Ballistic transformation of Caenorhabditis elegans

A novel method to transform the nematode Caenorhabditis elegans is described. DNA coprecipitated with gold particles is shot at worms by means of a helium beam. Transformed worms are either identified by a dominant visible marker or selected by a conditional lethal system.     

Actin and myosin-linked calcium regulation in the nematode Caenorhabditis elegans. Biochemical and structural properties of native filaments and purified proteins.

Calcium regulation of actomyosin activity in the nematode, Caenorhabditis elegans, has been studied with purified proteins and crude thin filaments. Actin and tropomyosin have been purified from C. elegans and shown to be similar in most respects to actin and tropomyosin from rabbit skeletal muscle. The actin comigrates with rabbit actin on polyacrylamide-sodium dodecyl sulfate gel electrophoresis, forms similar filaments and paracrystals, and activates the Mg2+-ATPase of rabbit myosin heads as efficiently as rabbit actin. Nematode tropomyosin has a greater apparent molecular weight (estimated by mobility on polyacrylamide-sodium dodecyl sulfate gels) than the rabbit protein, yet it forms Mg2+-paracrystals with a slightly shorter periodicity. Native thin filaments extracted from nematodes activate rabbit myosin subfragment 1 Mg2+-ATPase in a calcium sensitive manner; the extent of activation is threefold greater in 0.2 mM CaCl2 than in the absence of calcium. This observation suggests that the thin filaments contain components which are functionally equivalent to vertebrate troponins. Calcium is also required for maximal activation of the Mg2+-ATPase of purified nematode myosin by pure rabbit F-actin. C. elegans therefore has both myosin and thin filament-linked calcium regulatory systems. The origin of the actin, tropomyosin, and myosin from different tissues and the use of genetic analysis to answer questions about assembly and function in vivo are discussed.     

Identification of Heterodera glycines (soybean cyst nematode [SCN]) cDNA sequences with high identity to those of Caenorhabditis elegans having lethal mutant or RNAi phenotypes

The soybean cyst nematode (SCN; Heterodera glycines) is a devastating obligate parasite of Glycine max (soybean) causing one billion dollars in losses to the US economy per year and over ten billion dollars in losses worldwide. While much is understood about the pathology of H. glycines, its genome sequence is not well characterized or fully sequenced. We sought to create bioinformatic tools to mine the H. glycines nucleotide database. One way is to use a comparative genomics approach by anchoring our analysis with an organism, like the free-living nematode Caenorhabditis elegans. Unlike H. glycines, the C. elegans genome is fully sequenced and is well characterized with a number of lethal genes identified through experimental methods. We compared an EST database of H. glycines with the C. elegans genome. Our goal was identifying genes that may be essential for H. glycines survival and would serve as an automated pipeline for RNAi studies to both study and control H. glycines. Our analysis yielded a total of nearly 8334 conserved genes between H. glycines and C. elegans. Of these, 1508 have lethal phenotypes/phenocopies in C. elegans. RNAi of a conserved ribosomal gene from H. glycines (Hg-rps-23) yielded dead and dying worms as shown by positive Sytox fluorescence. Endogenous Hg-rps-23 exhibited typical RNA silencing as shown by RT-PCR. However, an unrelated gene Hg-unc-87 did not exhibit RNA silencing in the Hg-rps-23 dsRNA-treated worms, demonstrating the specificity of the silencing.     

Virulence of Leucobacter chromiireducens subsp. solipictus to Caenorhabditis elegans: characterization of a novel host-pathogen interaction

We describe the pathogenic interaction between a newly described gram-positive bacterium, Leucobacter chromiireducens subsp. solipictus strain TAN 31504, and the nematode Caenorhabditis elegans. TAN 31504 pathogenesis on C. elegans is exerted primarily through infection of the adult nematode uterus. TAN 31504 enters the uterus through the external vulval opening, and the ensuing uterine infection is strongly correlated with a significant reduction in host life span. Young worms can feed and develop on TAN 31504, but not preferably over the standard food source. C. elegans worms reared on TAN 31504 as the sole food source develop into thin adults with little intestinal fat stores, produce few progeny, and subsequently cannot persist on the pathogenic food source. Within 12 h of exposure, adult worms challenged with TAN 31504 alter the expression of a number of C. elegans innate immunity-related genes, including nlp-29, which encodes a neuropeptide-like protein. C. elegans worms exposed briefly to TAN 31504 develop lethal uterine infections analogous to worms exposed continuously to pathogen, suggesting that mere contact with the pathogen is sufficient for the host to become infected. TAN 31504 produces a robust biofilm, and this behavior is speculated to play a role in the virulence exerted on the nematode host. The interaction between TAN 31504 and C. elegans provides a convenient opportunity to study bacterial virulence on nematode tissues other than the intestine and may allow for the discovery of host innate immunity elicited specifically in response to vulva-uterus infection.     

Activity of novel nicotinic anthelmintics in cut preparations of Caenorhabditis elegans

The free-living nematode Caenorhabditis elegans is a useful model for studying the pharmacology of anthelmintics. Currently approved anthelmintics have various mechanisms of action, including activity at nematode nicotinic acetylcholine receptors (nAChRs). Classical anthelmintic agonists of these receptors (nicotine, levamisole, pyrantel and bephenium) caused intact specimens of C. elegans to undergo contracted paralysis. The nAChR antagonist mecamylamine paralysed intact worms and blocked the actions of the agonists. The time to onset of effects of these drugs was enhanced when worms bisected between the mid- and anterior-portions were tested. The novel anthelmintic nAChR antagonist derquantel (2-desoxoparaherquamide, 2-DOPH) was weakly active in intact specimens of C. elegans at concentrations of 50 μM over several days. No antagonism of the nAChR agonists was observed with this drug in intact worms. However, derquantel had direct and marked effects on motility in cut worms and blocked the effects of nAChR agonists in this preparation. A representative of the new amino-acetonitrile derivative (AAD) class of nAChR agonists was not antagonised by derquantel in cut C. elegans, suggesting that these two anthelmintics may not demonstrate unfavourable drug-drug interactions at the receptor level if used to treat livestock infected with parasitic nematodes. The permeability properties of the C. elegans cuticle may be more restrictive than those of adult parasites, calling into question primary anthelmintic screening strategies that rely on this model organism.     

First identification of a phosphorylcholine-substituted protein from Caenorhabditis elegans: isolation and characterization of the aspartyl protease ASP-6

Caenorhabditis elegans is a widely accepted model system for parasitic nematodes, drug screening and developmental studies. Similar to parasitic worms, C. elegans expresses glycosphingolipids and glycoproteins carrying, in part, phosphorylcholine (PCho) substitutions, which might play important roles in nematode development, fertility and, at least in the case of parasites, survival within the host. With the exception of a major secretory/excretory product from Acanthocheilonema viteae (ES-62), no protein carrying this epitope has been studied in detail yet. Here we report on the identification, characterization and localization of the aspartyl protease ASP-6 of C. elegans, which is excreted by the nematode in a PCho-substituted form. Within the worm, most prominent expression of the protein is observed in the intestine, while muscle and epithelial cells express asp-6 to a lesser extent. In animals harboring an ASP-6::GFP fusion protein, diffuse fluorescence throughout the body cavity of adult worms indicates that the chimeric protein is secreted.     

Nematode Memory: Now,Where Was I?

The nematode Caenorhabditis elegans is able to use tastes, smells and temperature to locate food. New data show that worms can also detect the level of oxygen in the environment and migrate towards an oxygen level associated with food.     

Myosin and paramyosin of Caenorhabditis elegans: biochemical and structural properties of wild-type and mutant proteins.

Myosin and paramyosin have been purified from the nematode, Caenorhabditis elegans. The properties of the myosin in general resemble those of other myosins. The native molecule is a dimer of heavy (210,000 dalton) polypeptide chains and contains 18,000 and 16,000 dalton light chains. When rapidly precipitated from solution, it forms small, bipolar aggregates, about 150 nm long, consistent with the expected molecular structure of a rigid rod with a globular head region at one end. Its ATPase activity is stimulated by Ca2+ and EDTA. The myosin binds to F actin in a polar and ATP-sensitive manner, and the Mg2+-ATPase is activated by either F actin or nematode thin filaments. Dialysis of myosin to low ionic strength produces very long filaments. When a myosin-paramyosin mixture is dialyzed under the same condtions, co-filaments form which consist of a myosin cortex, surrounding a paramyosin core. Some properties of myosin from the mutants E675 and E190, which have functionally and structurally altered body wall muscles, are compared with those of wild-type myosin. These myosins of these results are discussed in terms of the myosin heavy chain composition.     

Identification of a platyhelminth neuropeptide receptor

We report the characterisation of the first neuropeptide receptor from the phylum Platyhelminthes, an early-diverging phylum which includes a number of important human and veterinary parasites. The G protein-coupled receptor (GPCR) was identified from the model flatworm Girardia tigrina (Tricladida: Dugesiidae) based on the presence of motifs widely conserved amongst GPCRs. In two different assays utilising heterologous expression in Chinese hamster ovary cells, the Girardia GPCR was most potently activated by neuropeptides from the FMRFamide-like peptide class. The most potent platyhelminth neuropeptide in both assays was GYIRFamide, a FMRFamide-like peptide known to be present in G. tigrina. There was no activation by neuropeptide Fs, another class of flatworm neuropeptides. Also active were FMRFamide-like peptides derived from other phyla but not known to be present in any platyhelminth. Most potent among these were nematode neuropeptides encoded by the Caenorhabditis elegans flp-1 gene which share a PNFLRFamide carboxy terminal motif. The ability of nematode peptides to stimulate a platyhelminth receptor demonstrates a degree of structural conservation between FMRFamide-like peptide receptors from these two distinct, distant phyla which contain parasitic worms.     

Influence of lactic acid bacteria on longevity of Caenorhabditis elegans and host defense against salmonella enterica serovar enteritidis

This study aimed to develop a convenient model to investigate the senescence of host defenses and the influence of food and nutrition. A small soil nematode, Caenorhabditis elegans, was grown for 3 days from hatching on a lawn of Escherichia coli OP50 as the normal food source, and subsequently some of the nematodes were fed lactic acid bacteria (LAB). The life spans of worms fed LAB were significantly longer than the life spans of those fed OP50. To investigate the effect of age on host defenses, 3- to 7-day-old worms fed OP50 were transferred onto a lawn of Salmonella enterica serovar Enteritidis for infection. The nematodes died over the course of several days, and the accumulation of salmonella in the intestinal lumen suggested that the worms were infected. The 7-day-old worms showed a higher death rate during the 5 days after infection than nematodes infected at the age of 3 days; no clear difference was observed when the worms were exposed to OP50. We then investigated whether the LAB could exert probiotic effects on the worms' host defenses and improve life span. Seven-day-old nematodes fed LAB from the age of 3 days were more resistant to salmonella than worms fed OP50 until they were infected with salmonella. This study clearly showed that LAB can enhance the host defense of C. elegans and prolong life span. The nematode appears to be an appropriate model for screening useful probiotic strains or dietetic antiaging substances.     

A peroxiredoxin specifically expressed in two types of pharyngeal neurons is required for normal growth and egg production in Caenorhabditis elegans

A family of antioxidant proteins, the peroxiredoxins, serve two purposes, detoxification of reactive oxygen species and cellular signaling. Among the three peroxiredoxins of Caenorhabditis elegans (CePrx1-3), CePrx2 was found to have a very unusual expression pattern, restricted to only two types of pharyngeal neurons; namely, the single pharyngeal interneuron I4 and the sensory interneuron I2. CePrx1 and CePrx3-depleted worms showed no obvious phenotypic alterations, whereas worms devoid of CePrx2 were retarded developmentally and had a significantly reduced brood size. Other features, such as lifespan, pharyngeal activity or defecation rates were indistinguishable from those of wild-type worms. Recombinant CePrx2 revealed antioxidant activity, as it was able to detoxify hydrogen peroxide and butylhydroperoxide (t-BOOH), and to protect glutamine synthetase from inactivation by thiol-dependent metal-catalyzed oxidation. In addition, the molecule was able to act as a terminal peroxidase in the thioredoxin system. Expression of ceprx2 in C.elegans was induced after short-term exposure of worms to t-BOOH but survival of ceprx2 knockout mutants in the presence of reactive oxygen or nitrogen species was not impaired. Thus, CePrx2 may protect specifically the two types of neurons from oxidative damage or, more likely, plays a critical role in peroxide signaling in this nematode.     

Ingestion of Cryptosporidium oocysts by Caenorhabditis elegans

Cryptosporidium parvum has been associated with outbreaks of human illness by consumption of contaminated water, fresh fruits, and vegetables. Free-living nematodes may play a role in pathogen transmission in the environment. Caenorhabditis elegans is a free-living soil nematode that has been extensively studied and serves as a good model to study possible transmission of C. parvum oocysts that may come into contact with produce before harvest. The objective of this study was to determine whether C. elegans could serve as a potential mechanical vector for transport of infectious C. parvum and Cyclospora cayetanensis in agricultural settings and whether C. elegans could ingest, excrete, and protect oocysts from desiccation. Seventy to 85% of worms ingested between 0 and 500 oocysts after 1 and 2 hr incubation with oocysts. Most of the nematodes ingested between 101 and 200 oocysts after 2 hr. Intact oocysts and empty shells were excreted by nematodes. Infectivity was determined by the neonatal assay with different treatments of worms (intact or homogenized) or oocysts or both. Adult C. elegans containing C. parvum kept in water were infective for mice. In conclusion, C. elegans adults can ingest and excrete C. parvum oocysts. Caenorhabditis elegans containing C. parvum oocysts can infect mice but does not seem to protect oocysts from extreme desiccation at 23 C incubation of a day or longer. Cyclospora oocysts were not ingested by C. elegans. The role of free-living nematodes in produce contamination needs to be further examined.     

Modulation of myosin assembly

Myosin self-assembly is generally considered to be the major process in thick filament formation within striated muscles. The biological assembly of myosin into thick filaments is being analysed by genetic dissection as well as biochemical and morphological experiments in the nematode Caenorhabditis elegans. This work shows that the assembly of myosin is modulated by its biosynthesis and interaction with non-myosin proteins. Assemblages which generate multiple nascent thick filaments may play a central role in a catalytic cycle of myosin assembly.     

Assembly-dependent phosphorylation of myosin and paramyosin of native thick filaments in Caenorhabditis elegans.

Phosphorylation of the thick filament proteins myosin and paramyosin was studied in Caenorhabditis elegans. We have incubated partially purified, native thick filaments with [gamma 32P] ATP in the presence of 50-750 mM NaCl, pH 6.5-8.0. Myosin heavy chain and paramyosin were phosphorylatable only upon solubilization at 450 mM and higher NaCl concentrations. Under conditions preserving native structures, no phosphorylation of these proteins occurred. The phosphorylation required Mg2+ but was unaffected by cAMP, cGMP or Ca2+. The specific inhibitor of cAMP and cGMP kinase catalytic subunits, H8, inhibits the activity. Sedimentation experiments show that the kinase may associate with but is not an intrinsic component of thick filaments. In C. elegans, phosphorylation by the thick filament associated activity of myosin and paramyosin is dependent upon the state of their assembly.     

The longevity of Caenorhabditis elegans in soil

Relatively simple model organisms such as yeast, fruit-flies and the nematode, Caenorhabditis elegans, have proven to be invaluable resources in biological studies. An example is the widespread use of C. elegans to investigate the complex process of ageing. An important issue when interpreting results from these studies is the similarity of the observed C. elegans mortality pattern in the laboratory to that expected in its natural environment. We found that the longevity of C. elegans under more natural conditions is reduced up to 10-fold compared with standard laboratory culture conditions. Additionally, C. elegans mutants that live twice as long as wild-type worms in laboratory conditions typically die sooner than wild-type worms in a natural soil. These results indicate that conclusions regarding extended longevity drawn from standard laboratory assays may not extend to animals in their native environment.     

Expression of chimeric genes in Caenorhabditis elegans

We have shown the expression of transformed genes in the nematode Caenorhabditis elegans using a new gene fusion system. Vectors consisting of the flanking regions of a collagen gene (col-1) or a major sperm protein gene of C. elegans fused to the Escherichia coli uidA gene, encoding beta-glucuronidase, were microinjected into worms and found to be propagated as high-copy extrachromosomal tandem arrays. We have detected beta-glucuronidase activity in transformed lines, and have shown that the activity is dependent upon the correct reading frame of the construction and on the presence of the worm sequences. The enzyme activity was shown to be encoded by the chimeric beta-glucuronidase gene by co-segregation analysis and by inactivation with specific antisera. Expression is at a very low level, and seems to be constitutive. We have used histochemical techniques to visualize the enzyme activity in embryos.     

Hsp90 is essential in the filarial nematode Brugia pahangi

The development of a compound with activity against filarial nematodes (a 'macrofilaricide') has been a long-standing goal of the World Health Organization. However, adult filariae have proved remarkably difficult to kill. To some extent this reflects a lack of understanding of key pathways and processes in filarial nematodes that may be suitable targets for chemotherapy. In this paper we show that geldanamycin (GA), a specific inhibitor of the activity of the heat shock protein 90 (Hsp90) family, kills adult worms and microfilariae (Mf) of Brugia pahangi at nanomolar concentrations. In addition, release of Mf from adult worms is inhibited within 24 h of exposure to GA and is not recoverable, demonstrating that GA effectively sterilises the worm. Similar results were obtained with a second filarial worm Acanthocheilonema viteae. In contrast GA has no effect on the free-living nematode Caenorhabditis elegans despite a high degree of conservation between the nematode Hsp90 sequences. In keeping with these findings, Brugia Hsp90 binds GA in a solid phase pull-down assay while the binding of C. elegans Hsp90 to immobilised GA is undetectable. In other eukaryotes, GA is known to bind in the N-terminal ATP pocket of Hsp90, disrupting its interactions with client proteins which are then targeted for degradation via the proteasome pathway. Thus, Hsp90 or some of its client proteins may provide novel targets for the chemotherapy of filarial infection.     

Anti-connectin monoclonal antibodies that react with the unc-22 gene product bind dense bodies of Caenorhabditis (Nematode) bodywall muscle cells

Monoclonal antibodies, 3B9 and 4C9, specific to connectin (also called titin), 3000 kDa elastic filamentous protein of vertebrate skeletal muscle, crossreacted with a high molecular weight protein (500 kDa) of the nematode Caenorhabditis elegans. However, its crossreactivity was weak to that of the unc-22 gene deficient mutant. Immunofluorescence showed that the antibodies stained both bodywall and pharynx muscles in the wild type, but only pharynx muscle in the mutant. Immunoelectron microscopy revealed that the antibodies bound to the dense bodies of bodywall muscle cells of the wild type but not to those of the mutants. In the pharynx muscles the localization of the antibodies was not clear in both normal and mutant worms. Moerman, D.G. et al. (Genes & Development 2, 93-105 (1988) reported that the unc-22 gene product (500 kDa) is located in the A band of the bodywall muscle cells of C. elegans. Taking this information into consideration, it is suggested that the unc-22 gene product may be a qfilamentous protein linking a dense body and myosin filaments in the bodywall muscles of C. elegans.