Caenorhabditis elegans is a useful model for anthelmintic discovery

Parasitic nematodes infect one quarter of the world's population and impact all humans through widespread infection of crops and livestock. Resistance to current anthelmintics has prompted the search for new drugs. Traditional screens that rely on parasitic worms are costly and labour intensive and target-based approaches have failed to yield novel anthelmintics. Here, we present our screen of 67,012 compounds to identify those that kill the non-parasitic nematode Caenorhabditis elegans. We then rescreen our hits in two parasitic nematode species and two vertebrate models (HEK293 cells and zebrafish), and identify 30 structurally distinct anthelmintic lead molecules. Genetic screens of 19 million C. elegans mutants reveal those nematicides for which the generation of resistance is and is not likely. We identify the target of one lead with nematode specificity and nanomolar potency as complex II of the electron transport chain. This work establishes C. elegans as an effective and cost-efficient model system for anthelmintic discovery

The MADD-3 LAMMER Kinase Interacts with a p38 MAP Kinase Pathway to Regulate the Display of the EVA-1 Guidance Receptor in <i>Caenorhabditis elegans</i>

<div><p>The proper display of transmembrane receptors on the leading edge of migrating cells and cell extensions is essential for their response to guidance cues. We previously discovered that MADD-4, which is an ADAMTSL secreted by motor neurons in <i>Caenorhabditis elegans</i>, interacts with an UNC-40/EVA-1 co-receptor complex on muscles to attract plasma membrane extensions called muscle arms. In nematodes, the muscle arm termini harbor the post-synaptic elements of the neuromuscular junction. Through a forward genetic screen for mutants with disrupted muscle arm extension, we discovered that a LAMMER kinase, which we call MADD-3, is required for the proper display of the EVA-1 receptor on the muscle’s plasma membrane. Without MADD-3, EVA-1 levels decrease concomitantly with a reduction of the late-endosomal marker RAB-7. Through a genetic suppressor screen, we found that the levels of EVA-1 and RAB-7 can be restored in <i>madd-3</i> mutants by eliminating the function of a p38 MAP kinase pathway. We also found that EVA-1 and RAB-7 will accumulate in <i>madd-3</i> mutants upon disrupting CUP-5, which is a mucolipin ortholog required for proper lysosome function. Together, our data suggests that the MADD-3 LAMMER kinase antagonizes the p38-mediated endosomal trafficking of EVA-1 to the lysosome. In this way, MADD-3 ensures that sufficient levels of EVA-1 are present to guide muscle arm extension towards the source of the MADD-4 guidance cue.</p></div

Mutations in the p38 MAP kinase pathway suppress the synthetic lethality of the <i>unc-54; madd-3</i> double mutant.

<p><b>A.</b> The 41 suppressors that we isolated allow the <i>unc-54(e190); madd-3(tr186)</i> double mutants to survive without a rescuing extra-chromosomal array. The average number of progeny thrown by a single adult of the indicated genotype is shown (n = 10). The color of the name of the allele is matched to the color of the MAP kinase components in B-E. <b>B-D.</b> Schematics of the indicated MAP kinase pathway components and the position of the mutations that we identified. The green box indicates the kinase domain. <b>E.</b> A schematic of the MAP kinase pathway. <b>F.</b> Deletion alleles of the p38 MAP kinase pathway suppress the synthetic lethality of the <i>unc-54; madd-3</i> double mutant. Shown are the fraction of progeny from individual <i>unc-54(e190); madd-3(tr186)</i> double mutants that harbour an extra-chromosomal array that expresses MADD-3A specifically in muscles (from the construct pPRSAD499). The genetic background is indicated. Note that all triple mutant strains survive and propagate without the presence of the extra-chromosomal array. An asterisk indicates a significant difference (<i>p</i><0.05) compared to the data point indicated with a closed circle of the same color as the asterisks.</p

The muscle arm extension defects of <i>madd-3</i> mutants.

<p><b>A.</b> A schematic of muscle arms and their motor neuron targets. <b>B-E.</b> The muscle arms of animals of the indicated genotype. All animals harbour the <i>trIs30</i> transgene. Scale bars are 25μm. <b>F.</b> The number of muscle arms that extend from muscle VL11 towards the ventral nerve cord. <b>G.</b> The number of muscle arms that extend towards MADD-4 that is ectopically expressed by the lateral CAN neuron. Numbers shown are for the left side of the animal. The right side behaves similarly. In both graphs an asterisks indicates a significant difference (p<0.01) compared to the data point indicated with a closed circle with the same color as the asterisks. <b>H.</b> Schematics of the genomic <i>madd-3</i> locus and resulting transcripts. The position of the <i>tr186</i> allele and the <i>ok678</i> deletion are indicated. The position where a YFP tag was inserted in the context of the fosmid reporter (see text) is shown. The region of DNA used to drive the madd-3a YFP transcriptional reporter is indicated with a blue line. Black boxes indicate exons and white boxes are untranslated regions.</p

The MADD-3 expression pattern.

<p><b>A-D.</b> Expression of a MADD-3A translational reporter (the placement of the YFP tag is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006010#pgen.1006010.g001" target="_blank">Fig 1H</a>). <b>A.</b> A young adult worm. An arrow indicates a body wall muscle within one of the ventral quadrants, the arrowhead indicates the position of the vulval slit. The animal is twisted due to a marker within the extra-chromosomal array that carries the reporter. <b>B.</b> Body wall muscles showing relatively less YFP in the nucleus (two nuclei are encircled). <b>C.</b> The vulva muscles (arrow). <b>D.</b> The anal depressor muscle (arrow). <b>E-H</b>. Expression of a <i>madd-3Ap</i>::YFP transcriptional reporter. <b>E.</b> A partial bright-field photograph showing that YFP is not expressed from the <i>madd-3A</i> promoter at the embryonic two fold stage. <b>F.</b> At the embryonic pretzel stage, YFP expression driven by the <i>madd-3A</i> promoter is evident in the body wall muscles (arrow). <b>G.</b> An L1 larva with the two rows of body wall muscles showing. <b>H.</b> A young adult worm. A single body wall muscle is indicated with an arrow and a vulva muscle is indicated with an arrowhead. Scale bars represent 25 μm.</p

Mutations in p38 MAP kinase components and the mucolipin ortholog CUP-5 suppress the decrease in abundance of RAB-7 in <i>madd-3</i> mutants.

<p><b>A-I.</b> The muscle cells of animals of the indicated genotype expressing the indicated vesicle marker. <b>A-B.</b> mCherry::RAB-5 is specifically expressed in muscles from an extrachromsomal array harboring the pPRSAD950 plasmid. <b>C-F.</b> mCherry::RAB-7 is specifically expressed in muscles from the <i>huIs89</i> integrated transgene. <b>G-I.</b> mCherry::RAB-11 is specifically expressed in muscles from the <i>huIs97</i> integrated transgene. All RAB reagents were kind gifts from Rik Korswagen. The scale bars in A-H represent 50 μm. <b>J.</b> Western blot analyses of whole worm lysate from a mixed stage population of the indicated genotype probed with anti-RFP antibodies, which recognize mCherry (top), and anti-tubulin antibodies (bottom) as a loading control. <b>K.</b> Quantification of the RAB-7 vesicle phenotype in the indicated genetic background. <b>L.</b> Quantification of the RAB-11 vesicle phenotype in the indicated genetic background. MADD-3A is specifically in muscles from the <i>myo-3</i> promoter from an extra-chromosomal array (from the pPRSAD499 plasmid). For K-L, the presence of a wild type or <i>tr186</i> allele of MADD-3A is indicated with a respective (+) or (-) below the bars of the graph. An asterisk indicates a significant difference (<i>p</i><0.05) compared to the data point indicated with a closed circle of the same color as the asterisks.</p

A model of the relationship between MADD-3, EVA-1 and the p38 MAP kinase pathway.

<p>The EVA-1 receptor, which is required to guide muscle arm extension towards the MADD-4 guidance cue, is indicated in orange. RE, recycling endosome; EE, early endosome; LE, late endosome.</p

Mutations in p38 MAP kinase components and the mucolipin ortholog CUP-5 suppress the decrease in abundance of EVA-1 in <i>madd-3</i> mutants.

<p><b>A-E.</b> Muscle cells from animals of the indicated genotype express EVA-1:: MYC::3XFLAG::CFP in muscles from the <i>trIs89</i> chromosomally integrated transgene. Each picture was taken with the same exposure time. <b>F-G.</b> Muscle cells from animals of the indicated genotype express UNC-40::YFP in muscles from the <i>trIs41</i> transgene. <b>H-I.</b> Muscle cells from animals of the indicated genotype express PAT-2::CFP in muscles from the <i>trIs72</i> integrated transgene. The scale bars in A-I represent 25μm. <b>J.</b> A western blot of whole worm lysate from a mixed stage population of the indicated genotype probed with an antibody against GFP (which recognizes CFP) (top) and an antibody against tubulin as a loading control (bottom). EVA-1::CFP is expressed from the integrated transgene <i>trIs89</i>. <b>K.</b> Quantification of fluorescent signal for the indicated transgene and genetic background. The transgenic protein whose abundance is being measured is indicated below the genotype at the bottom of the panel. An asterisk indicates a significant difference (<i>p</i><0.01) compared to the data point indicated with a closed circle of the same color as the asterisks.</p

Genetic interaction analyses reveals a synthetic lethal interaction between <i>madd-3</i> and <i>unc-54</i>.

<p><b>A.</b> Double mutant analyses reveals that <i>madd-3(tr186)</i> enhances the muscle arm defects of all mutants shown. Counts for muscle VL11 are shown (n = 30). <b>B.</b> The viability of <i>unc-54(e190); madd-3(tr186)</i> double mutants is dependent on a rescuing array that expresses either MADD-3 or UNC-54. Shown for each genotype are the fraction of F1 progeny that carry the indicated extra-chromosomal (Ex) array from parents that also carried the same array. All double homozygotes are dependent on the array to become viable fertile adults (see text), while double heterozygotes do not. The array carrying the endogenous promoter expresses YFP::MADD-3A within the context of a 29 kb fosmid clone (construct pPRSAD539- see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006010#sec011" target="_blank">methods</a>); the <i>myo-3</i> promoter was used to express MADD-3A specifically in muscles (construct pPRSAD499); UNC-54 was expressed in the muscles from construct pPD5.41 (a gift from Andrew Fire). <b>C</b>. <i>madd-3(tr186)</i> is synthetic lethal with a temperature sensitive allele of <i>unc-54(e1157)</i>. Strains were grown at the indicated temperature (Celsius) for at least 3 days before individual L4s were cloned on to separate plates (n = 8). Progeny of the cloned animals were counted when they reached L4. For all graphs shown, an asterisk indicates a significant difference (<i>p</i><0.05) with respect to the data point indicated with a closed circle of the same color as the asterisks.</p

Deletion alleles of MAP kinase pathway components suppress the muscle arm extension defects of <i>madd-3</i> mutants.

<p><b>A.</b> Shown are the muscle arm defects of deletion alleles (as indicated by the <i>ok</i>, <i>gk</i>, or <i>tm</i> prefix) of the indicated MAP kinase component. <b>B.</b> Muscle-specific expression of SEK-3 (from the <i>him-4</i> promoter from construct pPRSAD936) is able to rescue <i>sek-3(ok1276)</i>’s suppression of <i>madd-3(tr186)</i>’s muscle arm defects. <b>C.</b> Muscle-specific over-expression of CEBP-1 (from the <i>him-4</i> promoter from construct pPRSAD961) phenocopies the <i>madd-3(tr186)</i> mutant phenotype from three distinct transgenic lines. For A-C, the presence of a wild type or <i>tr186</i> allele of MADD-3A is indicated with a respective (+) or (-) below the bars of the graph. In graphs A and C, counts for ventral left muscle 11 are shown, and in B, counts for dorsal right 15 is shown. In all graphs, an asterisk indicates a significant difference (p<0.01) compared to the data point indicated with a closed circle of the same color as the asterisks.</p