Zahreddine Fahs, Hala et al. (2021) International Worm Meeting "Multi-species nematode screening uncovers a new broad-spectrum class of anthelmintic compounds targeting mitochondrial lipid metabolism"

Gopinadhan, Suma, Refai, Fathima, White, Robert, Pearson, Yanthe, Kremb, Stephan, Butterfoss, Glenn, Gunsalus, Kristin C., Zahreddine Fahs, Hala, Xie, Xin, Cipriani, Patricia, Page, Anthony, Piano, Fabio

[International Worm Meeting, 2021]

Parasitic worms infect more than 1.5 billion people and cause significant losses in livestock and crops. New anthelmintic drugs are urgently needed, as resistance to the existing drugs is emerging. We built a high-throughput (HTP) and high-content robotic screening platform to identify bioactive small molecules and their molecular targets, focusing on novel disease therapeutics and broad spectrum anthelmintics. The platform is capable of screening compound collections and genetic libraries in model organisms and mammalian cells in a fully integrated manner. To find novel anthelmintics we screened a ~2300 compound library containing most of the FDA-approved approved drugs, plus natural products and bioactive compounds for broad spectrum toxic effects on two distantly related free living nematodes C. elegans and P. pacificus as model animals while having low toxicity on human cell lines. The screen confirmed the effects of most known anthelmintics on these nematode models. Additionally, among a set of new compounds, we found six natural lipid molecules that exhibit broad-spectrum anthelmintic activity. These novel compounds kill C. elegans and P. pacificus when applied at various developmental stages, including the dauer and embryonic stages, in a dose-dependent manner. Importantly, these compounds cause mortality in all three veterinary parasite species we tested: the hookworm Haemonchus contortus (ruminants), Teladorsagia circumcincta (sheep and goat) and Heligmosomoides polygyrus (rodents). Further characterization of their mode of action using C. elegans revealed defects associated with mitochondrial function and lipid metabolism. The lethality phenotype in C. elegans is partially rescued by knocking down the enzymes acs-2, cpt-4, dif-1 and cpt-2, which facilitates mitochondrial lipid transfer, while drug inhibitors of CPT1 and CPT2 enhance the phenotype, suggesting that these compounds perturb specifically fatty acid oxidation in nematodes. These compounds constitute a new class of broad-spectrum anthelmintics targeting lipid metabolism.

Marra MA et al. (1991) International C. elegans Meeting "CHARACTERIZATION OF THE COSMID C11 F2.II. FRAGMENT 'R' ENCODES A DECARBOXYLASE."

Clark DV, Prasad SS, Marra MA, Baillie DL

[International C. elegans Meeting, 1991]

We are attempting to identify and characterize all genes in the 100 kb interval between /et-56(1V) and unc-22(1V). Currently, we are focussing on the cosmid C1 1F2, which has been shown to rescue two alleles of let-56. This cosmid contains four Pst1 fragments we call P, Q, R, and S. The 18 kb fragment Q contains sequences homologous to the human Na+ / Ht antiporter (see other abstract Marra et al.). Immediately adjacent to this 18 kb fragment is the 8 kb fragment 'R'. Fragment R detects three messages on Northern blots. These messages are 1.9, 2.7 and 3.5 kilobases in length. The use of fragment R as a probe to screen B. Barstead's cDNA library in lambda ZAP resulted in the identification of a single positive clone out of approximately 300, 000 plaques screened. This clone was determined to be 1.9 kb in length. Sequence analysis of this clone revealed a long open reading frame of approximately 1940 nucleotides. Five hundred amino acids predicted from this long open reading frame were used to search the latest releases of the SWISS (rel.16) and translated EMBL(rel.25) databanks using the FASTA program of Pearson and Lipmann. Significant similarities to a variety of decarboxylases were detected, including rat and Drosophila melanogaster dopa decarboxylase, (40 % and 36% amino acid identity, respectively) and rat and human histidine decarboxylase (39% amino acid identity for both proteins). We are now sequencing genomic DNA from fragment R in order to elucidate the structure and function of this C. elegans decarboxylase-homologous gene. This work has been supported by an NSERC predoctoral scholarship to M.A.M. and an NSERC grant to D.L.B.

Jensen, Victor L. et al. (2013) International Worm Meeting "Analysis of developmental RNA-Seq libraries reveals signature profile for cilia-related genes."

Jensen, Victor L., Leroux, Michel R., Li, Chunmei, Timbers, Tiffany A., Morin, Ryan D.

[International Worm Meeting, 2013]

Ciliogenesis in C. elegans hermaphrodites occurs during late embryogenesis, with most cilia being formed within hours of each other. This makes C. elegans well suited to expression analyses to identify novel cilia genes. Using the available RNA-Seq libraries we identify a signature expression pattern specific to cilia-related genes, which includes a large spike in expression at late embryo with a reduction at mid-L1 (first larval stage) and low levels of expression at all other stages. P-values for Pearson correlation for each gene in the genome are compared to 41 known ciliary genes and each gene is then assigned the minimum calculated p-value. Using a 1E-5 cutoff and only including genes with human orthologues, we identify 635 genes with the specific pattern. Comparisons to CilDb (an online database of previously published ciliary genomics/proteomics experiments) reveals an 15-fold enrichment of genes with greater than seven or more cilia references. Further clustering of this list based on individual expression patterns increases the enrichment by greater than 40 times. Extending this expression analysis method, we also find a shared-but different from the above-expression pattern among serpentine olfactory G-protein-coupled receptors, many of which are known to be targeted to cilia. Using this approach, we identify evolutionarily-conserved proteins that function within three distinct ciliary regions or modules. One, a recently-characterized protein linked to retinitis pigmentosa, is found in the proximal-most segment of the axoneme, which is implicated as a ciliary gate needed for effective signalling. Another protein is present at the basal body, which is required for the formation of cilia. Finally, a third protein represents a previously undescribed component of the intraflagellar transport (IFT) machinery, which plays a critical role in ciliogenesis and delivering cargo (e.g. signalling proteins) to cilia.

Todd Lamitina et al. (2007) International Worm Meeting "Mutations in secreted proteins and cuticle collagens activate an osmosensitive transcriptional program in C. elegans."

Yana Miteva, Todd Lamitina

[International Worm Meeting, 2007]

C. elegans adapts to hypertonic stress by accumulating the solute glycerol via transcriptional upregulation of the glycerol biosynthetic enzymes gpdh-1 and gpdh-2. gpdh-1 expression is undetectable under isotonic conditions but is rapidly induced in the intestine and hypodermis following exposure to hypertonicity. Recently, we identified several mutants (osm-7, osm-11, dpy-9, & dpy-10) that cause constitutive expression of gpdh-1 in the absence of hypertonicity (regulators of gpdh-1 expression (rgpd) mutants). We and other have shown that rgpd mutants survive normally lethal levels of hypertonicity (the osmotic stress resistance (osr) phenotype) and contain glycerol levels that are >100-fold higher than wild type animals. We hypothesized that the rgpd mutant phenotype results from the accumulation of glycerol. To test this hypothesis, we used RNAi to inhibit osm-7, osm-11, dpy-9, & dpy-10 in both wild type and in gpdh-1;gpdh-2 double mutants. In all cases, gpdh-1;gpdh-2 mutants suppressed glycerol accumulation but failed to suppress the osr phenotypes. Therefore, osr phenotypes do not result from increased glycerol levels. The rgpd genes may transcriptionally activate other genes in addition to the gpdh enzymes to mediate osmotic stress resistance. To identify such genes, we used Affymetrix Genechips to compare the transcriptional profiles of rgpd mutants to those of wild type animals exposed to isotonic or hypertonic conditions (0.25, 1, 6, or 96 hours, 200 mM NaCl). 379 genes were significantly regulated >3-fold by hypertonic stress in wild type animals. Most of these 379 genes are concordantly regulated in at least one of the rgpd mutants. Based on Pearson Correlations, osm-7 and osm-11 mutants exhibit the most global similarity to wild type animals exposed to hypertonicity. Our data suggest that mutations in osm-7 and osm-11, which encode secreted hypodermal proteins, and dpy-9 and dpy-10, which encode cuticle collagens, specifically activate the signaling pathways that control the global transcriptional responses to osmotic stress. Currently, we are exploring the spatial and temporal qualities of this transcriptional program by examining Promoter::GFP fusions for osmotically regulated genes. Additionally, we are testing the functional significance of these transcriptional events in the osmotic stress response using RNAi based screening. Together, the combination of microarray data, in vivo expression patterns, and RNAi functional data in C. elegans should provide the first integrated description of the animal osmotic stress response.