Functional genomics in Brugia malayi reveal diverse muscle nAChRs and differences between cholinergic anthelmintics

Abstract

Many techniques for studying functional genomics of important target sites of anthelmintics have been restricted to Caenorhabditis elegans because they have failed when applied to animal parasites. To overcome these limitations, we have focused our research on the human nematode parasite Brugia malayi, which causes elephantiasis. Here, we combine single-cell PCR, whole muscle cell patch clamp, motility phenotyping (Worminator), and dsRNA for RNAi for functional genomic studies that have revealed, in vivo, four different muscle nAChRs (M-, L-, P-, and N-). The cholinergic anthelmintics had different selectivities for these receptors. We show that motility and patch-clamp responses to levamisole and pyrantel, but not morantel or nicotine, require the unc-38 and/or unc-29 genes. Derquantel behaved as a competitive antagonist and distinguished M-nAChRs activated by morantel (K_b 13.9 nM), P-nAChRs activated by pyrantel (K_b 126 nM), and L-nAChRs activated by levamisole (K_b 0.96 µM) and bephenium. Derquantel was a noncompetitive antagonist of nicotine, revealing N-type nAChRs. The presence of four diverse nAChRs on muscle is perhaps surprising and not predicted from the C. elegans model. The diverse nAChRs represent distinguishable drug targets with different functions: Knockdown of unc-38+unc-29 (L- and/or P-receptors) inhibited motility but knockdown of acr-16+acr-26 (M- and/or N-receptors) did not.

Keywords: Brugia; dsRNA; filaria; nAChR; patch clamp.

Fig. 1.

(A) Concentration response plots of the effects of levamisole, pyrantel, morantel, and nicotine on motility of whole adult female worms. Each point is the mean ± SE of four worms, each from a different batches of worms. Curves were fitted with variable slope nonlinear regression model analysis in GraphPad Prism 6. Concentration response curves for different agonists: LEV, levamisole 99.2 ± 2.3 nM; PYR, pyrantel 516 ± 3 nM; MOR, morantel 3.7 ± 0.7 µM; and NIC, nicotine 4.8 ± 0.8 µM (n = 4 for each concentration of agonist, four biological replicates). (B) Effects over time on motility of whole B. malayi of: 10 µM derquantel (Deq 10 µM); derquantel pretreatment followed by 10 µM levamisole (Deq 10 µM + Lev 10 µM, levamisole at the arrow); 10 µM levamisole (Lev 10 µM, levamisole at the arrow). Note that derquantel only has a modest effect on motility by itself, but clearly, it reduces the inhibitory effect of levamisole. n = 12, four biological replicates, two-way ANOVA and Bonferroni post hoc tests: ***P < 0.001; **P < 0.01.

Fig. 2.

(A) Single muscle cell PCR demonstrating the presence of different nAChR subunits expressed in single muscle cells. Representative gel pictures show the presence of mRNA for acr-16, acr-21, unc-29, unc-38, unc-63, acr-8, and acr-26. GAPDH was the internal control for each cell (example of n = 6, all positive for each gene). (B) Concentration-dependent effect of nicotinic agonists on B. malayi muscle cell whole cell patch-clamp recordings. Representative trace illustrating effects of increasing concentrations of levamisole on inward current, response is normalized to ACh 100 µM (first application) for EC₅₀ calculation. (C) Concentration-response curves for different agonists. ACH, acetylcholine; BEP, bephenium; LEV, levamisole; MOR, morantel; NIC, nicotine; PYR, pyrantel; (n = 7 for each agonist).

Fig. S1.

Single muscle cell PCR demonstrating the presence of ACR-27 nAChR amplicon expressed in single muscle cell (detected in two of eight single muscle cell PCR experiments). Representative gel picture shows acr-27. GAPDH was the internal control for each cell. M, single-cell muscle cell.

Fig. S2.

Derquantel is a potent antagonist. (A) Representative whole cell patch clamp recordings showing inward currents induced by different nAChR agonists in the absence and the presence of derquantel. Agonist concentrations were 30 µM for all except pyrantel, which was 10 µM. The agonists were acetylcholine (ACh), levamisole (Lev), pyrantel (Pyr), nicotine (Nic), bephenium (Bep), and morantel (Mor) in the absence and then presence (d) of derquantel (10 µM was used against all agonists except for morantel, where 1 µM was used). (B) Bar chart quantifying the average reduction of inward currents induced by different agonists in the presence of derquantel. All agonists were inhibited significantly (two-way ANOVA and Bonferroni post hoc tests, P < 0.001, n = 5 for each agonist). Ach-induced currents reduced from −1,483 ± 195.8 to −330.5 ± 64.5 pA; levamisole respponse reduced from −1,338 ± 252.9 pA to −437.5 ± 72.3 pA; pyrantel response reduced from −1,401 ± 224.6 to −645.4 ± 46.4 pA; nicotine-induced currents reduced from −419 ± 63.01 to –91.04 ± 11.5 pA and bephenium-induced inward currents were suppressed from −291.7 ± 42.8 to −109.7 ± 19.8 pA, respectively. *P < 0.05; **P < 0.01.

Fig. 3.

Selective antagonist effects of derquantel on nAChR agonist concentration response relationships. Control concentration response relationship in the absence (solid line) and then in the presence (broken line) of derquantel. (A) Morantel antagonism by 1 µM derquantel. (B) Pyrantel was antagonized in the presence of 10 µM derquantel. (C) Levamisole concentration–response relationship was antagonized by 10 µM derquantel. (D) Bephenium concentration response curve was antagonized in the presence of 10 µM derquantel. (E) Derquantel suppressed the nicotine concentration response relationship in noncompetitive manner, n = 5 worms for each set of experiments (CR, concentration ratio). (F) Diagrammatic representation of the four types of nAChR: P, L, M, and N. Also shown is their selective agonists with derquantel being the most potent inhibitor on the M-receptor. The L-receptor is shown as composed of the subunits ACR-63 (63), UNC-38 (38), UNC-29 (29), and ACR-8 (8); the P-receptor is shown as composed of the subunits ACR-63 (63), UNC-38 (38), and UNC-29 (29); the M-receptor is shown as composed of the subunits ACR-26 (26) and another (+), possibly ACR-27 subunit; the N-receptor is proposed to be composed of ACR-16-like (16-like) subunits and may include ACR-21. Bep, bephenium; CR, concentration ratio; Deq, derquantel; Mor, morantel; Nic, nicotine.

Fig. 4.

Effects of dsRNA unc-38+unc-29 on quantitative PCR and motility analysis demonstrate a selective reduction in unc-38 and unc-29 transcript levels and motility in dsRNA mixture soaked B. malayi. (A) Bar chart demonstrating quantitative PCR results showing a significant reduction in transcript levels of unc-29 (by 95 ± 2.8%, 29 red bar) and unc-38 (by 80 ± 6.9%, 38 red bar) from the control transcript levels in lacZ dsRNA-treated worms, unc-29 (by 7.3 ± 3.1%, 29 blue bar) and unc-38 (by 7.8 ± 2.9%, 38 filled blue bar). Quantitative PCR results are also shown for GAPDH (hatched bars) and for the GABA receptor subunit transcript unc-49 (open bars). n = 8, one-way ANOVA and Bonferroni post hoc tests; ***P < 0.001. (B) Time series of motility for dsRNA-soaked worms. Black color data points (circles) representing control, blue color data points (squares) lacZ (nonspecific dsRNA), and red (triangles) color representing the worms treated with mixture of unc-29 and unc-38 dsRNA. n = 38 worms for all treatments, two-way ANOVA and Bonferroni post hoc tests; ***P < 0.001.

Fig. S3.

Bar chart demonstrating % reduction in transcript level for whole worm quantitative PCR results produced by the 29+38 dsRNA mixture (red bars) and lacZ dsRNA (blue bars)-soaked B. malayi for: acr-26 (14.3 ± 9.6% and 15.0 ± 8.5%); acr-27 (10.2 ± 7.4% and 17.9 ± 6.3%); acr-16 (17.7 ± 4.6% and 16.2 ± 5.1%), and GAPDH (9.9 ± 3.6% and 6.4 ± 1.7%). GAPDH (n = 8 worms each), acr-26 (n = 5 worms each), acr-27 (n = 5 worms each), and acr-16 (n = 5 worms each). There was no significant difference between the dsRNA lacZ and dsRNA 29+38 values, P = 0.77, two-way ANOVA.

Fig. S4.

Effects of dsRNA acr-16+acr-26 on motility and quantitative PCR demonstrates no effect on motility following soaking for 5 d but a reduction in acr-16 and acr-26 mRNA. The effect of dsRNA acr-16+acr-26 contrasts with the effect on motility of dsRNA unc-38+unc-29. (A) Time series of motility for dsRNA soaked worms. Black (circles) representing control, blue (squares) lacZ (nonspecific dsRNA), red (triangles) (dsRNA unc-29 + unc-38), and green (triangles) (dsRNA acr-16+acr-26) color data points representing the different treatments of worms (n = 10, four biological replicates for each treatment, two-way ANOVA and Bonferroni post hoc tests; ***P < 0.001). (B) Bar chart demonstrating quantitative PCR results showing a significant reduction in transcript levels of acr-16 (hatched green, by 93 ± 2.0%) and acr-26 (filled green by 92 ± 1.1%) compared with the control transcript levels in lacZ dsRNA-treated worms, acr-16 (hatched blue by 27 ± 2.4%) and acr-27 (filled blue by 17 ± 4.9%). n = 6 worms. (unpaired t tests: ***P < 0.001; **P < 0.01; *P < 0.05). ds, double stranded.

Fig. 5.

Levamisole, pyrantel and bephenium currents are sensitive to unc-29 and unc-38 knockdown. (A) Representative traces demonstrating the inward current responses to acetylcholine, levamisole, nicotine, pyrantel, bephenium and GABA at -40 mV in control untreated and dsRNA-treated worms under whole-cell patch clamp (n = 8). (B) Bar chart illustrating the agonist current responses of 29+38 dsRNA-treated worm normalized against mean current response of each agonist in control untreated worms. n = 8 worms for each agonist. Only levamisole, pyrantel, and bephenium responses were significantly inhibited when 29+38 dsRNA worm’s responses were compared with untreated control dsRNA-treated worm currents: one-way ANOVA and Bonferroni post hoc tests: ***P < 0.001; **P < 0.01; *P < 0.05. Ach, acetylcholine; Bep, bephenium; Lev, levamisole; Mor, morantel; Nic, nicotine; Pyr, pyrantel.

Fig. S5.

Bar chart showing the mean ± SE agonist current responses of dsRNA unc-29+unc-38-treated worms normalized against their mean control (lacZ-treated) currents. n = 8 worms for each agonist. Only levamisole, pyrantel, and bephenium responses were significantly inhibited when dsRNA unc-29+unc-38 worm’s responses were compared with untreated control or lacZ dsRNA-treated worms. Two-way ANOVA and Bonferroni post hoc tests: ***P < 0.001; *P < 0.05. ACh, acetylcholine; Bep, bephenium; Lev, levamisole; Mor, morantel; Nic, nicotine; Pyr, pyrantel.

Fig. S6.

Effect of monepantel on ACh and choline responses. (A) Representative voltage clamp recording illustrating inward currents induced by ACh (10 µM) and choline (1 mM) in absence and presence of monepantel (1 µM). There is little or no effect. (B) Bar chart demonstrating that monepantel had no significant effect on the acetylcholine (ACh) and choline-induced inward currents (n = 5). Cho, choline; Mon, monepantel.