Oligonucleotide sequences for generation of ECA485 <i>cct-8</i>(<i>ean8</i>) in a N2 background and ECA601 <i>cct-8</i>(<i>ean39</i>) in a JU775 background.

Oligonucleotide sequences for generation of ECA485 cct-8(ean8) in a N2 background and ECA601 cct-8(ean39) in a JU775 background.</p

Primer sequences for qPCR.

Anthelmintic drugs are used to treat parasitic roundworm and flatworm infections in humans and other animals. Caenorhabditis elegans is an established model to investigate anthelmintics used to treat roundworms. In this study, we use C. elegans to examine the mode of action and the mechanisms of resistance against the flatworm anthelmintic drug praziquantel (PZQ), used to treat trematode and cestode infections. We found that PZQ inhibited development and that this developmental delay varies by genetic background. Interestingly, both enantiomers of PZQ are equally effective against C. elegans, but the right-handed PZQ (R-PZQ) is most effective against schistosome infections. We conducted a genome-wide association mapping with 74 wild C. elegans strains to identify a region on chromosome IV that is correlated with differential PZQ susceptibility. Five candidate genes in this region: cct-8, znf-782, Y104H12D.4, Y104H12D.2, and cox-18, might underlie this variation. The gene cct-8, a subunit of the protein folding complex TRiC, has variation that causes a putative protein coding change (G226V), which is correlated with reduced developmental delay. Gene expression analysis suggests that this variant correlates with slightly increased expression of both cct-8 and hsp-70. Acute exposure to PZQ caused increased expression of hsp-70, indicating that altered TRiC function might be involved in PZQ responses. To test if this variant affects development upon exposure to PZQ, we used CRISPR-Cas9 genome editing to introduce the V226 allele into the N2 genetic background (G226) and the G226 allele into the JU775 genetic background (V226). These experiments revealed that this variant was not sufficient to explain the effects of PZQ on development. Nevertheless, this study shows that C. elegans can be used to study PZQ mode of action and resistance mechanisms. Additionally, we show that the TRiC complex requires further evaluation for PZQ responses in C. elegans.</div

Praziquantel affects <i>C</i>. <i>elegans</i> development.

Dose response for the N2 (orange) and JU775 (purple) strains in praziquantel (PZQ), (S)-PZQ, and (R)-PZQ. The y-axis represents development measured as median optical density normalized for animal length. Linetype indicates the type of drug: solid = PZQ racemate, long dash = (S)-PZQ, and short dash = (R)-PZQ.</p

Two-way ANOVA results for dose response assay of racemate praziquantel (PZQ), (<i>S</i>)-PZQ, and (<i>R</i>)-PZQ.

Two-way ANOVA results for dose response assay of racemate praziquantel (PZQ), (S)-PZQ, and (R)-PZQ.</p

Fig 3 -

A) Expression of cct-8, hsp-16.2, and hsp-70 in control conditions (black) and PZQ (gray) in the strains N2 and JU775. The biological samples were spread over two 96-well plates per gene (triangles and circles). B) Expression of hsp-16.2 and hsp-70 after treatment with RNAi or praziquantel (PZQ) in the strain N2. *, **, and *** indicate p-values of less than 0.05, 0.01, and 0.001, respectively.</p

Tissue-selective induction of the <b><i>phsp70::gfp</i></b><b> reporter by knockdown of negative regulators.</b>

<p>Nomarski and fluorescent images corresponding to whole animals and fluorescent images of the spermatheca, intestine, and muscle tissue are shown. The boundary of the animals, intestine and spermatheca taken from Nomarski images are added as a visual aide to some images. (A–E) RNAi knockdown of <i>daf-21</i> leads to induction of the reporter in all three tissues. (F–J) RNAi knockdown of <i>cct-1</i> causes induction only in muscle. (K–O) knockdown of <i>F38A1.8</i> causes induction in the intestine and spermatheca. Images are taken at different exposures to maximize fluorescence of each image. Scale bars of whole animal images correspond to 100 µm, while scale bars of the images depicting specific tissues correspond to 50 µm. Asterisks denote only autofluorescence.</p

Two-way-ANOVA results for <i>cct-8</i>, <i>hsp-16</i>.<i>2</i>, and <i>hsp-70</i> expression in the strains N2 and JU775 in control (1% DMSO) and PZQ conditions (1 mM PZQ in 1% DMSO).

Two-way-ANOVA results for cct-8, hsp-16.2, and hsp-70 expression in the strains N2 and JU775 in control (1% DMSO) and PZQ conditions (1 mM PZQ in 1% DMSO).</p

Validation of tissue-selective effects using small molecules and mutants.

<p>A) Incubation with 100 µM MG132, a small molecule inhibitor of the proteasome, but not DMSO alone, causes tissue-selective induction of the <i>phsp70::gfp</i> reporter in the intestine and spermatheca (arrows), similar to RNAi knockdown of proteasomal subunits. The scale bar corresponds to 200 µm. Asterisks denote only autofluorescence. B) Mutations in <i>T24H7.2, sgt-1, cyn-11, and unc-45</i> cause induction of the HSR in whole worms measured using qRT-PCR. C) Mutation of <i>T24H7.2</i>, but not <i>unc-45</i>, causes induction in the intestine relative to N2 control animals, measured by qRT-PCR analysis of <i>hsp70</i> in dissected intestinal tissue. Averages shown are from at least two biological replicates.</p

Positive Regulators of the HSR.

<p>Positive Regulators of the HSR.</p

Genome-wide RNAi screen for HSR regulators.

<p>(A–D) Nomarski and fluorescent images corresponding to the <i>phsp70::gfp</i> reporter strain. (A) Control animals show little reporter expression and only faint autofluorescence of the intestine. (B) Reporter induction in animals exposed to heat shock at 33°C for 1 hour. (C) RNAi knockdown of <i>hsf-1</i> decreases induction of the reporter by heat shock. (D) RNAi knockdown of <i>hsp-1</i> causes constitutive induction of the reporter in the absence of heat shock. The scale bar corresponds to 100 µm. (E–F) Quantitation of the effects on endogenous <i>hsp70</i> and <i>hsp-16.2</i> genes using qRT-PCR. (E) HSR positive regulators normalized to the heat shocked empty vector control. (F) HSR negative regulators normalized to empty vector control. Averages are from at least three biological replicates and error bars represent SEM.</p