Figure 1.
Phylogenetic tree of SLO-1 amino acid sequences and related potassium channels.
The tree was calculated using Neighbour Joining method. Numbers at the branches indicate bootstrap values (in %, 1000 replicates). The bar shows number of substitutions per mutation site. The selected sequences (GenBank accession numbers in brackets) are as follows: C. elegans SLO-1a (AAL28102); C. elegans SLO-1b (AAL28103); C. elegans SLO-1c (AAL28104); C. briggsae hypothetical protein CBG12923 (XP_001675579.1); A. caninum SLO-1 (EU828635); C. oncophora SLO-1 (EF494185); H. contortus SLO-1 (EF494184); Ixodes scapularis putative calcium-activated potassium channel (EEC10339.1); Cancer borealis calcium-activated potassium channel (AAZ80093.4); Manduca sexta calcium-activated potassium channel alpha subunit (AAT44358.1); Pediculus humanus corporis putative calcium-activated potassium channel alpha subunit (EEB13088.1); Drosophila melanogaster slowpoke, isoform P (NP_001014652.1); Tribolium castaneum predicted protein similar to slowpoke CG10693-PQ (XP_968651.2); Aplysia californica high conductance calcium-activated potassium channel (AAR27959.1); Xenopus laevis potassium large conductance calcium-activated channel, subfamily M, alpha member 1 (NP_001079159.1); Danio rerio novel calcium activated potassium channel (CAX13266.1); Trachemys scripta calcium-activated potassium channel (AAC41281.1); Gallus gallus calcium-activated potassium channel alpha subunit (AAC35370.1); Monodelphis domestica predicted protein similar to large conductance calcium-activated potassium channel subfamily M alpha member 1 (XP_001367795.1); Mus musculus mSlo (AAA39746.1); Homo sapiens potassium large conductance calcium-activated channel, subfamily M, alpha member 1, isoform CRA_d (EAW54600.1); Bos taurus BK potassium ion channel isoform C (AAK54354.1); Canis familiaris calcium-activated K+ channel, subfamily M subunit alpha-1 (Q28265.2); Strongylocentrotus. purpuratus predicted protein similar to calcium-activated potassium channel alpha subunit (XP_783726.2).
Figure 2.
Behavioural phenotype of transgenic C. elegans.
(A) number of reversals in 3 min were counted on NGM agar without bacteria for N2 Bristol, slo-1(js379) and the indicated transgenic lines derived from slo-1(js379). All values are means + SD. An asterisk (*) marks significant differences to all other lines (p<0.001) determined by One-Way-ANOVA followed by Tukey's test. (B) number of body bends per minute counted on NGM agar without bacteria. One-Way-ANOVA revealed no significant differences between different lines. N2, N2 Bristol wild-type strain; js379, slo-1(js379) mutant strain; Cel-snb-1::Aca-slo-1, line expressing A. caninum slo-1 from the C. elegans snb-1 promoter; Cel-snb-1::Con-slo-1, line expressing C. oncophora slo-1 from the C. elegans snb-1 promoter; Cel-slo-1::Aca-slo-1, line expressing A. caninum slo-1 from the C. elegans slo-1 promoter; Cel-slo-1::Cel-slo-1, line expressing C. elegans slo-1 from the C. elegans slo-1 promoter; Aca-slo-1::Aca-slo-1, line expressing A. caninum slo-1 from the A. caninum slo-1 promoter; Con-slo-1::Con-slo-1, line expressing C. oncophora slo-1 under control of the C. oncophora slo-1 promoter.
Figure 3.
Parasite SLO-1 expressed from C. elegans promoters recover emodepside susceptibility in resistant slo-1 loss-of-function mutants.
Body bend activity in percent (relative to the highest number of body bends in that group) of young adults after 24 h exposure to emodepside. Comparison of wild-type N2, emodepside-resistant strain slo-1(js379), and transformed slo-1(js379) lines. Error bars represent standard errors of the mean. Dots (•) represent transformed lines, squares (▪) N2 Bristol wild-type strain, triangles (▴) js379(slo-1) mutant strain. (A) Cel-snb-1::Aca-slo-1, line expressing A. caninum slo-1 from the C. elegans snb-1 promoter. (B) Cel-snb-1::Con-slo-1, line expressing C. oncophora slo-1 from the C. elegans snb-1 promoter. (C) Cel-slo-1::Aca-slo-1, line expressing A. caninum slo-1 from the C. elegans slo-1. (D) Cel-slo-1::Cel-slo-1, C. elegans slo-1 expressed from C. elegans slo-1 promoter.
Table 1.
A summary of the pharmacological response to emodepside in transgenic lines expressing either C. elegans or parasite slo-1 under the control of C. elegans-derived promoters.
Figure 4.
Parasite SLO-1 expressed from parasite-derived slo-1 promoters partially recover emodepside susceptibility in resistant slo-1 loss-of-function mutants.
Body bend activity (relative to the highest number of body bends in each group) of young adults after 24 h exposure to emodepside. Comparison of wild-type N2, emodepside-resistant strain slo-1(js379), and transformed slo-1(js379) lines. Dots (•) represent Aca-slo-1::Aca-slo-1 lines (expressing A. caninum slo-1 from the putative A. caninum slo-1 promoter); inverted triangles (▾) Con-slo-1::Con-slo-1 lines (expressing C. oncophora slo-1 from the putative C. oncophora slo-1 promoter); squares (▪) N2 Bristol wild-type strain, triangles (▴) js379(slo-1) mutant strain.
Table 2.
Hill slope, EC50 and bottom value with 95% confidence intervals for transgenic lines expression slo-1 under control of a parasitic nematode-derived promoter.
Figure 5.
Effect of penitrem A and emodepside on nematode locomotion.
(A) Migration of infective A. caninum larvae (relative to negative control without vehicle) through a 20 µm precision sieve after incubation in different concentrations of emodepside in presence or absence of penitrem A. (B) Body bend activity of C. elegans (relative to negative control without vehicle). (A, B) Negative control (black bar), without vehicle or substance; vehicle control (light grey bars), with 28 mM DMSO, 170 mM ethanol, and the indicated emodepside concentrations; 1 µM penitrem A (dark grey bars), with 1 µM penitrem A, 170 mM ethanol and the indicated emodepside concentrations. Error bars represent standard deviations. Asterisks mark a significant difference between vehicle controls and the experiments with 1 µM penitrem A at the same emodepside concentration (*** p<0.001) determined by One-Way-ANOVA followed by a Tukey's pairwise comparison.