The uptake of avermectins in Caenorhabditis elegans is dependent on Intra-Flagellar Transport and other protein trafficking pathways

Parasitic nematodes are globally important and place a heavy disease burden on infected humans, crops and livestock, while commonly administered anthelmintics used for treatment are being rendered ineffective by increasing levels of resistance. Although the modes of action and resistance mechanisms caused by detoxification and target site insensitivity for these compounds is well documented, the mechanisms for uptake, which can also cause resistance, are still poorly defined. It has recently been shown in the model nematode Caenorhabditis elegans that the avermectins or macrocyclic lactones such as ivermectin and moxidectin gain entry though the sensory cilia of the amphid neurons. This study interrogated the molecular mechanisms involved in the uptake of avermectins using a combination of forward genetics and targeted resistance screening approaches along with visualising a BODIPY labelled ivermectin analog and confirmed the importance of intraflagellar transport in this process. This approach also identified the protein trafficking pathways used by the downstream effectors and the components of the ciliary basal body that are required for effector entry into these non-motile structures. Mutations in many of the genes under investigation also resulted in resistance to the unrelated anthelmintic drugs albendazole and levamisole, giving insights into the potential mechanisms of multidrug resistance observed in field isolates of the parasitic nematodes that are a scourge of ruminant livestock. In total 50 novel C. elegans anthelmintic survival associated genes were identified in this study, three of which (daf-6, rab-35 and inx-19) are associated with broad spectrum cross resistance. When combined with previously known resistance genes, there are now 53 resistance associated genes which are directly involved in amphid, cilia and IFT function. Author Summary Nematodes represent significant pathogens of man and domestic animals and control relies heavily on limited classes of Anthelminitic drugs. Single and multi-drug resistance is a growing problem however mechanisms of anthelmintic drug resistance and drug uptake by nematodes remain to be clearly elucidated. In Caenorhabditis elegans there has been an association between amphid and dye filling defects with resistance to avermectins however the effector and causal mechanisms remain elusive. This study uses a combination of fluorescently labelled anthelmintics and anthelmintic resistance screens to probe the uptake mechanisms for these drugs. The role of the sensory amphids in the uptake of avermectins was confirmed. The avermectins enter the distal segment of the cilia using an effector which is delivered by the UNC-119 and UNC-33/UNC-44 transport systems to the base of the cilia, followed by distal appendage dependent entry and transport along the cilia by the intraflagellar transport pathway. Of the genes investigated, three (daf-6, rab-35 and inx-19) were linked to cross resistance against all the anthelmintics tested (Ivermectin, Moxidectin, Albendazole and Levamisole). This study gives further insight into how important classes of anthelmintics enter nematodes and highlights the potential for this process to give rise to anthelmintic resistance.


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Abstract: 22 Parasitic nematodes are globally important and place a heavy disease burden on infected humans, 23 crops and livestock, while commonly administered anthelmintics used for treatment are being 24 rendered ineffective by increasing levels of resistance. Although the modes of action and resistance 25 mechanisms caused by detoxification and target site insensitivity for these compounds is well 26 documented, the mechanisms for uptake, which can also cause resistance, are still poorly defined. It 27 has recently been shown in the model nematode Caenorhabditis elegans that the avermectins or 28 macrocyclic lactones such as ivermectin and moxidectin gain entry though the sensory cilia of the 29 amphid neurons. This study interrogated the molecular mechanisms involved in the uptake of 30 avermectins using a combination of forward genetics and targeted resistance screening approaches 31 along with visualising a BODIPY labelled ivermectin analog and confirmed the importance of 32 intraflagellar transport in this process. This approach also identified the protein trafficking pathways 33 used by the downstream effectors and the components of the ciliary basal body that are required for 34 effector entry into these non-motile structures. Mutations in many of the genes under investigation 35 also resulted in resistance to the unrelated anthelmintic drugs albendazole and levamisole, giving 36 insights into the potential mechanisms of multidrug resistance observed in field isolates of the 37 parasitic nematodes that are a scourge of ruminant livestock. In total 50 novel C. elegans 38 anthelmintic survival associated genes were identified in this study, three of which (daf-6, rab-35 39 and inx-19) are associated with broad spectrum cross resistance. When combined with previously 40 known resistance genes, there are now 53 resistance associated genes which are directly involved in 41 amphid, cilia and IFT function.

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Nematodes represent significant pathogens of man and domestic animals and control relies heavily 44 on limited classes of Anthelminitic drugs. Single and multi-drug resistance is a growing problem 45 however mechanisms of anthelmintic drug resistance and drug uptake by nematodes remain to be 46 clearly elucidated. In Caenorhabditis elegans there has been an association between amphid and dye 47 filling defects with resistance to avermectins however the effector and causal mechanisms remain 48 elusive. This study uses a combination of fluorescently labelled anthelmintics and anthelmintic 49 resistance screens to probe the uptake mechanisms for these drugs. The role of the sensory amphids 50 in the uptake of avermectins was confirmed. The avermectins enter the distal segment of the cilia 51 using an effector which is delivered by the UNC-119 and UNC-33/UNC-44 transport systems to the 52 base of the cilia, followed by distal appendage dependent entry and transport along the cilia by the 53 intraflagellar transport pathway. Of the genes investigated, three (daf-6, rab-35 and inx-19) were 54 linked to cross resistance against all the anthelmintics tested (Ivermectin,Moxidectin,Albendazole 55 and Levamisole). This study gives further insight into how important classes of anthelmintics enter 56 nematodes and highlights the potential for this process to give rise to anthelmintic resistance.

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Introduction: 59 Parasitic nematodes place a highly significant and heavy disease burden on infected plants and 60 animals causing annual global yield and productivity losses in excess of $100 billion (1, 2) and in 61 addition requires over $20 billion annually to treat with anthelmintics (3). Currently available broad 62 spectrum anthelmintics are from a limited range of chemical families (3) and resistance to one or 63 more classes is becoming widespread in field populations (4) jeopardizing food security and human 72 avermectins is determined by saccharide groups on C-13 (eg. ivermectin), methoxime on C-23 (eg. 73 moxidectin) and alkyl groups on C-25 of the lactone ring (6, 7). Since subunit interactions vary 74 between different avermectins, commonly occurring target site insensitivity mutations in one 75 subunit binding site do not necessarily confer cross resistance to other macrocyclic lactones (8). As 76 nematodes have limited capacity for phase I detoxification of macrocyclic lactones (9, 10), resistance 77 relies on increased phase II conjugation and efflux (11), target site insensitivity or reduced drug 78 uptake (12). However, all identified and candidate resistance genes that interact directly with 79 avermectins or their metabolites function downstream of macrocyclic lactone uptake (11)(12)(13). The 80 macrocyclic lactones lack the chemical properties that would allow them to spontaneously cross 81 biological membranes (14) meaning that uptake is dependent on the ability of biological systems of 82 the organism to accumulate appropriate concentrations in the target tissues however, the 83 mechanism and associated genes involved in uptake are still unknown or poorly defined. 84 There is a high degree of conservation in the layout of the central nervous system between 85 nematode species, which consists of around 200-300 neurons, with sensory inputs from sensilla 86 being processed by the nerve ring to output motor neuron mediated responses (15)(16)(17). The amphid 87 sensilla function as the primary sensory organ for environmental stimuli (chemical, ion and osmotic 88 gradients, temperature, pheromones and noxious compounds). The sensilla consist of two pairs of 89 12-13 neurons (12 in Caenorhabditis elegans) which have non-motile cilia enriched in G protein-90 coupled receptors on the dendrites that are exposed to the environment through pores in the cuticle 91 (16,(18)(19)(20). Ciliogenesis of sensory cilia utilise assembly pathways that are conserved throughout 92 Eukaryota where a centriole derived basal body anchors to the cell membrane restricting the local 93 diffusion of proteins and lipids and organises microtubules (21). These microtubules are then used 94 for the delivery of lipids and proteins to the growing cilia by intraflagellar transport (IFT) complexes 95 that travel along the microtubules using dynein and kinesin motors (21).

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There has been an observed correlation between macrocyclic lactone resistance caused by 97 reduced uptake and defects in amphid morphology in Caenorhabditis elegans with several causative 98 genes being associated with dye-filling, chemosensation, osmosensation, dauer formation and 99 mechanosensation defective phenotypes (12,22,23 Table S1.

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The cilia axoneme is composed of polarised tubulins (including DYF-10 and DYF-12 (58) (90) 582 so it was surprising that these phenotypes were unable to be replicated in mutants for any of the exocyst complex genes tested (exoc-7, exoc-8 and sec-6) indicating this pathway only plays a minor 584 role during chronic exposure. Despite having a known interaction with IFT-20 (82), the BLOC-1 585 complex was not associated with survival against any of the anthelmintics tested. The UNC-119 586 secretion pathway of myristoylated and laurylated acyl-anchored membrane proteins (91) is known 587 to deliver ARL-3 and ARL-13 to the cilia facilitating regulation of the assembly and disassembly of the 588 IFT complexes (92). The observed resistance to avermectins in the Unc-119(e2498) and Arl-589 13(tm1745) mutants may therefore be explained by the impaired delivery of proteins involved in 590 cilia maintenance resulting in truncated cilia (49).

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The ciliary gate of the basal body ( Fig 5B) acts as an impermeable barrier to proteins and 592 macromolecules over 70 kDa in size (93)

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The protein DYF-19 is known to play an important role in facilitating the passage of IFT components across the transition zone of the basal body (95) a fact that explains why both  610 mutants and those for Hyls-1(tm3067), which connects DYF-19 to the mother centriole of the basal 611 body (106, 107), were also found to be resistant to avermectins and showed impaired uptake of FBI. amphid cilia proteins and the downstream effectors for ivermectin susceptibility would become disorganised in these mutants. In an attempt to identify which kinesins are responsible for delivering 662 these proteins to the end of the axon, the axonal kinesins KLC-1, 124,125) 663 were investigated but found not to influence ivermectin tolerance, indicating either functional 664 redundancy or that ciliary proteins are transported by other cytoplasmic kinesin family members. 665 Unlike the other kinesins tested, the kinesin-13 family, of which KLP-7 is a member, has roles in 666 microtubule depolymerisation and primary ciliogenesis (126) plays an important role in lumen formation and the morphogenesis of the amphidial sheath but is 719 also present in other tubular lumens, such as the intestine, which would also potentially be exposed 720 to anthelmintics (141, 142). DAF-6 is predicted to function by inhibiting endocytosis of the 721 extracellular matrix (142), a determining factor in apical-basal polarity of the lumen (143) anthelmintics. The use of a fluorescently labelled ivermectin probe allowed the hypothesis that the 754 amphids are the tissue responsible for ivermectin uptake to be visually confirmed while the labelled 755 albendazole probe has shown that albendazole enters via the gut. Differences in the intensity of 756 absorbed probe fluorescence compared to wild type also corresponded well with observed 757 resistance in the tested mutant strains. As the intensity of fluorescence is proportional to 758 concentration at the steady state the question remains open as to whether a reduction in uptake or 759 increase in efflux is responsible for the reduced intensity observed in many of the tested mutants, 760 however, given the functions of the mutated genes it is probably being caused by reduced uptake.

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The reduced primary toxicity observed in the FBI probe was to be expected as even small Whole genome sequencing of forward genetics screen mutants 777 The avermectin resistance causing genes uncovered by random mutagenic screens and identified by 778 whole genome sequencing were all determined to be involved in IFT. The dynein heavy chains che-3 779 and dhc-3 were found to be mutated more commonly than other genes. This is similar to 780 TP238(ka32) and TP239(ka33) from the previous study looking for ivermectin resistance (23) and 781 other studies looking for dye-filling defects (49,151). This overrepresentation of dyneins in forward 782 genetic screens is probably caused by dynein heavy chains having very long coding sequences 783 (12,516nt and 9,828nt for che-3 and dhc-3 respectively) making them more prone to mutation by 784 EMS as the rate of mutation for a loss of function mutation is proportional to gene size (152). Having 785 found genes involved in ciliogenesis and IFT is not surprising as it is a complex process requiring the 786 interaction of multiple genes to produce a functional structure and lacks redundancy. Therefore, for 787 the aforementioned reasons, genes involved in IFT are statistically more likely to be mutated by EMS 788 than single downstream effectors that rely on functional cilia.

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Conclusion: 790 The findings of this study not only provide strong evidence that the avermectin compounds 791 ivermectin and moxidectin are taken up via the amphid cilia as has been shown previously (23)