Fig 1.
Maximum likelihood tree showing relationships of ACR-26 and ACR-27 acetylcholine receptor (AChR) subunits from parasitic nematodes with C. elegans AChR subunits.
Tree was built upon an alignment of AChR subunit sequences excluding the predicted signal peptide and the highly variable region between TM3 and TM4. Potential homologs of ACR-26 identified in the molluscs, Aplysia californica, Crassostrea gigas, Lottia gigantean, the annelid Capitella telata and the arthropods Ixodes scapularis and Daphnia pulex were also included in the analysis. The tree was rooted with DEG-3 group subunit sequences. Branch lengths are proportional to the number of substitutions per amino acid. Scale bar represents the number of substitution per site. Red dots at the nodes indicate bootstrap values >70%. Accession numbers for sequences used in the phylogenetic analysis are provided in Materials and Methods section. C. elegans AChR subunit groups were named as proposed by Mongan et al. [35]. The three letter prefixes in AChR subunit gene names, Cel, Tci, Hco, Aca, Tca, Peq, Ovo, Dim, Llo, Bma, Sra and Ptr refer to Caenorhabditis elegans, Teladorsagia circumcincta, Haemonchus contortus, Ancylostoma caninum, Toxocara canis, Parascaris equorum, Onchocerca volvulus, Dirofilaria immitis, Loa loa, Brugia malayi, Strongyloides ratti and Parastrogyloides trichosuri respectively. Parasitic nematode species belonging to Clade III, VI and V are highlighted in red, green and blue respectively.
Fig 2.
Amino-acid alignments of ACR-26 and ACR-27 AChR subunit sequences from Haemonchus contortus and Parascaris equorum.
acr-26 and acr-27 deduced amino-acid sequences from Haemonchus contortus and Parascaris equorum were aligned using the MUSCLE algorithm [36] and further processed using GeneDoc. Predicted signal peptide sequences are shaded in grey. Amino acids conserved between ACR-26 and ACR-27 sequences are highlighted in red. Amino acids specifically shared by ACR-26 homologs are highlighted in dark blue. Amino acids specifically shared by ACR-27 homologs are highlighted in light blue. The cys-loop, the four transmembrane regions (TM1–TM4) and the primary agonist binding (YxCC) which is present in ACR-26 homologs but absent in ACR-27 homologs are noted above the sequences. Hco (Haemonchus contortus), Peq (Parascaris equorum).
Fig 3.
Expression monitoring of Hco-acr-26 and Hco-acr-27 in Haemonchus contortus.
(A) Transcription of Hco-acr-26 and Hco-acr-27 throughout the H. contortus lifecycle as revealed by RT-PCR experiments. ω: embryonated egg; L2: second stage larvae; L3: ensheated third stage larvae; XL3: in vitro exsheated third stage larvae, L4: fourth stage larvae; m: adult males; f: adult females. Integrity of cDNA preparations was verified by PCR using primers designed to amplify a 380 bp fragment of the H. contortus gapdh cDNA. (B) Relative mRNA expression levels of Hco-acr-26 and Hco-acr-27 in H. contortus L3 and adult males. Real-time RT-PCR experiments were performed in triplicate for third stage larvae (L3) and adult males corresponding to free-living and parasitic stage respectively. Each set of experiments was repeated twice using two independent cDNAs templates. The mRNA expression level for Hco-acr-26 was normalized to 1. The mRNA fold changes were calculated using three distinct reference genes encoding for gapdh, actin and β-tubulin. The data are presented as fold changes in mean ± SEM of mRNA expression. No difference was observed between relative mRNA expression of Hco-acr-26 and Hco-acr-27 in L3 and adult males (paired Student’s t-test).
Fig 4.
In-situ hybridizations of Hco-acr-26 and Hco-acr-27 mRNAs in H. contortus.
XL3 larvae of H. contortus were fixed and hybridized with digoxygenin labeled antisense cDNA probes to monitor the localization of Hco-acr-26 (panel A) and Hco-acr-27 mRNAs (panel B) within the worm. Sense probes were used as negative control. cDNA/mRNA hybridomes were detected using primary anti-digoxygenin antibodies in combination with secondary Alexa 594-labeled antibodies (red). Body wall muscular cells were stained using primary antibodies raised against the myosin protein and further revealed with secondary Alexa 488-labeled antibodies (green). The scale bars correspond to 20 μm.
Fig 5.
Immunolocalization of Hco-ACR-26 and Hco-ACR-27 in H. contortus.
H. contortus L2 larval stage and adult males were fixed and incubated with affinity-purified antibodies raised against Hco-ACR-26 and Hco-ACR-27 subunits. Alexa 594- and Alexa 488- labeled secondary antibodies were used to determine the localization of Hco-ACR-26 (red) and Hco-ACR-27 (green) respectively. (A and B) Transmitted light and corresponding fluorescent apotome imaging performed on H. contortus L2 stage. Nematode’s nuclei were stained in blue using DAPI. Panel A shows a staining for Hco-ACR-26 and Hco-ACR-27 in striated cells of the nematode. Panel B shows that in a deeper focal section, Hco-ACR-26 and Hco-ACR-27 were also found to be expressed in another tissue potentially corresponding to the nerve ring (white arrows). (C, D and E) Confocal microscopy performed on L2 stage (C and E) and cross section of adult males (D). Body wall muscle cells were stained using primary antibodies raised against the myosin protein and further revealed with secondary Alexa 350-labeled antibodies (blue). (E) Negative control performed with anti-Hco-ACR-26 and anti-Hco-ACR-27 pre-adsorbed with their respective peptide antigen. For A, B, C and E anterior region is to the right. The scale bars correspond to 20 μm.
Fig 6.
Identification of the minimal subunit combination and ancillary proteins required for the functional expression in Xenopus oocyte of AChR containing ACR-26 and ACR-27 from H. contortus and P. equorum.
For both parasitic nematode species, the robust expression of functional AChRs required the co-injection of cRNAs corresponding to ACR-26 and ACR-27 AChR subunits in combination with the three ancillary factors RIC-3.1; UNC-50 and UNC-74. Currents were measured at the plateau. (A) Average plateau values for co-injection of all cRNAs were 161.4±18.67 nA (n = 41) for H. contortus receptor. (B) Average plateau values for co-injection of all cRNAs were 246.8±33.39 nA (n = 37) for P. equorum receptor. Results are shown in mean ± SEM.
Fig 7.
Pharmacological profiles of Hco-26/27 and Peq-26/27.
(A and B) Representative recording traces from a single oocyte expressing Hco-26/27 (A) or Peq-26/27 (B) challenged with 100 μM ACh and 100 μM of different anthelmintic compounds (morantel (Mor), pyrantel (Pyr), oxantel (Oxa), levamisole (Lev), bephenium (Beph) and nicotine (Nic)). The bars indicate the time period of the agonist application. (C and D) Scatter plot (mean ± SEM) of normalized currents elicited by 100 μM of anthelmintic compounds on Hco-26/27 (C) or Peq-26/27 (D). Currents have been normalized to and compared with 100 μM ACh currents. Paired Student’s t-test, ***p<0.001. (E) Dose-response relationships of Hco-26/27 for the agonists ACh (black circle, n = 6), Mor (red squares, n = 8) and Pyr (blue triangles, n = 6). Responses are all normalized to the response to 1 mM acetylcholine. EC50 are 80.1±1.1 μM, 29.0±1.3 μM and 6.8±1.3 μM for ACh, Mor and Pyr respectively. (F) Dose-response relationships of Peq-26/27 for the agonists ACh (black circle, n = 7), Mor (red squares, n = 9), Pyr (blue triangles, n = 10) and Lev (green lozenges, n = 6). Responses are all normalized to the response to 1 mM acetylcholine. EC50 are 34.9±1.1 μM; 0.32±0.26 μM; 0.98±0.26 μM and 16.7±1.3 μM for ACh, Mor, Pyr and Lev respectively.
Fig 8.
Co-expression of ACR-26 and ACR-27 in C. elegans increases its morantel sensitivity.
Thrashing assays were performed during 30 minutes with t0 corresponding to basal movements. For each C. elegans expressing parasitic nematode ACR-26 or ACR-27 alone or in combination, two independent lines were used with >12 worms per lines. Thrashing assays performed with 40 μM morantel on N2 (WT) and transformed worms expressing ACR-26 and ACR-27 subunits (alone or in combination) from H. contortus (A) or P. equorum (B). All results are expressed as mean ± SEM. Using C. elegans N2 as reference, statistical analysis was performed using the One-Way ANOVA with Tukey’s Multiple Comparison Test with ***p<0.001, NS, not significant.