Functional investigation of conserved glutamate receptor subunits reveals a new mode of action of macrocyclic lactones in nematodes

Glutamate-gated chloride channels receptors (GluCls) are involved in the inhibition of neurotransmission in invertebrates and represent major molecular targets for therapeutic drugs. Among these drugs, macrocyclic lactones (MLs) are widely used as anthelmintic to treat parasitic nematodes impacting both human and animal health. Despite massive use of MLs since the 80’s, the exact molecular targets of these drugs are still unknown in many important parasite species. Among the GluCl subunit encoding genes, avr-14, glc-2, glc-3 and glc-4 are highly conserved throughout the nematode phylum. Using the Xenopus oocyte as an expression system, we pharmacologically characterized these GluCl subunits from the model nematode Caenorhabditis elegans, the human filarial nematode Brugia malayi and the horse parasitic nematode Parascaris univalens. In contrast with C. elegans, expression of parasitic nematode subunits as homomeric receptors was not reliable and needed glutamate application at the mM range to induce low currents at the nA range. However, the co-expression of GLC-2 and AVR-14B lead to the robust expression of ML-sensitive receptors for the three nematode species. In addition, we demonstrated that for C. elegans and P. univalens, GLC-2 co-assembled with GLC-3 to form a new GluCl subtype with distinct pharmacological properties. Whereas 1μM ivermectin, moxidectin and eprinomectin acted as agonist of the GLC-2/GLC-3 receptor from C. elegans, they did not directly activate GLC-2/GLC-3 of P. univalens. In contrast, these MLs potentialized glutamate elicited currents thus representing a unique pharmacological property. Our results highlight the importance of GLC-2 as a key subunit in the composition of heteromeric channels in nematodes and demonstrate that MLs act on novel GluCl subtypes that show unusual pharmacological properties, providing new insights about MLs mode of action. Author summary The filarial and ascarid parasitic nematodes include some of the most pathogenic or invalidating species in humans, livestock and companion animals. Whereas the control of these worms is critically dependent on macrocyclic lactones (MLs) such as ivermectin, the mode of action of this anthelmintic class remains largely unknown in these parasites. In the model nematode Caenorhabditis elegans, MLs target GluCl pentameric glutamate-sensitive chloride channels (GluCl). Because MLs are potent anthelmintics on C. elegans, ascarid and filarial nematodes, in the present study we investigated GluCl subunits highly conserved between these distantly related worms. Using the Xenopus oocyte as a heterologous expression system, we identified and performed the pharmacological characterization of novel GluCl receptors from C. elegans, the human filarial parasite Brugia malayi and the horse parasite Parascaris univalens. Our results highlight heteromeric GluCls from parasites as molecular targets for a wide range of MLs. We report an original mode of action of MLs on a new GluCl subtype made of the GLC-2/GLC-3 subunit combination. This study brings new insights about the diversity of GluCl subtypes in nematodes and opens the way for rational drug screening for the identification of next generation anthelmintic compounds.


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C. elegans, they did not directly activate GLC-2/GLC-3 of P. univalens. In contrast, these MLs 27 potentialized glutamate elicited currents thus representing a unique pharmacological property. 28 Our results highlight the importance of GLC-2 as a key subunit in the composition of 29 heteromeric channels in nematodes and demonstrate that MLs act on novel GluCl subtypes that 30 show unusual pharmacological properties, providing new insights about MLs mode of action.

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Author summary 32 The filarial and ascarid parasitic nematodes include some of the most pathogenic or The phylum Nematoda is divided into five major clades (I to V) that include free living 49 and parasitic species impacting both human and animal health (1,2). Among these parasitic 4 74 The molecular targets of the MLs as well as the mechanisms involved in resistance 75 remains unclear for most of the parasitic nematodes. In this context, a better understanding of 76 MLs mode of action is essential for the control of resistance and the development of novel 77 therapeutical strategies (35). 78 In the free-living model nematode Caenorhabditis elegans, MLs act as allosteric  Interestingly, in Cooperia oncophora (27) and Haemonchus contortus (50), GLC-2 also 96 combined with AVR-14B to form a heteromeric GluCl subtype sensitive to ivermectin. 97 Whereas GluCls investigations were mainly focused on clade V nematodes, the GluCl diversity 5 98 and the mode of action of MLs remains poorly understood in human and animal parasitic 99 nematodes from the clade III. 100 In the present study, we describe new GluCl subtypes made of highly conserved subunits 2), the first cys-loop domains specific of ligand-gated ion channels (LGIC) which is composed 118 of two cysteines that are 13 amino acid residues apart, the second cys-loop domain found in 119 GluCls and four transmembrane domains (TM1-4) ( Fig. S2 and Fig. S3). 120 In comparison with C. elegans, the GluCl deduced amino-acid sequences of B. malayi 121 and P. univalens respectively show an identity of 78.4% and 80.3% for AVR-14B, 64.1% and 122 67.2% for GLC-2, 66.5% and 67.8% for GLC-3 and 64.8% and 66.6% for GLC-4 (S1 Table). 123 A phylogenetic analysis including GluCl sequences from C. elegans (Cel), B. malayi (Bma), H. 124 contortus (Hco) and P. univalens (Pun) confirmed the orthologous relationship of the parasitic 125 subunit sequences with their respective counterparts in C. elegans (Fig. 1) In order to test the ability of C. elegans, B. malayi and P. univalens GluCl subunits to form 132 functional homomeric receptors, their respective cRNA (avr-14b, glc-2, glc-3 and glc-4) were 133 injected singly in the Xenopus laevis oocyte. Three to five days post injection, currents elicited 134 by 1 mM glutamate were recorded using the two-electrode voltage-clamp technic ( Fig. 2A). 135 Here, the C. elegans AVR-14B, GLC-2 and GLC-3 GluCl subunits were used as positive 136 controls as their ability to form functional glutamate-sensitive homomeric channels has been 137 previously reported (26,28,36,45). In our hands, expression of Cel-AVR-14B or Cel-GLC-2 138 alone formed functional homomeric receptors with robust glutamate-elicited currents in the µA 139 range: 2.1 ± 0.4 µA (n = 16) and 9.1 ± 0.5 µA (n = 32) for Cel-AVR-14B and Cel-GLC-2 140 respectively ( Fig. 2A). Whereas Cel-GLC-3 was also able to form functional receptors, the 141 glutamate application (1 mM) induced significantly smaller peak currents (285 ± 54 nA, n = 142 50) in comparison with homomeric channels composed of Cel-AVR14B (p < 0.001) or Cel-143 GLC-2 (p < 0.001) ( Fig. 2A). These results are in agreement with previous studies (26,36,45).

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In order to evaluate the involvement of GLC-2/GLC-3 as putative molecular targets for 256 MLs, we decided to focused our attention on ivermectin, moxidectin and eprinomectin 257 representing commonly used anthelmintic compounds (i.e. ivermectin and moxidectin used for 258 Parascaris spp. treatment; eprinomectin used for clade V parasites on lactating animals).
265 Surprisingly, for P. univalens, none of the tested MLs induced a current (Fig. 6D). This result was further confirmed using a wider panel of MLs (Fig. S4A). In order to investigate if the drug 267 application time could potentially impact the ML agonist effect, 1 µM ivermectin was perfused 268 during 90 s on Pun-GLC-2/GLC-3. Even with this long-lasting application, ivermectin showed 269 a weak agonist effect with a current amplitude corresponding to 3.56 ± 0.54 % (n = 7) of the 270 maximum response obtained with glutamate (Fig. S4B). 271 Because of this unexpected lack of activity as agonists, we hypothesized that MLs could   Table. 285 286

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Among the six distinct genes encoding GluCl subunits in C. elegans, AVR-14, GLC-2, 288 GLC-3 and GLC-4 are highly conserved in distantly related nematode species from different phylogenetic clades (52). Therefore, we reasoned that receptors including these subunits could 290 be involved in the broad-spectrum activity of MLs on nematodes.

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In the present study, using the xenopus oocyte as a heterologous expression system, we 292 identified a panel of functional homomeric and heteromeric receptors made of the GLC-2, 293 GLC-3 and AVR-14B subunits from the free-living model nematode C. elegans and B. malayi 294 and P. univalens, two parasitic nematodes presenting a major impact for human and equine 295 health respectively.  In the present study, our hypothesis that GLC-2 could combine with other GluCls and Bma-GLC-3 (Fig. S5, n = 12), confirmed that Bma-GLC-3 was a functional subunit.

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Therefore, we could only speculate that additional subunits combining to GLC-3 alone or GLC- In summary, our results highlight that subunit combination is critical for clade III 339 parasites to form functional glutamate-sensitive receptor in Xenopus oocyte expression system 340 but might also contribute to the diversity of GluCl subtypes in clade V nematodes.  In addition, we showed that this GluCl subtype is also sensitive to a wide range of MLs  Distinct pharmacology between C. elegans and parasites 373 In the present study, we reported that GLC-2/GLC-3 of C. elegans and P. univalens  since all eggs had a single pair of chromosomes (Fig. S1).