The relationship between intraflagellar transport and upstream protein trafficking pathways and macrocyclic lactone resistance in Caenorhabditis elegans

Abstract

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. It has recently been shown in the model nematode Caenorhabditis elegans that the sensory cilia of the amphid neurons play an important role in resistance toward macrocyclic lactones such as ivermectin (an avermectin) and moxidectin (a milbemycin) either through reduced uptake or intertissue signaling pathways. This study interrogated the extent to which ciliary defects relate to macrocyclic lactone resistance and dye-filling defects using a combination of forward genetics and targeted resistance screening approaches 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 nonmotile structures. In total, 24 novel C. elegans anthelmintic survival-associated genes were identified in this study. When combined with previously known resistance genes, there are now 46 resistance-associated genes that are directly involved in amphid, cilia, and intraflagellar transport function.

Keywords: Caenorhabditis elegans; amphids; anthelmintic resistance; ciliogenesis; intraflagellar transport; ivermectin; macrocyclic lactone; moxidectin; xenobiotic resistance.

Fig. 1.

Representative images of DiI phenotypes at 250× magnification. DiI, 1,1ʹ-dioctadecyl-3,3,3ʹ,3ʹ-tetramethylindocarbocyanine perchlorate. a) N2: DiI dye-filling positive, b) ifta-1(nx61): weak DiI dye-filling positive, c) dyf-2(m160): DiI dye-filling negative and c14h10.2(tm10737): novel Dyf mutant that has variable DiI dye filling with d) weak positive individuals in a predominantly e) negative population. Individuals were photographed using a DIC filter (lower right inset image) to highlight the position and orientation of the worm and a FITC filter (main image) to visualize fluorescence. Areas of fluorescence for weak phenotypes are highlighted with arrows.

Fig. 2.

Position of novel and tested alleles in resistance genes identified by whole-genome sequencing. Transcript structures and positions of genes were obtained from WormBase (https://wormbase.org) (JBrowse version: WS281; genome build WBcel235). Arrows above alleles point to their location in the genomic sequence. Solid lines directly above alleles span the length of deletions. Alleles featured (name = chr-number: position nt-change [aa-change]) are e1124 = I: 8,071,718 G > A (Q > Stop); ka30 = IV: 3,797,404 G > A (Q > Stop); ka32 = I: 8,070,133 C > T (G > R); ka33 = V: 13,150,172–13,150,276 deletion; ka35 = I: 8,058,869–8,079,083 deletion; ka64 = I: 8,075,488 A > T (L > Stop); ka66 = I: 8,072,572 C > T (E > K); ka200 = X: 16,544,813 C > T (Q > Stop); ka201 = V: 13,150,224 AGG > AG frameshift; ka202 = III: 13,676,892 G > A (Q > Stop); ka203 = I: 8,077,873 G > A splice site acceptor change; ka204 = X: 5,550,502 A > T (C > Stop); m160 III: 13,686,367 G > A (R > Stop); nx61 = X: 5,545,532–5,547,540 deletion; p802 = IV: 3,797,722 G > A (Q > Stop); p808 = X: uncharacterized; p816 = X: uncharacterized ∼600-bp deletion.

Fig. 3.

IFT in C. elegans and resistance patterns in the IFT protein–protein interaction network. a) Summary of ciliary cargo transport in C. elegans during IFT. Line = protein/complex–protein/complex interaction; small arrow = change in protein or complex localization or interaction; large arrow = direction of IFT particle travel. b) A simplified version of predicted IFT protein–protein interaction network in C. elegans showing resistances found in mutants of each node. Box = group of proteins from the same complex or with the same function; line = predicted protein/complex–protein/complex interaction; small arrow = protein self-interaction. c) Summary of IFT complex interactions during IFT C. elegans. Line = protein/complex–protein/complex interaction; small arrow = change in protein or complex localization or interaction; large arrow = direction of IFT particle travel.

Fig. 4.

Ciliary protein trafficking pathways in C. elegans and resistance patterns in the ciliary gate protein–protein interaction network. a) Protein trafficking pathways used to deliver and remove ciliary proteins. Small arrow = show directionality of protein trafficking between cellular locations or organelles with key proteins and complexes involved in trafficking listed next to the arrow (placed before junctions if merging into a common secretion pathway); large arrow = directionality of axonal transport or passive diffusion. b) A simplified version of predicted basal body protein–protein interaction network in C. elegans showing resistances found in mutants of each node. Box = group of proteins from the same complex or with the same function; line = predicted protein–protein interaction; / = multiple (2–4) candidate genes with homology to a node found in other species (if gene IDs differ only by the last digit, then only the last digit is shown to the right of the candidate with a similar ID); Ce (node name of vertebrate ortholog) = multiple (>4) candidate genes with homology to the node found in other species.

Fig. 5.

Potential routes of macrocyclic lactone entry into C. elegans tissues and possibilities eliminated. Summary of routes that would facilitate macrocyclic lactone entry into C. elegans. Red star = macrocyclic lactone molecule; small arrow = show directionality of transport; large arrows = carrier protein cycling between membrane surfaces; gold circle = endosome; red cross = possible entry route that has been eliminated; green question mark = potential entry route remaining.