Characterization of the Ca2+-gated and voltage-dependent k+-channel slo-1 of nematodes and its interaction with emodepside

The cyclooctadepsipeptide emodepside and its parent compound PF1022A are broad-spectrum nematicidal drugs which are able to eliminate nematodes resistant to other anthelmintics. The mode of action of cyclooctadepsipeptides is only partially understood, but involves the latrophilin Lat-1 receptor and the voltage- and calcium-activated potassium channel Slo-1. Genetic evidence suggests that emodepside exerts its anthelmintic activity predominantly through Slo-1. Indeed, slo-1 deficient Caenorhabditis elegans strains are completely emodepside resistant. However, direct effects of emodepside on Slo-1 have not been reported and these channels have only been characterized for C. elegans and related Strongylida. Molecular and bioinformatic analyses identified full-length Slo-1 cDNAs of Ascaris suum, Parascaris equorum, Toxocara canis, Dirofilaria immitis, Brugia malayi, Onchocerca gutturosa and Strongyloides ratti. Two paralogs were identified in the trichocephalids Trichuris muris, Trichuris suis and Trichinella spiralis. Several splice variants encoding truncated channels were identified in Trichuris spp. Slo-1 channels of trichocephalids form a monophyletic group, showing that duplication occurred after the divergence of Enoplea and Chromadorea. To explore the function of a representative protein, C. elegans Slo-1a was expressed in Xenopus laevis oocytes and studied in electrophysiological (voltage-clamp) experiments. Incubation of oocytes with 1-10 µM emodepside caused significantly increased currents over a wide range of step potentials in the absence of experimentally increased intracellular Ca2+, suggesting that emodepside directly opens C. elegans Slo-1a. Emodepside wash-out did not reverse the effect and the Slo-1 inhibitor verruculogen was only effective when applied before, but not after, emodepside. The identification of several splice variants and paralogs in some parasitic nematodes suggests that there are substantial differences in channel properties among species. Most importantly, this study showed for the first time that emodepside directly opens a Slo-1 channel, significantly improving the understanding of the mode of action of this drug class

Involvement of neuronal receptors in anthelmintic modes of action analysed in Caenorhabditis elegans and parasitic nematodes

Auf Grund der wachsenden Resistenzproblematik bei der Bekämpfung von parasitischen Nematoden ist die Entdeckung neuer Wirkstoffe, die Aufklärung ihrer Wirkmechanismen und die Identifikation möglicher Wechselwirkungen von entscheidender Bedeutung. In dieser Arbeit konnte eine Beteiligung des γ Aminobuttersäure (GABA)-Rezeptors unc-49 im Wirkmechanismus des Anthelminthikums Emodepsid anhand von Caenorhabditis elegans Bewegungs- und Entwicklungsassays gezeigt werden. Die getesteten GABAA-Rezeptor unc-49 "loss of function" C. elegans Stämme erwiesen sich in den durchgeführten Assays als signifikant geringer empfindlich gegenüber der Wirkung von Emodepsid als der Wildtyp. Dies wurde durch ein "rescue" Experiment überprüft, für welches die unc 49B cDNA des zoonotischen Ascariden Toxocara canis vollständig identifiziert wurde. Die T. canis unc 49B cDNA wurde in ein Expressions- Plasmid gemeinsam mit dem UNC-49 C. elegans Promotor und einer C. elegans 3' UTR eingebaut und in die Gonaden der GABAA-Rezeptor unc-49 "loss of function" C. elegans Stämme injiziert. Auf diese Weise konnte der Wildtyp Phänotyp wieder hergestellt werden. Außerdem konnte noch eine verminderte Emodepsidempfindlichkeit bei C. elegans Mutanten, denen die Acetylcholin Rezeptoruntereinheiten lev-1 und unc-38 fehlten, festgestellt werden. Desweiteren wurden die Wirkmechanismen der anthelminthischen Wirkstoffe Piperazin, Tribendimidin und HLR-9291 anhand von C. elegans Assays überprüft und Wechselwirkungen mit Emodepsid, sowie das Vorkommen von "negative cross resistance" untersucht. Es konnte für Piperazin weder eine Beteiligung des GABAA Rezeptors unc-49 noch eine Beteiligung der untersuchten Acetylcholin Rezeptoruntereinheiten lev-1, lev-8, unc-38 und unc-63 am Piperazinwirkmechanismus festgestellt werden, obwohl es dafür Hinweise aus früheren Studien gab. Die Beteiligung der Acetylcholin-Rezeptoruntereinheiten lev-1, lev-8, unc-38 und unc-63 am Tribendimidinwirkmechanismus konnte dagegen bestätigt werden, denn lev-1, lev-8, unc-38 und unc-63 "loss of function" C. elegans Stämme zeigten sich im C. elegans Entwicklungsassay als resistent gegen Tribendimidin. Dem Tribendimidinderivat HLR-9291 liegt vermutlich der selbe Wirkmechanismus zu Grunde wie Tribendimidin. Auch hier sind die lev-1, lev-8, unc-38 und unc-63 "loss of function" C. elegans Stämme vollständig unempfindlich gegenüber HLR-9291. Allerdings ist die Wirkung von HLR-9291 deutlich schwächer als die Wirkung von Tribendimidin. Es wurden mögliche Wechselwirkungen zwischen Emodepsid, Tribendimidin und HLR-9291 überprüft und dabei festgestellt, dass es keinen Synergismus zwischen den Substanzen gibt. Allerdings konnte in mehreren Fällen das Auftreten von "negative cross resistance" festgestellt werden. Die Emodepsid-resistente Mutante slo-1 js379 zeigte sich deutlich empfindlicher als der Wildtyp bei Behandlung mit Piperazin und Tribendimidin bzw. HLR-9291. Die geringer Emodepsid- empfindlichen GABAA-Rezeptor unc-49 "loss of function" C. elegans Stämme waren ebenfalls sensibler als der Wildtyp bei Behandlung mit Tribendimidin und HLR-9291. Dies lässt darauf schließen, dass "negative cross resistance" ein bei Nematoden oft vorkommendes Ereignis ist und näher untersucht werden sollte. Die vorliegende Arbeit leistet einen Beitrag zur Aufklärung der Wirkmechanismen der anthelminthischen Wirkstoffe Emodepsid, Piperazin, Tribendimidin und HLR-9291 und liefert außerdem wichtige Erkenntnisse und neue Ansatzpunkte für zukünftige Forschungen im Bereich der anthelminthischen Wechselwirkungen.Due to the growing resistance problem in the control of parasitic nematodes the discovery of new drugs, the elucidation of their mechanisms of action and the identification of possible anthelmintic interactions are very important. In this research work the involvement of the γ-aminobutyric acid (GABA) receptor unc-49 in the mechanism of action of the anthelmintic emodepside was shown in Caenorhabditis elegans movement and development assays. In these assays the tested GABAA receptor unc-49 loss of function C. elegans strains were found to be significantly less susceptible to emodepside than the wildtype. The Toxocara canis unc 49B cDNA sequence was completely identified and used in a rescue experiment. The T. canis unc-49B cDNA was inserted into a plasmid together with the C. elegans UNC-49 promoter and a C. elegans 3' UTR. This plasmid was injected in the gonads of the GABAA receptor unc-49 loss of function C. elegans strains. Using this approach the wild-type phenotype was successfully restored. Interestingly, there was a lower susceptibility against emodepside in C. elegans mutants which have lost the acetylcholine receptor subunit lev-1 and unc-38. Furthermore, the mechanisms of action of the anthelmintic drugs piperazine, tribendimidine and HLR-9291 were examined using C. elegans the above mentioned assay. Possible interactions between emodepside and other anthelminthic substances were investigated as well as the presence of negative-cross-resistance. In contrast to evidence from previous studies, the current study did not indicate an involvement of the GABAA receptor unc-49 or the acetylcholine receptor subunit lev 1, lev-8, unc-38 and unc-63 in the mechanism of action of piperazine. However, lev-1 lev 8, unc-38 and unc-63 loss of function C. elegans strains were shown to be resistant to tribendimidine in the C. elegans development assay, therefore confirming the involvement of those acetylcholine receptor subunits in the mechanism of action of tribendimidine. The tribendimidine derivative HLR-9291 has obviously the same mechanism of action as tribendimidine since the lev-1 lev-8, unc-38 and unc-63 loss of function C. elegans strains were completely resistant to HLR-9291. However, the effect of HLR-9291 was significantly weaker than the effect of tribendimidine. Possible interactions between emodepside, tribendimidine and HLR-9291 were examined and no synergism between the substances was found. However, it was shown that negative cross resistance occurs often. The emodepside resistant mutant slo-1 js379 was significantly more susceptible than the wildtype when treated with piperazine, tribendimidine or HLR-9291. The GABAA receptor unc-49 loss of function mutants with reduced susceptibility to emodepside were also significantly more ssusceptible than the wildtype when treated with tribendimidine or HLR-9291.This suggests that "negative cross resistance" is an often occurring event in nematodes and should be further investigated. This work contributes to the elucidation of the mechanisms of action of the anthelmintic drugs emodepside, piperazine, tribendimidine and HLR 9291 and also provides important insights and new approaches for future research in the field of anthelminthic interactions

Calibration of tactile pressure sensing mats for static geotechnical centrifuge applications

The use of tactile pressure sensing mats has been gaining popularity among geotechnical centrifuge modellers. Tactile sensing systems such as Tekscan allow experimenters to obtain profiles of soil-structure contact pressures and visualise the results. This report builds upon previous work on the calibration of such pressure mats and describes how they were used to measure slab-soil and wall-soil contact pressures on basements models subject to heave movements in clay, for the benefit of future researchers who want to use tactile pressure mats for static geotechnical centrifuge applications. Each Tekscan sheet should be waterproofed by lamination and then calibrated. Known loads were applied onto Tekscan sheets using the Enerpac hydraulic frame in Schofield Centre. This produced individual calibration curves for each sensel. In contrast to previous work which fit a linear calibration relationship to measure cyclic load changes, large changes in pressure were expected in the basement heave centrifuge tests, with pressures sometimes dropping to near-zero values. Therefore, a quadratic fit with a forced zero intercept was applied to each sensel to capture the non-linearity of sensitivity. The dead weight of the basement slab and heavy fluid during spin-up and in-flight reconsolidation provided an independent check of the calibration factors. This check also generates a calibration adjustment factor which may account for the influence of centrifuge gravity on the tactile sensors’ sensitivity. The data was processed using Matlab with filtering in both time (averaging over 10 frames, typically) and space (taking special averages, typically over a 3×3 grid), and then presented as graphs and heat maps

Phylogenetic characterization of β-tubulins and development of pyrosequencing assays for benzimidazole resistance in cattle nematodes.

Control of helminth infections is a major task in livestock production to prevent health constraints and economic losses. However, resistance to established anthelmintic substances already impedes effective anthelmintic treatment in many regions worldwide. Thus, there is an obvious need for sensitive and reliable methods to assess the resistance status of at least the most important nematode populations. Several single nucleotide polymorphisms (SNPs) in the β-tubulin isotype 1 gene of various nematodes correlate with resistance to benzimidazoles (BZ), a major anthelmintic class. Here we describe the full-length β-tubulin isotype 1 and 2 and α-tubulin coding sequences of the cattle nematode Ostertagia ostertagi. Additionally, the Cooperia oncophora α-tubulin coding sequence was identified. Phylogenetic maximum-likelihood analysis revealed that both isotype 1 and 2 are orthologs to the Caenorhabditis elegans ben-1 gene which is also associated with BZ resistance upon mutation. In contrast, a Trichuris trichiura cDNA, postulated to be β-tubulin isotype 1 involved in BZ resistance in this human parasite, turned out to be closely related to C. elegans β-tubulins tbb-4 and mec-7 and would therefore represent the first non-ben-1-like β-tubulin to be under selection through treatment with BZs. A pyrosequencing assay was established to detect BZ resistance associated SNPs in β-tubulin isotype 1 codons 167, 198 and 200 of C. oncophora and O. ostertagi. PCR-fragments representing either of the two alleles were combined in defined ratios to evaluate the pyrosequencing assay. The correlation between the given and the measured allele frequencies of the respective SNPs was very high. Subsequently laboratory isolates and field populations with known resistance status were analyzed. With the exception of codon 167 in Cooperia, increases of resistance associated alleles were detected for all codons in at least one of the phenotypically resistant population. Pyrosequencing provides a fast, inexpensive and sensitive alternative to conventional resistance detection methods

Relative abundance of cDNA amplicons encoding full-length or truncated <i>Trichuris muris</i> Slo-1 channels.

<p>Relative abundance of cDNA amplicons encoding full-length or truncated <i>Trichuris muris</i> Slo-1 channels.</p

Estimation of splice variant abundances encoding truncated or full-length versions of <i>Trichuris muris</i> Slo-1.1.

<p><b>A</b>) Primers flanking the introns retained in the cDNAs encoding the truncated proteins (<i>Tmu</i>Slo-1.1c and <i>Tmu</i>Slo-1.1d) were used in RT-PCR (lanes 1, 2, 5, and 6) and genomic PCR (lanes 4 and 8). Template was either derived from the Monheim (lanes 1–4) or the Edinburgh Zoo (lanes 5–8) isolate. Lanes 3 and 7 show no reverse transcription controls. M, 100 bp ladder. <b>B</b>) Representative samples separated on the Bioanalyzer. The upper panel shows a genomic PCR product, the middle panel the control without reverse transcription and the bottom panel the RT-PCR.</p

Phylogenetic analysis of nematode Slo-1 channels.

<p><b>A</b>) Phylogram obtained by maximum likelihood analysis from full-length Slo-1 channels. Slo-1 protein sequences from the nematode species <i>Caenorhabditis elegans</i> (<i>Cel</i>), <i>Caenorhabditis briggsae</i> (<i>Cbr</i>), <i>Caenorhabditis remanei</i> (<i>Cre</i>), <i>Pristionchus pacificus</i> (<i>Pca</i>), <i>Haemonchus contortus</i> (<i>Hco</i>), <i>Cooperia oncophora</i> (<i>Con</i>), <i>Ancylostoma caninum</i> (<i>Acan</i>), <i>Onchocerca gutturosa</i> (<i>Ogu</i>), <i>Brugia malayi</i> (<i>Bma</i>), <i>Dirofilaria immitis</i> (<i>Dim</i>), <i>Toxocara canis</i> (<i>Tca</i>), <i>Parascaris equorum</i> (<i>Peq</i>), <i>Ascaris suum</i> (<i>Asu</i>), <i>Meloidogyne incognita</i> (<i>Min</i>), <i>Strongyloides ratti</i> (<i>Sra</i>), <i>Trichuris muris</i> (<i>Tmu</i>) and <i>Trichuris suis</i> (<i>Tsu</i>) were aligned together with orthologs from <i>Drosophila melanogaster</i> (<i>Dme</i>), <i>Anopheles gambiae</i> (<i>Aga</i>), <i>Pediculus humanus corporis</i> (<i>Phu</i>), <i>Daphnia pulex (Dpu)</i>, <i>Aplysia callifornica</i> (<i>Acal</i>), <i>Gallus gallus</i> (<i>Gal</i>), <i>Bos taurus</i> (<i>Bta</i>) and <i>Homo sapiens</i> (<i>Hsa</i>), which were used as outgroup, using ClustalX2. For <i>B. malayi</i> only the experimentally identified splice variant Slo-1f and for <i>C. elegans</i> only the variants Slo-1a-c were included. The JTT model of amino acid substitutions was used and PhyML was set to optimize the number of invariable sites while amino acid frequencies were based on the model. The number of Γ distributed substitution rate categories was set to 16 and PhyML optimized the Γ shape parameter. Support for individual nodes was calculated using the Shimodaira-Hasegawa modification and a Bayesian transformation of the approximate likelihood ratio test and results are shown close to the nodes before and after the slash, respectively. For those cases where support values were not shown next to the node they are shown on the right and refer to the most terminal node on the same vertical position. The scale bar represents 0.2 substitutions per site. C, Crustacea; G, Gastropoda; Vert, Vertebrata; Arthropod, Arthropoda, M, Mollusca; L, Lophotrophora; Deut, Deuerostomia. <b>B</b>) Phylogenetic tree calculated on an alignment of the conserved alternative exons from all Ecdysozoa included in the tree in A). In addition, four alternative exons identified in <i>Bma</i>Slo-1h and in the genome sequences of <i>Onchocerca volvulus</i> (<i>Ovo</i>Slo-1 and <i>Ovo</i>Slo-1 alt. exon) and <i>A. suum</i> (<i>Asu</i>Slo-1 alt. exon) were included. Parameters were identical to those used to calculate the tree from full-length sequences.</p