. developmental retardation seen in the wild-type. unc-63 Transformants at the egg-laying stage were subsequently mutants seemed to grow faster than wild-type animals shown to thrash at similar rates to non-bombarded unc-38 (Fig. 2); this enhanced development may also have negated controls and were unaffected by the anthelmintics (Fig. 5), the developmental retardation caused by oxantel. suggesting that microparticle bombardment had no signifi- Contrary to wild-type, unc-63 mutant behaviours all cant effect on thrashing rate. Of note, the unc-38 controls appeared refractory to the anthelmintics. This was also were not significantly affected by the anthelmintics as they true for the unc-29 mutants, excluding the significantly were in the previous thrashing assay (Fig. 3). This is most reduced thrashing rate in levamisole (Fig. 3B). In contrast, likely due to a lack of age-synchronization and small unc-38 thrashing frequency was significantly reduced by sample sizes (n ¼ 4). Now that a transgenic control line levamisole, morantel and pyrantel (Fig. 3C). This perhaps has been established, this can easily be rectified. implies that both UNC-29 and UNC-63 are more important for binding of the various L-type agonists than UNC-38. Conclusion When UNC-29 and UNC-63 were present and UNC-38 was absent, mutant thrashing rates were significantly It has been shown that the nicotinic agonist oxantel has little reduced. This reduction in thrashing and not the other phe- effect on adult wild-type C. elegans locomotory behaviour, notypes suggests that restricted movement is the consequence which may reflect differential nAChR subtype expression. It of exposure to anthelmintics. Thrashing is most vulnerable to has also been demonstrated that the C. elegans nAChR subunits the anthelmintics and as such is the most sensitive assay to UNC-29 and UNC-63 are potentially more important for detect weak/mild drug susceptibilities. Eight genes are anthelmintic binding than UNC-38. The generation of baseline required for functional reconstruction of the L-type receptor data on C. elegans nicotinic receptors in combination with the in a Xenopus oocyte expression system; however, the sig- establishment of an important transgenic control line provides nificant reduction of thrashing rates of unc-38 mutants in a solid base for future rescue experiments with parasitic nema- response to levamisole suggests that some semblance of an tode DNA. This will undoubtedly aid in the functional analysis L-type receptor is still partially functional in vivo. of nematode nicotinic receptors and potentially elucidate The conductance dose – response curves of morantel and mechanisms involved in parasitic drug resistance. pyrantel are ‘bell-shaped’, such that past a certain concen- tration, increasing their molarities will cause a reduction in Acknowledgements receptor opening. This phenomenon may serve to explain why morantel and pyrantel did not reduce unc-29 thrashing I would like to thank Susan Thawley and Sally Williamson rates like levamisole at 1 mM. Additional structural for their guidance on my final year project and for ......................................................................................................................................................................................................................................... 37 Research article Bioscience Horizons † Volume 3 † Number 1 † March 2010 ......................................................................................................................................................................................................................................... 10. Aubry ML, Cowell P, Davey MJ et al. (1970) Aspects of the pharmacology of withstanding my incessant questions. I am also very grateful new anthelmintics: pyrantel. Br J Pharmacol 38: 332–344. to Adrian Wolstenholme for allowing me to work in his lab 11. Fleming JT, Squire MD, Barnes TM et al. (1997) Caenorhabditis elegans leva- and granting me the freedom to make the project my own. In misole resistance genes lev-1, unc-29, and unc-38 encode functional nic- addition, I would like to voice my gratitude to all of the aca- otinic acetylcholine receptor subunits. J Neurosci 17: 5843–5857. demic and administrative staff in the Department of Biology 12. Lewis JA, Wu CH, Berg H et al. (1980) The genetics of levamisole resistance in and Biochemistry at the University of Bath for invaluable the nematode Caenorhabditis elegans. Genetics 95: 905–928. help throughout my undergraduate career. Finally, I am 13. Culetto E, Baylis HA, Richmond JE et al. (2004) The Caenorhabditis elegans very grateful to Chris Sleigh for helping with my figures. unc-63 gene encodes a levamisole-sensitive nicotinic acetylcholine receptor alpha subunit. J Biol Chem 279: 42476–42483. 14. 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Bioscience Horizons – Oxford University Press
Published: Mar 13, 2010
Keywords: parasite resistance nicotinic acetylcholine receptor (nAChR) Caenorhabditis elegans parasitic nematode anthelmintic drug nicotinic agonist
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