Lustigman S et al. (1998) East Coast Worm Meeting "Cysteine Proteases of the Parasitic Nematode Onchocerca volvulus"

Liu J, Guiliano DB, Lustigman S, Huima T

[East Coast Worm Meeting, 1998]

We have shown previously that an endogenous cysteine protease is essential for the molting of the Onchocerca volvulus third-stage larvae (L3). The full length cDNA clone encoding the O. volvulus cysteine protease, LOVCP, was found to have a significant identity (22-35% ) to many members of the papain super-family, but mostly to cathepsin C. LOVCP is unique in that it has in its composition a 5 amino acid repeat, CGSCW, which is part of the cysteine active site motif, CGSCWAF. Antibodies directed against the recombinant enzyme detected the protein in cuticle regions where the separation between cuticles occurs during molting, the hypodermis of molting larvae inside secretory vesicles, the basal lamina lining the pseudocoelom, and the epidermal cord. Interestingly, the density of staining is highest in larvae after one day in culture before the separation between the cuticles starts, and was absent from L3. In comparison, antibodies to a recently cloned cysteine protease, a cathepsin L-like enzyme, reacted only with the granules of the glandular esophagus of L3. We are in the process of establishing the function of both enzymes during molting. The cloning of cysteine proteases required for the molting of O. volvulus L3 provides us with the opportunity to study the structure-function relations between the onchocerca enzymes and their endogenous inhibitor, onchocystatin, and the regulation of their activities during molting. The identification of cysteine proteases as essential enzymes for the molting process of O. volvulus L3 and the development of L4, and probably other stages of the parasite as well, opens up important new avenues for drug development.

Lustigman S et al. (1998) East Coast Worm Meeting "C. elegans as a model for studying the role of proteins in the molting and development of human parasitic nematode, Onchocerca volvulus and development"

Hashmi S, Lustigman S

[East Coast Worm Meeting, 1998]

The nematode, Onchocerca volvulus causes Onchocerciasis or river blindness in human. The parasite is transmitted in human by bites of Simulium black flies. For the past few years, Lustigman's group initiated a search for the enzymes involved in the molting process of this nematode. Our emphasis is on the transition from L3 to L4, which can be obtain in vitro as well. The molting involves separation of the cuticle from the underlying epidermis. The formation of a new cuticle arises from the outermost surface of the epidermis, and the shedding of the old cuticle. Previously Lustigman et al shown the involvement of cysteine protease and their endogenous inhibitors as well as a lectin binding protein and other proteins with unknown function in the molting process of infective larvae L3 and successful development of O. volvulus L4. Many of these proteins relevant to the molting and development of O. volvulus have been cloned and characterized. We are using C. elegans as a model to study the function of these proteins. Such studies could provide means for preventing infection and diseased associated with O. volvulus infection.

Radice AD et al. (1995) International C. elegans Meeting "MOLECULAR ANALYSIS OF THE C. ELEGANS GLUTAMATE TRANSPORTER"

Radice AD, Lustigman S

[International C. elegans Meeting, 1995]

We are investigating whether L-glutamate, the major excitatory transmitter in vertebrates, is utilized by neurons that regulate development and behavior in nematodes. In addition, we are interested in the processes regulating signalling at excitatory synapses. Initially, we identified and cloned the glutamate transporter, which is essential for removing glutamate from the synaptic clef. This transporter will be used as a marker to identify neurons that synaptically release glutamate and aspartate. The full length cDNA, CeGlu-1, is 1804 bp and contains an ORF of 1476 bp which encodes a polypeptide of 492 amino acids. The CeGlu-1 ORF is 57-50% identical to the vertebrate glutamate transporters. Southern blot analysis indicates that CeGlu-1 is a single copy gene. The cDNA hybridizes to the overlapping YACs Y43A10, Y43D5 and Y60B6 and maps to the left arm of the X chromosome close to vit-3. The cosmids R01F10, C12D12, M02D2, and B0015 were used to identify a 7 kb genomic fragment containing the CeGlu-1 gene. Flanking DNA upstream of the CeGlu-1 sequence will be used to construct GFP-reporter plasmids to analyze the spatial and temporal expression. To determine which cells express the native CeGlu-1 protein, we have raised antibodies against three distinct regions of the recombinant protein. Affinity purified antibodies recognized a 63 kDa protein on Western blots containing C. elegans extracts.

Radice AD et al. (1993) International C. elegans Meeting "Cloning and characterization of neurotransporters in Caenorhabditis elegans and Onchocerca volvulus."

Radice AD, Lustigman S

[International C. elegans Meeting, 1993]

Liu J et al. (2000) East Coast Worm Meeting "The C. elegans cysteine protease genes Ce-cpz-1 and Ce-cpl have a role in worm's Development"

Liu J, Lustigman S, Hashmi S

[East Coast Worm Meeting, 2000]

We are currently characterizing two C. elegans cysteine proteases, Ce-cpz-1 (cathepsin Z) and Ce-cpl (cathepsin L) family of enzymes. Previously we have reported that Ce-cpz-1 dsRNA blocked embryogenesis and was lethal for the injected worm. While Ce-cpl dsRNA blocked egg production. To understand the potential distinct function of both enzyme, we initiated to study the temporal and spatial pattern of gene expression by creating several stable lines of transgenic C. elegans carrying Ce-cpz-1: gfp and Ce-cpl:gfp genes. All lines of both genes displayed similar expression patterns in all developmental stages of the worm. The Ce-cpz-1 expression was observed along the length of the worm throughout worm's development and was particularly strong in the hypodermal region. The expression of cpz-1 in the hypodermal region suggest that this gene possibly responsible in regulating activities such as molting and cuticle remodeling in C. elegans. In addition, to hypodermal region, the expression of cpz-1 was also observed in the posterior bulb of pharynx. Few clusters of cells also expressed cpz-1 in the vulval region. Intense cpl:gfp expression was observed in the pharyngeal bulb, pharyngeal muscles and hypodermal region throughout the nematode's body in all developmental stages. However, the hypodermal expression was more intense in the head and the tail. The expression of cpl in the hypodermal region suggests that the gene may also be involved in regulating activities such as molting, and cuticle remodeling in C. elegans. Furthermore, robust expression of cpl was also observed in the vulva, eggshell, uterus and spermatheca. Thus, cpl may also be involved in the control of egg production, egg laying and the maintenance of tissues that concentrates sperms and mediates fertilization of oocytes. Results of RNAi experiments with Ce-cpl have shown that injection of dsRNA prepared from Ce-cpl cDNA interfered with normal egg production. It is possible that CPL enzyme is also required for normal hermaphrodite fertility.

Pinan-Lucarre, Berangere et al. (2009) International Worm Meeting "Transgenic C. elegans expressing the fungal prion protein HET-s as a model for the study of amyloid infectivity and toxicity."

Qiao, Yujie, Saupe, Sven, Pinan-Lucarre, Berangere, Driscoll, Monica

[International Worm Meeting, 2009]

Amyloids are protein fibers rich in beta sheet structure that are associated with numerous neurodegenerative diseases, including Alzheimer disease. Amyloids have two major biological properties. First, they can be infectious--meaning that they can spread the altered amyloid conformation to the same protein or even among proteins of distinct primary sequence (the latter phenomenon is known as cross-polymerization). Infectious amyloids are called prions. Second, amyloids can be toxic, whether they are infectious or not. Both properties have tremendous fundamental and biomedical impact. [Het-s] is a prion of the fungus Podospora anserina involved in a defense cell suicide mechanism named heterokaryon incompatibility, which disables mixing of different strains by somatic fusion, an ubiquitous feature in filamentous fungi. The HET-s protein exists in a soluble state or in an aggregated, prion state named [Het-s]. The [Het-s] prion is not toxic by itself in P. anserina or in yeast. However cell death is rapidly triggered by the lethal interaction between 2 alleles of the het-s locus: het-S (het-"large S") and het-s (het-"small s") when the HET-s protein adopts the prion form. This prion has been extensively characterized and it is the only prion for which tridimensional structure is available to date. The critical point is that in the [Het-s] system, transgenic expression of het-s allows propagating a non-toxic prion while co-expression of het-s and het-S generates toxicity. We constructed strains expressing het-s or the prion domain only het-s(218-289) (the 218-289 fragment of the protein) as GFP fusions under the control of the ubiquitous promoter dpy-30. The fluorescence of pdpy-30het-s::gfp appears to be mainly diffuse in the cytoplasm while it shows strong aggregation in pdpy-30het-s(218-289)::gfp strains. We constructed strains expressing het-s(218-289)::gfp in the touch neurons using mec-4 promoter and observed that the fusion protein is mostly aggregated into a large fiber spanning the cell body and the proximal part of the axon. These aggregates remain to be shown as infectious [Het-s] amyloids. In future experiments, we will aim to generate neuronal toxicity through the co-expression of het-s(218-289) and het-S. This C. elegans model for prion studies, filling a gap between simple fungal systems and highly complex mammalian systems, might allow a better understanding of the molecular and cellular basis of amyloid infectivity and toxicity.

Dana L. Miller et al. (2007) International Worm Meeting "Adaptation to hydrogen sulfide increases thermotolerance and lifespan."

Mark B. Roth, Dana L. Miller

[International Worm Meeting, 2007]

Hydrogen sulfide (H₂S), which is naturally produced in animal cells, has been shown to effect physiological changes that improve the capacity of mammals to survive environmental changes. We have investigated the physiological response of C. elegans to H₂S to begin to elucidate the molecular mechanisms of H₂S action. We show that nematodes exposed to H₂S are apparently healthy and do not exhibit phenotypes consistent with metabolic inhibition. However, we observed that animals exposed to H₂S had increased thermotolerance and lifespan and survived subsequent exposure to otherwise lethal concentrations of H₂S. Increased thermotolerance and lifespan is not observed in the sir-2.1(ok434) deletion mutant exposed to H₂S. However, mutants in the insulin signaling pathway (both daf-2 and daf-16), animals with mitochondrial dysfunction (isp-1 and clk-1) and a genetic model of caloric restriction (eat-2) all exhibit H₂S-induced increased thermotolerance. These data suggest that H₂S activates a pathway including SIR-2.1 that is separate from dietary restriction and insulin signaling that results in increased lifespan. Moreover, these studies suggest that SIR-2.1 activity may translate environmental change into physiological alterations that improve survival. It is interesting to consider the possibility that the mechanisms by which H₂S increases thermotolerance and lifespan in nematodes are conserved, and that studies using C. elegans may help explain beneficial effects observed in mammals exposed to H₂S.

Oppenheimer AJ et al. (1995) International C. elegans Meeting "G-PROTEIN SIGNALING IN C. ELEGANS"

Kaplan JM, Oppenheimer AJ

[International C. elegans Meeting, 1995]

We are interested in determining how G-proteins modulate the activity of ion channels in the C. elegans nervous system. To generate behavioral phenotypes dependent on G-protein signaling, we targeted expression of mammalian G-proteins to a set of neurons including those controlling foraging and locomotion (RMD, AVA, AVB, AVD, and PVC). Ectopic expression of several G-proteins under the control of the not-3 promoter results in behavioral defects. Expression of constitutively active rat G-alpha-i1 causes uncoordinated forward and backward locomotion. Expression of wild-type rat G-alpha-s impairs backward locomotion, while constitutively active rat G-alpha-s causes defective backward locomotion and lethargy. In order to identify components of the G-alpha-s signaling pathway, we have initiated a screen for mutations that suppress the behavioral effects of activated G-alpha-s expression. In a screen of approximately 7000 haploid genomes, we have identified 11 independent mutations that improve backward locomotion. Several of these mutations result in a hyperactive phenotype in the absence of activated G-alpha-s expression. Mapping and complementation tests will determine whether the suppressors are allelic to other hyperactive mutants isolated by D. Elkes and J. Kaplan. By cloning the suppressor genes, we hope to identify ion channels regulated by G-alpha-s as well as other components of the G-alpha-s signaling pathway. We also plan to determine whether the behavioral effects of G-alpha-s expression are mediated by known second messenger systems. We are first testing the role of cAMP-dependent protein kinase (PKA), because it is activated by G-alpha-s in other systems and has been shown to phosphorylate ion channels. If G-alpha-s signals through PKA, increasing the level of PKA activity should mimic the effect of activated G-alpha-s expression, and inhibiting PKA should suppress the effect of activated G- alpha-s expression.

Oppenheimer AJ et al. (1996) East Coast Worm Meeting "G-alpha-s signaling in C. elegans"

Kaplan JM, Oppenheimer AJ

[East Coast Worm Meeting, 1996]

We are interested in determining how G-proteins modulate the activity of ion channels in the C. elegans nervous system. To generate phenotypes dependent on G-protein signaling, we expressed mammalian G-proteins under the control of the glr-1 promoter, which drives expression in a set of neurons including those controlling foraging and locomotion (RMD, AVA, AVB, AVD, and PVC). Expression of wild-type rat G-alpha-s impairs backward locomotion, while consitutively active rat G-alpha-s causes paralysis and neuronal degeneration in a subset of G-alpha-s-expressing cells, primarily the PVC and AVD neurons. Our model for the neurodegeneration is that activated G-alpha-s is misregulating an ion channel in AVD and PVC, causing osmotic imbalance and consequent swelling. Degenerating neurons appear enlarged and vacuolated, similar to those in worms carrying gain-of-function mutations in deg-1 and other degenerins. We tested whether activated G-alpha-s is killing cells by misregulating known ion channels. Loss-of-function mutations in deg-1, mec-6, unc-36, unc-2, and egl-19 were unable to suppress the degenerations, suggesting that G-alpha-s is not acting through these degenerins or calcium channels. Mutations in ced-3 and ced-4 do not prevent G-alpha-s-induced degenerations, indicating that G-alpha-s is not killing through apoptosis. To identify the components of the signaling pathway downstream of G-alpha-s, we screened for mutations that suppress the paralysis caused by activated G-alpha-s. In a screen of 7,760 haploid genomes, we identified 8 mutations that suppress paralysis and neurodegeneration and 8 mutations that suppress only paralysis. Five of the degeneration suppressors map to chromosome III and may be allelic. These mutations are semidominant, and several cause a hyperactive phenotype in the absence of activated G-alpha-s. Because hyperactivity also results from mutations in the Go signaling pathway, our suppressor mutations may identify a gene invovled in both the Gs and Go signaling pathways. We are continuing to map and characterize the suppressor genes.

Costi D. Sifri et al. (2007) International Worm Meeting "Immune evasion by staphylococcal exopolysaccharide during C. elegans infection."

Jakob Begun, Dietrich Mack, Costi D. Sifri, Jessica M. Gaiani, Frederick M. Ausubel, Holger Rohde, Stephen B. Calderwood

[International Worm Meeting, 2007]

We have previously shown that C. elegans can serve as a model host for the study of S. aureus pathogenesis and host innate immunity. Here we characterize nematode killing by S. epidermidis. C. elegans fed S. epidermidis survive 3-5 days, which is ~1 day longer than their lifespan on S. aureus. Inactivation of the ica operon (the biofilm exopolysaccharide [PIA] biosynthetic operon and Yersinia hms homolog) in S. epidermidis strain M10 greatly reduces nematode killing compared to the isogenic parental strain 9142 and a plasmid complemented mutant. In S. aureus, overproduction of PIA due to derepression of ica augments nematocidal activity. FITC-WGA labeling confirmed the presence of PIA within the digestive tracts of worms fed 9142 but not M10. Attachment of bacteria or bacterial matrix to the nematode cuticle was not observed. Nematodes briefly exposed to 9142 could not be rescued by transfer to M10, and worms fed dilutions of 9142 in M10 at ratios as low as 1:10e4, respectively, still die. Pulse/chase experiments using 9142, M10, and a second PIA-deficient strain, ATCC 12228, showed that durable colonization requires an intact ica operon. Disruption of N2 worms fed 1:100 9142/M10 mixtures for 20h confirmed significant enrichment of 9142 within the digestive tract of N2 but not sek-1 animals. Moreover, sek-1 and pmk-1 animals were found to be equally sensitive to M10- and 9142-mediated killing, in contrast to wild-type N2 worms. Several conclusions can be drawn from these studies. (a) S. epidermidis can infect and kill worms. (b) Disruption of PIA biosynthesis abrogates S. epidermidis virulence, while overproduction of PIA in S. aureus augments virulence. (c) PIA-producing S. epidermidis has a competitive advantage over PIA-deficient S. epidermidis within the nematode digestive tract. (d) Genetic analysis suggests that PIA may protect luminal staphylococci from SEK-1 p38 MAPK-regulated immune responses.