Recent advances in candidate-gene and whole-genome approaches to the discovery of anthelmintic resistance markers and the description of drug/receptor interactions

Abstract

Anthelmintic resistance has a great impact on livestock production systems worldwide, is an emerging concern in companion animal medicine, and represents a threat to our ongoing ability to control human soil-transmitted helminths. The Consortium for Anthelmintic Resistance and Susceptibility (CARS) provides a forum for scientists to meet and discuss the latest developments in the search for molecular markers of anthelmintic resistance. Such markers are important for detecting drug resistant worm populations, and indicating the likely impact of the resistance on drug efficacy. The molecular basis of resistance is also important for understanding how anthelmintics work, and how drug resistant populations arise. Changes to target receptors, drug efflux and other biological processes can be involved. This paper reports on the CARS group meeting held in August 2013 in Perth, Australia. The latest knowledge on the development of molecular markers for resistance to each of the principal classes of anthelmintics is reviewed. The molecular basis of resistance is best understood for the benzimidazole group of compounds, and we examine recent work to translate this knowledge into useful diagnostics for field use. We examine recent candidate-gene and whole-genome approaches to understanding anthelmintic resistance and identify markers. We also look at drug transporters in terms of providing both useful markers for resistance, as well as opportunities to overcome resistance through the targeting of the transporters themselves with inhibitors. Finally, we describe the tools available for the application of the newest high-throughput sequencing technologies to the study of anthelmintic resistance.

Keywords: Anthelmintic drugs; Anthelmintic resistance; Anthelmintic targets; Molecular markers; Receptors.

Graphical abstract

Fig. 1

Schematic representation of principal known anthelmintic resistance pathways, and their relevance to each of the current anthelmintic drug classes. The ability of the drug to enter the worm and interact with its target receptor in order to trigger a harmful physiological effect (shown at top for a drug- susceptible worm) is diminished through four principal mechanisms. These mechanisms apply to varying degrees to the major anthelmintic drug classes, as indicted by the relative font of the drug class names at the base of the figure; ML = macrocyclic lactones, TCBZ = triclabendazole, Lev = levamisole (as a representative of the nicotinic agonist drug class), BZ = benzimidazoles, AAD = amino-acetonitrile derivatives; ^∗denotes that resistance to the AADs is only characterised in laboratory-selected isolates.

Fig. 2

Mechanistic and structural features of muscle nematode somatic muscle ion channels. (A) Diagram of the putative pentameric subunit composition of the levamisole receptor in Oesophagostomum dentatum composed of one or more subunits of UNC-63, UNC-29, UNC-38, and ACR-8. (B) Two-micropipettes used for a two electrode voltage-clamp oocyte recordings of expressed nAChRs from O. dentatum. (C) Diagram of a proposed mechanism of calcium entry and muscle contraction in Ascaris suum muscle, with entry through the sarcolemma via calcium permeable nicotinic acetylcholine receptors (nAChRs activated by levamisole) and voltage-activated calcium channels (VACCs) which produce the biggest component of contraction (ryanodine-insensitive), and another component of contraction (ryanodine-sensitive) mediated by a calcium-induced calcium-release via the ryanodine receptors in the sarcoplasmic reticulum.

Fig. 3

Effects of derquantel and abamectin on Ascaris suum muscle strips. (A) Isometric contraction of Ascaris suum muscle strips produced by application of increasing concentrations of acetylcholine, and antagonism by 1 μM derquantel (red bar), 1 μM derquantel+0.3 μM abamectin (green bar), and wash (blue bar). Note that derquantel decreases the responses to acetylcholine and that the addition of abamectin increases the inhibition. (B) The concentration-depolarizing-response plot of acetylcholine showing mean ± S.E. (n = 11). Control (black); in the presence of 1 μM derquantel (red); 1 μM derquantel+0.3 μM abamectin (green) and wash (blue). Note that abamectin increases the inhibition produced by derquantel (Figure modified from Puttachary et al., 2013). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Expression pattern of monepantel receptor in C. elegans, and model for the interaction of the drug and its receptor in H. contortus. (A) Expression of ACR-23. Transgenic L4 larva containing an integrated array expressing the acr-23 open reading frame fused to the green fluorescent protein gene. Transgene expression was mainly visible in the body wall muscle bundles (white arrows), and in two unidentified cells, which are neither the PLM neurons nor body wall muscle cells nuclei (white arrowheads in the inset, which shows a magnification of the tail). Gut granules emit yellow autofluorescence. (B) Image taken by differential interference contrast microscopy. Black arrows and arrowhead indicate the pharyngeal bulbs and the position of the developing vulva, respectively. The inset shows a detail of the tail, ventral view. The rectal opening (asterisk) is immediately anterior to the two GFP labelled cells in A. Bar, 50 μm. (C) Hypothetical model for the interaction of monepantel with its target receptor in H. contortus, Hco-MPTL-1. In the resting situation, the MPTL-1 receptor is closed and no ion is flowing through the channel. The neurons or muscle cells are silent respectively not contracted. When the receptor-agonist (e.g. choline or betaine) is released from a presynaptic or potentially an epidermal cell, it binds to the MPTL-1 receptors present at the postsynaptic nerve cell or at the body wall muscle cell. An inflow of Na⁺ ions enters the cell through the pore formed by the opened receptor, creating a depolarization of the cell membrane. This leads to the stimulation of the nerve cell or to the pulse contraction of the muscle cell and finally a controlled movement. The interaction of monepantel with MPTL-1 results in a permanent stimulation or contraction creating a spastic paralysis of the nematode and its expulsion from the host. The ancillary protein RIC-3, which is resident in the endoplasmic reticulum (ER), may play a role for the assembly of the receptor containing MPTL-1. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Utility of high-throughput sequencing (HTS) to explore the development of anthelmintic resistance. Four separate HTS applications shown as coloured boxes in columns A–D, with single horizontal black box representing generalized features of library construction, sequencing and post-sequencing quality control applicable to most HTS applications (^∗fragmentation not required for small RNA sequencing). (A) Blue boxes present the detection of genetic mutations through de novo assembly and subsequent comparative alignment, or in comparison with a reference genome. Analyses relevant to genomic sequencing include the identification of single nucleotide polymorphisms (SNP), as well as, structural (SV) and gene copy number variation (CNV). (B) Green boxes outline the use of RNA-seq to define differentially expressed genes (DEG) through de novo transcriptomic assembly, or a referenced transcriptome assembly through alignment to an existing genome. (C) Orange boxes demonstrate the identification of small non-coding RNAs by their mapping to a reference genome and exploration of their potential role in regulating differentially expressed genes. These analyses include the identification of micro-RNAs (miRNAs) by prediction of their hair-pin precursor, and their annotation using data in miRBase (www.miRBase.org). Additional small RNAs can be explored by identifying ncRNA precursor transcripts (represented by large, contiguous clusters of small-RNA reads mapping to a reference genome) and their annotation through comparisons [e.g., by hidden Markov modeling (HMM)] with the Rfam database (rfam.sanger.ac.uk); ^α small-RNAs are also identified by their length, sequence and relative mapping position (e.g., with respect to coding genes, gene regulatory and/or transposable elements). (D) Purple boxes describe the use of ChIP-seq and bisulfite sequencing (BSS) to explore epigenetic changes, including histone modification and DNA methylation respectively, that might impact on gene regulation. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)