Fig 1.
Maximum likelihood tree showing relationships of the ACR-16 related acetylcholine receptor (nAChR) subunits from Trichuris spp., with other C. elegans and T. spiralis nAChR subunits.
The tree was built upon an alignment of nAChR subunit deduced amino-acid sequences. The tree was rooted with the Parascaris equorum ACR-26 and ACR-27 sequences that are absent from C. elegans and clade I nematode species [34]. Scale bar represents the number of substitutions per site. Bootstrap values are indicated on branches. Accession numbers for sequences used in the phylogenetic analysis are provided in the Material and Methods section. C. elegans nAChR subunit groups are named as proposed by Mongan et al. [31], Cel, Tsu, Ttr, Tmu, Tsp and Peq refer to Caenorhabditis elegans, Trichuris suis, Trichuris trichiura, Trichuris muris and Parascaris equorum, respectively.
Fig 2.
Amino acid alignment of ACR-16-(like) and ACR-19 subunit sequences from the Clade I parasitic nematodes Trichuris suis, T. trichiura, T. muris, Trichinella spiralis and Caenorhabditis elegans.
Predicted signal peptide sequences are shaded in grey, the Cys-loop, the transmembrane regions (TM1-TM4), and the YxCC motif that characterize an α-subunit are indicated above the sequences. Conserved amino acids between ACR-16-(like) and ACR-19 sequences (dark blue), conserved amino acids between all ACR-16-(like) sequences (red), conserved amino acid between all ACR-19 sequences (light green), conserved amino acid between ACR-16-like sequences of Clade I parasitic nematodes and ACR-19 of C. elegans (light blue).
Fig 3.
Effect of the ancillary protein Resistance-to-cholinesterase (RIC-3) from Xenopus laevis (Xla-RIC-3), Ascaris suum (Asu-RIC-3), Haemonchus contortus isoform 1 (Hco-RIC-3.1), Caenorhabditis elegans (Cel-RIC-3) on the functional expression of the ACR-16-like nAChR from Trichuris suis (Tsu-ACR-16-like receptor).
Representative sample traces of inward current in response to 100 μM ACh are shown together with a scatter dot plot presenting the relative currents (mean ± SEM). Non-injected oocytes and oocytes injected with Tsu-acr-16-like- or Asu-ric-3 cRNA alone did not respond to 100 μM ACh which was significantly different from oocytes co-injected with Tsu-acr-16-like and either of the tested ric-3 cRNAs (**P < 0.01). The relative currents of oocytes co-injected with Tsu-acr-16-like and either of the tested ric-3 cRNAs were not significantly different (P > 0.9) as indicated, Kruskal-Walis Test.
Fig 4.
The effect of 9 agonists on Tsu-ACR-16-like receptor.
Representative sample traces and a bar chart (mean ± SEM) show the rank order potency series of 4 cholinergic anthelmintics: oxantel (oxa), pyrantel (pyr), morantel (mor), levamisole (lev) and 5 nAChR agonists: epibatidine (epi), nicotine (nic), 3-bromocytisine (3-bc), dimethylphenylpiperazinium (DMPP) and cytisine (cyt). P < 0.05; significantly different as indicated; Turkey’s multiple comparison test.
Fig 5.
A: Chemical structure of oxantel and pyrantel.
Oxantel: free drawing after https://pubchem.ncbi.nlm.nih.gov/compound/oxantel#section=2D-Structure. Pyrantel: free drawing after https://pubchem.ncbi.nlm.nih.gov/compound/pyrantel#section=2D-Structure B: Dose-response curves for oxantel (oxa), pyrantel (pyr) and acetylcholine (ACh). The current response on Tsu-ACR-16-like receptor is normalised to current responses induced by 300 μM ACh and given as mean ± SEM. The EC50 ± SD values were 9.48 ± 1.15 μM for oxa, 152.7 ± 1.20 μM for pyr and 14.5 ± 1.03 for ACh, the relative maximum current responses, Imax were 86.85 ± 4.63% and 29.41 ± 1.95%, and the Hill slope, nH, 2.51 ± 1.30, 3.13 ± 1.07 and 2.14 ± 0.1 for oxa, pyr and ACh respectively.
Fig 6.
Effect of the antagonists: dihydro-β-erythroidine (DHβE) and derquantel (der) on Tsu-ACR-16-like receptor mediated 100 μM ACh current response.
Results are given as normalized mean ± SEM inhibition of the initial current response of 100 μM ACh. DHβE produced an almost insignificant block of the Tsu-ACR-16-like receptor mediated ACh response (i.e. 7.60 ± 1.61%) and no effect was observed for der (0.16 ± 1.61%).
Fig 7.
A and B: Effect of the antagonists α-bungarotoxin (α-BTX) on Tsu-ACR-16-like receptor mediated 100 μM ACh current response. The first ACh current response (ACh1) is set to 100 for both test- (Fig 7A) and control oocytes (Fig 7B), and subsequent current responses are given as normalized mean ± SEM inhibition of ACh1. P < 0.05; significantly different as indicated, Dunnett’s test.
Fig 8.
Heterologous expression of Tsu-ACR-16-like receptor in Caenohabditis elegans confer oxantel sensitivity to the recombinant worms Boxplot depicts number of thrashes/30 sec of worms in M9 media prior to incubation (0 h) and after 24 h incubation with (+) 500 μM oxantel.
The number of thrashes were significantly higher for wildtype C. elegans; N2 (red, n = 34) than each of the recombinant C. elegans N2;pmyo3::Tsu-acr-16-like lines: line 1 (yellow, P <0.0001, n = 28), line 2 (blue, P <0.0001, n = 30), line 3 (green, P <10−4, n = 34) when incubated in M9 media with 500 μM oxantel.