George-Raizen, J.B. et al. (2013) International Worm Meeting "Dynamically-expressed prion-like proteins form a cuticle in the pharynx of Caenorhabditis elegans."

Shockley, K.R., Lamb, A.L., George-Raizen, J.B., Raizen, D.M.

[International Worm Meeting, 2013]

In all molting animals, a cuticular extracellular matrix forms the first barrier to infection and other environmental insults. In C. elegans there are two types of cuticle, a well-studied collagenous cuticle that lines the body, and a poorly-understood chitinous cuticle that lines the pharyngeal lumen and buccal cavity. By performing a transcriptional profiling experiment in precisely staged developing larvae, we find strong induction during the molt of abu/pqn genes encoding secreted prion-like (P) glutamine (Q) and asparagine (N) rich PQN proteins. 32 cysteine-rich PQN proteins form an ABU/PQN paralog group and of the 30 abu/pqn genes represented on the affymetrix array, 27 are transcriptionally induced during periods of cuticular synthesis. Transcriptional reporters for eight randomly selected abu/pqn genes are expressed in pharyngeal muscle. Translational fluorescent reporters for ABU/PQN proteins are expressed on the cuticular apical side of pharyngeal muscle and are shed during the molt. Disrupting abu/pqn gene function results in abnormal pharyngeal cuticular structures and abnormal feeding. Prior reports of modest transcriptional activation of abu/pqn genes in response to genetic, infectious, and chemical perturbations, can be explained by the unappreciated presence of molting animals in those experiments. Our findings suggest that the expression of the abu/pqn genes is regulated by a developmental timing program in the C. elegans pharynx where they promote the assembly and function of unique cuticular structures.

Sugimoto A et al. (2000) Japanese Worm Meeting "PAF-acetylhydrolase type II is essential for hypodermal cell epithelialization in C. elegans"

Sugimoto A, Tsujimoto M, Arai H, Aoki J, Yamamoto M, Inoue K, Inoue T

[Japanese Worm Meeting, 2000]

PAF-acetylhydrolase (PAF-AH), which hydrolyzes the acetyl group, is distributed widely in tissues and plasma. In mammals, tissue cytosol contains two types of PAF-AH, type I and type II. Type II PAF-AH (PAF-AH (II)) is a 40-kDa monomeric enzyme and N-myristoylated at the N-terminus. Since PAF-AH (II) effectively hydrolyzes oxidized phospholipids as well as PAF, it is suggested that PAF-AH (II) functions to protect cells against oxidative stress by removing oxidized phospholipids. In this study, we discovered two PAF-AH (II) genes from C. elegans and disrupted those genes to elucidate the biological function in this organism. Amino acid sequences of two PAF-AH (II) paf-1 and paf-2 exhibited 70% identity with each other and 35% identity with mammalian PAF-AH (II)s. Both paf-1 and paf-2 concerved a Gly-X-Ser-X-Gly catalytic center and the N-myristoylation sequence. As mammalian PAF-AH (II), C elegans PAF-AH (II)s expressed in E. coli hydrolyzed PAF and oxidized phospholipid but not phospholipids with long acyl chains. Then we made knockout C. elegans and analyzed their phenotypes. paf-2 (-/-) was lethal at the embryonic stage, whereas paf-1 (-/-) grew normally. paf-2 (-/-) embryo was examined using a scanning electron microscope and immuno-staining. paf-2 (-/-) embryo had a profound defect in the formation of epitherial cells, especially in hypodermal cells possibly because of very low expression level of a certain adhesion molecule at this stage. We also examined the expression of paf-2 by injecting paf-2-GFP fusion gene in wild type. paf-2 was expressed in the epithelial cells and in gut at high levels during embryogenesis. At adult stage paf-2 was expressed in the phrynx, vulva and intestine. These data demonstrate that PAF-AH (II) plays an important role in the formation of epithelial cells and suggest that oxidative stress against cell membranes is increased during epithelialization.

Takao Inoue et al. (2003) International Worm Meeting "Type II PAF-AH, paf-2, is essential for hypodermal organization in C. elegans"

Takao Inoue, Masayuki Yamamoto, Yuka Suzuki, Junken Aoki, Hiroyuki Arai, Asako Sugimoto

[International Worm Meeting, 2003]

PAF-acetylhydrolase (PAF-AH) is an enzyme that inactivates PAF (Platelet-activating factor) by hydrolyzing the acetyl group attached at the sn-2 position of the glycerol backbone. Type II PAF-AH (PAF-AH (II)) is a 40-kDa monomeric enzyme and myristoylated at the N-terminus. PAF is not detected in C. elegans, however, orthologous genes of PAF-AH (II) are conserved in many species, including C. elegans. The physiological function and substrate(s) of this enzyme have not been fully understood so far. In this study, we cloned two PAF-AH (II) genes in C. elegans (paf-1, paf-2) and disrupted these genes to elucidate the biological function. Amino acid sequences of PAF-1 and PAF-2 exhibited 70% identity with each other and 35% identity with mammalian PAF-AH (II)s. Both PAF-1 and PAF-2 conserved a Gly-X-Ser-X-Gly catalytic center and the N-myristoylation sequence, and exhibited similar substrate specificity as mammalian PAF-AH (II)s in vitro. Next we generated deletion mutants of these two enzymes and examined their phenotypes. paf-2(-/-) was lethal during embryogenesis, whereas paf-1(-/-) grew normally. All paf-2(-/-) embryos failed to elongate and the development was arrested at the lima bean stage. Analysis using SEM (scanning electron microscope) and immuno-staining revealed that the organization of the hypodermis in the paf-2(-/-) mutant embryo is severely obstructed. To further analyze the function of paf-2 during elongation process and following postembryonic development, we generated transgenic animals in which heat-shock induces paf-2 RNAi, producing both senses of paf-2 RNAs in vivo. Heat-shocked hsp::paf-2 RNAi animals showed negligible PAF acetylhydrolase activity, indicating that PAF-2 is a major isoform for enzyme activity in C. elegans. These worms exhibited variable abnormal morphology (Vab) at the late embryonal and early larval stages including ectopic protrusions due to epithelial organization defects. These observations demonstrate that PAF-AH (II) is required for hypodermal formation and suggest that particular lipids, the substrates or products of PAF-2, are critically involved in epithelial morphogenesis.

Mohan Viswanathan et al. (2007) International Worm Meeting "A Role for C. elegans sir-2.1 in Regulating Endoplasmic Reticulum Homeostasis."

Leonard Guarente, Mohan Viswanathan

[International Worm Meeting, 2007]

Members of the Sir2 family of NAD+-dependent protein deacetylases regulate life span in several model organisms including yeast, nematodes, and fruit flies. Overexpression of the C. elegans Sir2 homolog, sir-2.1, extends nematode life span and requires the function of the forkhead transcription factor daf-16. sir-2.1, but not daf-16, is required for life span extension following treatment with the compound resveratrol, indicating the existence of two different sir-2.1-dependent pathways that regulate worm life span. Resveratrol treatment induces the expression of multiple C. elegans abu/pqn gene family members implicated in endoplasmic reticulum (ER) stress response to incorrectly folded or incorrectly modified ER client proteins. Resveratrol likely extends life span by inhibiting sir-2.1-mediated transcriptional repression of abu-11 and other abu/pqn genes. Overexpression of abu-11 by transgene extends life span while down-regulation of expression by RNAi shortens life span and abolishes resveratrol-mediated life span extension. These results suggest a novel link between ER stress response function and life span. To elucidate if there is a direct role for sir-2.1 in ER homeostasis, we have performed genetic analysis combining loss of function mutants in sir-2.1 and components of the ER unfolded protein response pathways. We will present data that indicate a role for sir-2.1 in mediating cellular response to ER-stress.

Sun, Jingru et al. (2011) International Worm Meeting "G-protein coupled receptor OCTR-1 controls innate immunity by regulating non-canonical unfolded protein response genes."

Kajino-Sakamoto, Rie, Singh, Varsha, Aballay, Alejandro, Sun, Jingru

[International Worm Meeting, 2011]

The endoplasmic reticulum (ER) has developed specific signaling pathways called the unfolded protein response (UPR) to cope with ER stress and restore ER homeostasis. Recent studies indicate that the increased demand on protein folding in the ER, that may occur during bacterial infections, must be successfully alleviated by canonical and non-canonical UPR pathways for a complete immune response to be mounted. We found that the nervous system controls the activity of a non-canonical UPR pathway required for innate immunity in Caenorhabditis elegans. OCTR-1, a G-protein coupled receptor, functions in ASH and ASI neurons to actively suppress innate immune responses by down-regulating the expression of non-canonical UPR genes pqn/abu in non-neuronal tissues. Pseudomonas aeruginosa exposure up-regulates pqn/abu genes to levels comparable to those induced by treatment with the ER stressor tunicamycin. Our studies highlight pathways that may play important roles at dealing with pathogens directly and with the damages caused by the infection.

Takao Inoue et al. (2002) Japanese Worm Meeting "paf-2 RNAi induces abnormal hypodermal organization during elongation process in C. elegans"

Masafumi Tsujimoto, Asako Sugimoto, Hiroyuki Arai, Takao Inoue, Junken Aoki, Masayuki Yamamoto, Yuka Suzuki

[Japanese Worm Meeting, 2002]

We previously discovered a unique N-myristoylated phospholipase [called type II platelet-activating factor-acetylhydrolase: PAF-AH (II)], and showed that paf-2, an orthologue of PAF-AH (II), is essential for hypodermal organization during embryogenesis. To further analyze the function of paf-2 during the elongation process and following postembryonic development, we generated transgenic animals in which heat-shock induces paf-2 RNAi, producing both senses of paf-2 RNAs in vivo. Heat-shocked hsp::paf-2 RNAi animals showed negligible PAF acetylhydrolase activity, indicating that PAF-2 is a major isoform for enzyme activity in C. elegans. In about 11% of the heat-shocked hsp::paf-2 RNAi transgenic animals, development was arrested during embryogenesis and in about 5% of these animals it was arrested at the early larval stages. The arrested hsp::paf-2 RNAi animals showed variable abnormal morphology (Vab) due to defects of hypodermal integration during the elongation process. These observations confirm PAF-AH (II) is required for hypodermal formation and suggest that particular lipids, the substrates or products of paf-2, are critically involved in epithelial morphogenesis. Identification of such lipid molecule(s) is in progress using hsp::paf-2 RNAi transgenic animals in our laboratory.

Sugimoto A et al. (2000) Japanese Worm Meeting "Targeted disruption of C.elegans lis-1, a causative gene for human type I lissencephaly"

Arai H, Inoue K, Koizumi H, Sugimoto A, Marubashi Y, Aoki J, Yamamoto M

[Japanese Worm Meeting, 2000]

Type I lissencephaly (Miller-Dieker syndrome and isolated lissencephaly sequence) is the human brain malformation caused by defective neuronal migration during embryogenesis. LIS1, a causative gene for type I lissencephaly encodes a 45 kDa protein containing G beta-like 7 WD repeats. LIS1 protein was also identified as a non-catalytic subunit (beta) of platelet-activating factor acetylhydrolase type I (PAF-AH (I)) from mammals. PAF-AH(I) forms G-protein like complex consisting of two mutually homologous catalytic subunits (alpha1 and alpha2) and a non-catalytic beta subunit, and hydrolyses PAF or PAF like phospholipids, suggesting an involvement of certain phospholipid(s) in neuronal migration in mammals. In an evolutional aspect, the LIS1 gene are ubiquitous and have been identified even in the lower eukaryote, yeast, fungi and Drosophila, but the genes for the catalytic subunits of PAF-AH(I) have been identified only in Drosophila. This indicates that the catalytic subunits of PAF-AH(I) have a role in modulating the LIS1/beta function in higher organisms. As other lower organisms, C.elegans possesses LIS1 gene (lis-1) but not the gene for the catalytic subunits. To understand the function of the lis-1, we used a reverse genetic strategy to isolate deletion alleles of the lis-1 locus in C. elegans. The C.elegans LIS1 protein showed about 60% identity in amino acid sequence to the human LIS1/beta. Although lis-1 heterozygous was fertile and indistinguishable from wild-type in the appearance, lis-1 homozygous was lethal at the comma stage of embryo. Immunofluorescence study using hypodermal cell specific monoclonal antibody, MH27, revealed abnormal morphology, decreased number of hypodermal cells and failure of body enclosure, possibly resulting from abnormal cell division. The results suggested a pivotal role of LIS1 in the cell division of dynamically migrating hypodermal cells during C.elegans embryogenesis.

Sahu, Surasri et al. (2011) International Worm Meeting "Genomic Analysis of Immune Response Against Vibrio cholerae Hemolysin in Caenorhabditis elegans ."

LeClerc, Joseph, Bozdag, Serdar, Lewis, Jada, Sahu, Surasri, Cinar, Hediye, Patel, Isha

[International Worm Meeting, 2011]

Vibrio cholerae, a natural inhabitant of aquatic ecosystem, causes acute severe gastroenteritis in large populations through epidemic and pandemic dissemination. The major virulence factor responsible for massive fluid loss via watery diarrhea is cholerae toxin (CT). V. cholerae strains, which lack CT, and V. cholerae vaccine strains with deleted CT locus are also capable of causing diarrhea, soft tissue infections, sepsis, inflammatory enterocholitis, in humans in a sporadic fashion through mechanisms that are currently unclear. V. cholerae cytolysin (VCC) is among the accessory V. cholerae virulence factors that may contribute to sporadic disease pathogenesis. VCC, encoded by hlyA gene, belongs to the most common class of bacterial toxins, pore-forming toxins (PFTs), which are important virulence factors. Vibrio cholerae infects and kills C. elegans via cholerae toxin independent manner. VCC is required for the lethality, growth retardation and intestinal cell vacuolation during the infection (1). Little is known, however about the gene expression responses against VCC. To address this question we performed a microarray study in C. elegans which was exposed to V. choleare strains with intact and deleted hlyA genes, for 18 hours. 2611 differentially expressed genes were identified when we compared expression in C. elegans exposed to wild type E7946 versus E7946 Dhly, and 758 differentially expressed genes when we compared expression in C. elegans exposed to nearly isogenic V. cholerae vaccine strains CVD109 (hlyA+) versus CVD110 (hlyA-) [considering fold change (+/-) 1.2, FDR=0.5 and P<0.01]. We found significant overlap between these two comparisons, such that 582 genes are common between the two. Many of these genes have been previously reported as mediators of innate immune response against other bacteria in C. elegans. Among the differentially expressed genes are: C-type lectins, such as clec-45, clec-174, clec-209, clec-17, clec-47, abu (activated in blocked unfolded protein response) genes, which contain Prion-like (Q/N-rich)-domain; pqn-5, abu-6, abu-7, abu-8, lipase related lips-6, tollish gene toh-1, genes regulated by daf-16; dod-22 and dod-24. Protective function of the subset of the differentially expressed genes against V. cholerae infection was confirmed using RNAi. Our results suggest that VCC is a major virulence factor, which induces wide variety of immune response related genes during V. cholerae infection in C. elegans. (1) Cinar et al. PLoS ONE, 2010, 5(7): e11558. doi:10.1371/journal.pone.0011558.

Rudgalvyte, M. et al. (2017) International Worm Meeting "Echoes of early life exposure: delayed methylmercury toxicity in Caenorhabditis elegans."

Peltonen, J., Nass, R., Wong, G., Rudgalvyte, M.

[International Worm Meeting, 2017]

Background. Methylmercury (MeHg) is a well-known environmental pollutant, causing adverse effects even in small concentrations. Exposure to MeHg may lead to physical disorders, impaired development, or/and neurobehavioral alterations. MeHg causes oxidative stress, disrupted Ca2+ homeostasis, mitochondrial dynamics changes, cell cycle alterations and cell death. MeHg may lead to delayed toxicity since the first symptoms of poisoning appear long after initial exposure. Clinical signs of exposure may also appear later in life as the result of additional stress, aging, or disease. Since time may pass until the emergence of the first signs of exposure, delayed toxicity is very challenging to study. Results. Age synchronized N2 worms were treated with MeHg (25uM) for 30 min at room temperature at L1 stage and allowed to develop until stage L4 at +200C on NGM/OP-50 plates. Animals were analyzed at L4 and at 12-day old adult stages to determine the delayed effect of MeHg treatment. We observed that acute MeHg treatment delays animal development and shortens lifespan. Moreover, a basal slowing response assay suggests that treated animals exhibit dopaminergic signaling impairment. Interestingly, early MeHg exposure had no effect on either brood size of P0 generation or development, brood size, and longevity of their progeny (F1). A single exposure to MeHg resulted in mitochondria membrane potential decrease and reduced oxygen consumption rate. Global RNA sequencing was performed and data analysis revealed 624 down-regulated and 35 up-regulated genes. During gene annotation analysis, enriched groups of protein modification and metabolic processes, as well as regulation of cell morphogenesis, phosphorylation, protein kinase activity and poly(A) RNA-binding were uncovered. The members of dpy and lpr gene families were further investigated. Knockdown of single genes from these families resulted in a changed animal tolerance to MeHg exposure. Among downregulated genes, abu-8 and abu-11 were observed suggesting the involvement of the unfolded protein response in MeHg-induced animal toxicity. Conclusions. Our study indicates that a single acute MeHg exposure during the development of C. elegans results in adverse effects that are observable later in life, and thus provide a model for delayed toxicity.

Fu Y et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Cooperative Regulation of Innate Immunity by HDA-4, MEF-2, DKF-2A and PMK-1"

Fu Y, Rubin C

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

Infection by the pathogen P. aeruginosa (PA14) elicits expression of microbicidal peptides and other defense related proteins in C. elegans intestinal cells. C. elegans protein kinase D (DKF-2A), which transmits regulatory signals downstream from EGL-8 and TPA-1, contributes to innate immunity by promoting induction of ~85 mRNAs. The mRNAs encode proteins that protect intestine against PA14 toxicity. Signals transmitted by DKF-2A also activate PMK-1, a key regulator of immunity. A complex of HDA-4 (histone deacetylase) and MEF-2 (transcription factor) is a target-effector for DKF-2A and PMK-1. HDACs repress MEF-2 by protein deacetylation and direct binding mechanisms. Non-phosphorylated HDA-4 is concentrated in nuclei of transfected cells. After DKF-2A phosphorylated 2 serines, HDA-4 was exported to cytoplasm. This should de-repress MEF-2 controlled genes. Consequently, HDA-4-GFP and mutant (regulatory Ser mutated to Ala) HDA-4-(AA)-GFP proteins were expressed in transgenic animals. HDA-4-GFP accumulated in both cytoplasm and nuclei. However, depletion of DKF-2A, or phosphorylation site mutation targeted HDA-4 exclusively to nuclei. A mutation in hda-4 (hda-4(oy57)), which de-represses MEF-2, potently increased PA14 resistance. Expression of HDA-4-GFP in the hda-4(oy57) background restored WT sensitivity to PA14; animals expressing HDA-4-(AA)-GFP were hypersensitive to PA14. DKF-2A and PMK-1 were indispensable for MEF-2 mediated PA14 resistance. DKF-2A acts upstream and downstream from HDA-4 in immune signaling; PMK-1 may regulate MEF-2 by direct phosphorylation. Animals lacking MEF-2 (WT or hda-4(oy57) background) were hyper-sensitive to PA14, confirming that MEF-2 and HDA-4 are in a common pathway. Effects of HDA-4, MEF-2, DKF-2A and PMK-1 on specific mRNAs were assessed. CNC-4, NLP-34, F10A3.1, ABU-1 and ABU-7 mRNAs increased 3-10-fold in HDA-4 deficient animals. Depletion of MEF-2, DKF-2A or PMK-1 suppressed mRNA induction in the hda-4(oy57) background.Micro-arrays revealed that MEF-2 induces many immune effector mRNAs that are unaffected by DKF-2A. Expression of HDA-4-GFP in intestine or neurons partly suppressed the hda-4(oy57) phenotype. However, expression in both cell types restored normal sensitivity to PA14. The observations suggest HDA-4-MEF-2 may regulate both innate immunity and pathogen avoidance. C. elegans may use partly overlapping and partly distinct groups of immune effector genes to confer various levels of protection against pathogens.