Correction: Antinematode Activity of Violacein and the Role of the Insulin/IGF-1 Pathway in Controlling Violacein Sensitivity in Caenorhabditis elegans.

[This corrects the article DOI: 10.1371/journal.pone.0109201.].

Caenorhabditis elegans employs innate and learned aversion in response to bacterial toxic metabolites tambjamine and violacein.

Bacteriovorus eukaryotes such as nematodes are one of the major natural predators of bacteria. In their defense bacteria have evolved a number of strategies to avoid predation, including the production of deterrent or toxic metabolites, however little is known regarding the response of predators towards such bacterial defenses. Here we use the nematode C. elegans as a model to study a predators' behavioral response towards two toxic bacterial metabolites, tambjamine YP1 and violacein. We found that C. elegans displays an innate avoidance behavior towards tambjamine YP1, however requires previous exposure to violacein before learning to avoid this metabolite. The learned avoidance of violacein is specific, reversible, is mediated via the nematode olfactory apparatus (aversive olfactory learning) and is reduced in the absence of the neurotransmitter serotonin. These multiple strategies to evade bacterial toxic metabolites represent a valuable behavioral adaptation allowing bacteriovorus predators to distinguish between good and bad food sources, thus contributing to the understanding of microbial predator-prey interactions.

Neuron-specific knock-down of SMN1 causes neuron degeneration and death through an apoptotic mechanism.

Spinal muscular atrophy is a devastating disease that is characterized by degeneration and death of a specific subclass of motor neurons in the anterior horn of the spinal cord. Although the gene responsible, survival motor neuron 1 (SMN1), was identified 20 years ago, it has proven difficult to investigate its effects in vivo. Consequently, a number of key questions regarding the molecular and cellular functions of this molecule have remained unanswered. We developed a Caenorhabditis elegans model of smn-1 loss-of-function using a neuron-specific RNA interference strategy to knock-down smn-1 selectively in a subclass of motor neurons. The transgenic animals presented a cell-autonomous, age-dependent degeneration of motor neurons detected as locomotory defects and the disappearance of presynaptic and cytoplasmic fluorescent markers in targeted neurons. This degeneration led to neuronal death as revealed by positive reactivity to genetic and chemical cell-death markers. We show that genes of the classical apoptosis pathway are involved in the smn-1-mediated neuronal death, and that this phenotype can be rescued by the expression of human SMN1, indicating a functional conservation between the two orthologs. Finally, we determined that Plastin3/plst-1 genetically interacts with smn-1 to prevent degeneration, and that treatment with valproic acid is able to rescue the degenerative phenotype. These results provide novel insights into the cellular and molecular mechanisms that lead to the loss of motor neurons when SMN1 function is reduced.

Antinematode activity of Violacein and the role of the insulin/IGF-1 pathway in controlling violacein sensitivity in Caenorhabditis elegans.

The purple pigment violacein is well known for its numerous biological activities including antibacterial, antiviral, antiprotozoan, and antitumor effects. In the current study we identify violacein as the antinematode agent produced by the marine bacterium Microbulbifer sp. D250, thereby extending the target range of this small molecule. Heterologous expression of the violacein biosynthetic pathway in E. coli and experiments using pure violacein demonstrated that this secondary metabolite facilitates bacterial accumulation in the nematode intestine, which is accompanied by tissue damage and apoptosis. Nematodes such as Caenorhabditis elegans utilise a well-defined innate immune system to defend against pathogens. Using C. elegans as a model we demonstrate the DAF-2/DAF-16 insulin/IGF-1 signalling (IIS) component of the innate immune pathway modulates sensitivity to violacein-mediated killing. Further analysis shows that resistance to violacein can occur due to a loss of DAF-2 function and/or an increased function of DAF-16 controlled genes involved in antimicrobial production (spp-1) and detoxification (sod-3). These data suggest that violacein is a novel candidate antinematode agent and that the IIS pathway is also involved in the defence against metabolites from non-pathogenic bacteria.

The G protein regulators EGL-10 and EAT-16, the Giα GOA-1 and the G(q)α EGL-30 modulate the response of the C. elegans ASH polymodal nociceptive sensory neurons to repellents.

BACKGROUND: Polymodal, nociceptive sensory neurons are key cellular elements of the way animals sense aversive and painful stimuli. In Caenorhabditis elegans, the polymodal nociceptive ASH sensory neurons detect aversive stimuli and release glutamate to generate avoidance responses. They are thus useful models for the nociceptive neurons of mammals. While several molecules affecting signal generation and transduction in ASH have been identified, less is known about transmission of the signal from ASH to downstream neurons and about the molecules involved in its modulation. RESULTS: We discovered that the regulator of G protein signalling (RGS) protein, EGL-10, is required for appropriate avoidance responses to noxious stimuli sensed by ASH. As it does for other behaviours in which it is also involved, egl-10 interacts genetically with the G(o)/(i)α protein GOA-1, the G(q)α protein EGL-30 and the RGS EAT-16. Genetic, behavioural and Ca²(+) imaging analyses of ASH neurons in live animals demonstrate that, within ASH, EGL-10 and GOA-1 act downstream of stimulus-evoked signal transduction and of the main transduction channel OSM-9. EGL-30 instead appears to act upstream by regulating Ca²(+) transients in response to aversive stimuli. Analysis of the delay in the avoidance response, of the frequency of spontaneous inversions and of the genetic interaction with the diacylglycerol kinase gene, dgk-1, indicate that EGL-10 and GOA-1 do not affect signal transduction and neuronal depolarization in response to aversive stimuli but act in ASH to modulate downstream transmission of the signal. CONCLUSIONS: The ASH polymodal nociceptive sensory neurons can be modulated not only in their capacity to detect stimuli but also in the efficiency with which they respond to them. The Gα and RGS molecules studied in this work are conserved in evolution and, for each of them, mammalian orthologs can be identified. The discovery of their role in the modulation of signal transduction and signal transmission of nociceptors may help us to understand how pain is generated and how its control can go astray (such as chronic pain) and may suggest new pain control therapies.

Mutation of SHOC2 promotes aberrant protein N-myristoylation and causes Noonan-like syndrome with loose anagen hair.

N-myristoylation is a common form of co-translational protein fatty acylation resulting from the attachment of myristate to a required N-terminal glycine residue. We show that aberrantly acquired N-myristoylation of SHOC2, a leucine-rich repeat-containing protein that positively modulates RAS-MAPK signal flow, underlies a clinically distinctive condition of the neuro-cardio-facial-cutaneous disorders family. Twenty-five subjects with a relatively consistent phenotype previously termed Noonan-like syndrome with loose anagen hair (MIM607721) shared the 4A>G missense change in SHOC2 (producing an S2G amino acid substitution) that introduces an N-myristoylation site, resulting in aberrant targeting of SHOC2 to the plasma membrane and impaired translocation to the nucleus upon growth factor stimulation. Expression of SHOC2(S2G) in vitro enhanced MAPK activation in a cell type-specific fashion. Induction of SHOC2(S2G) in Caenorhabditis elegans engendered protruding vulva, a neomorphic phenotype previously associated with aberrant signaling. These results document the first example of an acquired N-terminal lipid modification of a protein causing human disease.

Efficient and cell specific knock-down of gene function in targeted C. elegans neurons.

The nematode C. elegans has become an important model for understanding how genes influence behavior. However, in this organism the available approaches for identifying the neuron(s) where the function of a gene is required for a given behavioral trait are time consuming and restricted to non essential genes for which mutants are available. We describe a simple reverse genetics approach for reducing, in chosen C. elegans neurons, the function of genes. The method is based on the expression, under cell specific promoters, of sense and antisense RNA corresponding to a gene of interest. By targeting the genes osm-10, osm-6 and the Green Fluorescent Protein gene, gfp, we show that this approach leads to efficient, heritable and cell autonomous knock-downs of gene function, even in neurons usually refractory to classic RNA interference (RNAi). By targeting the essential and ubiquitously expressed gene, gpb-1, which encodes a G protein beta subunit, we identify for the first time two distinct sets of neurons in which the function of gpb-1 is required to regulate two distinct behaviors: egg-laying and avoidance of repellents. The cell specific knock-downs obtained with this approach provide information that is complementary to that provided by the cell specific rescue of loss-of-function mutations and represents a useful new tool for dissecting the role that genes play in selected neurons.

A new competitive fluorescence assay for the detection of patulin toxin.

Patulin is a toxic secondary metabolite of a number of fungal species belonging to the genera Penicillum and Aspergillus. It has been mainly isolated from apples and apple products contaminated with the common storage-rot fungus of apples, Penicillum expansum, but it has also been extracted from rotten fruits, moldy feeds, and stored cheese. Human exposure to patulin can lead to serious health problems, and according to a long-term investigation in rats, the World Health Organization has set a tolerable weekly intake of 7 ppb body weight. The content of patulin in foods has been restricted to 50 ppb in many countries. Conventional analytical detection methods involve chromatographic analyses, such as HPLC, GC, and, more recently, techniques such as LC/MS and GC/MS. However, extensive protocols of sample cleanup are required prior to the analysis, and to accomplish it, expensive analytical instrumentation is necessary. An immunochemical analytical method, based on highly specific antigen-antibody interactions, would be desirable, offering several advantages compared to conventional techniques, i.e., low cost per sample, high selectivity, high sensitivity, and high throughput. In this paper, the synthesis of two new derivatives of patulin is described, along with their conjugation to the bovine serum albumin for the production of polyclonal antibodies. Finally, a fluorescence competitive immunoassay was developed for the on-line detection of patulin.

UMODL1/Olfactorin is an extracellular membrane-bound molecule with a restricted spatial expression in olfactory and vomeronasal neurons.

The olfactory system provides a unique model for developmental neurobiology. Precise targeting of axonal projections from sensory neurons located in the olfactory epithelium to specific neurons in the olfactory bulb establishes a highly refined spatial sensory map. Distinctively, this process is not restricted to embryonic stages, but continues during the entire life of mammals. A number of secreted and membrane molecules have been implicated in guidance and targeting of olfactory sensory neurons. Here we describe olfactorin, the protein product of the mouse Umodl1 gene, as a potential new element in this process. Olfactorin is a secreted modular protein containing several domains typically present in extracellular matrix proteins (EMI, WAP, FNIII, Ca2+ -binding EGF-like, SEA and ZP domains). By in situ hybridization we find that during embryonic development expression of the Umodl1 gene is detectable only in the olfactory epithelium and vomeronasal organ starting at embryonic day 16.5. At this stage, Umodl1 expression within the olfactory epithelium is punctate, and is restricted to only some of the sensory neurons. At birth and postnatally, expression in these organs continues and involves more neurons. Kallmann syndrome is a genetic disease in which olfactory axons fail to connect to target neurons in the bulb. We tested whether olfactorin might be responsible for an autosomal form of this disease and show that this is not the case. However, based on its domain composition and on the expression in olfactory neurons we suggest that olfactorin may play a role in correct olfactory axon navigation to the brain.

The Zona Pellucida domain containing proteins, CUT-1, CUT-3 and CUT-5, play essential roles in the development of the larval alae in Caenorhabditis elegans.

The alae, longitudinal ridges of the lateral cuticle, are the most visible specialization of the Caenorhabditis elegans surface. They are present only in L1 and dauer larvae and in adults. Little is known about the mechanisms through which at the appropriate stages secretion of cuticle components by the seam cells results in the formation of the alae. Here we show that three proteins, each containing a Zona Pellucida domain (ZP), are components of the cuticle necessary for larval alae development: CUT-1 and CUT-5 in dauer larvae and CUT-3 and CUT-5 in L1s. Transcriptional regulation of the corresponding genes contributes to the stage-specific role of these proteins. Larvae with reduced cut-1, cut-3 or cut-5 function not only lack alae but are also larger in diameter due to an increase in the width of the lateral cuticle. We propose a model in which reduction of the body diameter, which occurs in normal L1 and dauer larvae, is the result of a dorso-ventral shrinking of the internal layer of the lateral cuticle and formation of the alae results from the folding of the external layer of the lateral cuticle over the reduced, internal one. Alae of adults appear to form through a different mechanism.

Glucose biosensors as models for the development of advanced protein-based biosensors.

Glucose sensing is used as a model to explore the advantages and problems deriving from the use of either enzymes or sugar binding proteins to develop stable fluorescence biosensors. We report on a novel approach to address the problem of substrate consumption by sensors based on enzymes, namely the utilization of apo-enzymes as non-active forms of the protein which are still able to bind the substrate/ligand. We also review studies in which derivatization of a naturally thermostable sugar-binding protein with a fluorescent probe allows quantitative monitoring of glucose binding even after immobilization on a solid support.

In vivo imaging of C. elegans ASH neurons: cellular response and adaptation to chemical repellents.

ASH sensory neurons are required in Caenorhabditis elegans for a wide range of avoidance behaviors in response to chemical repellents, high osmotic solutions and nose touch. The ASH neurons are therefore hypothesized to be polymodal nociceptive neurons. To understand the nature of polymodal sensory response and adaptation at the cellular level, we expressed the calcium indicator protein cameleon in ASH and analyzed intracellular Ca(2+) responses following stimulation with chemical repellents, osmotic shock and nose touch. We found that a variety of noxious stimuli evoked strong responses in ASH including quinine, denatonium, detergents, heavy metals, both hyper- and hypo-osmotic shock and nose touch. We observed that repeated chemical stimulation led to a reversible reduction in the magnitude of the sensory response, indicating that adaptation occurs within the ASH sensory neuron. A key component of ASH adaptation is GPC-1, a G-protein gamma-subunit expressed specifically in chemosensory neurons. We hypothesize that G-protein gamma-subunit heterogeneity provides a mechanism for repellent-specific adaptation, which could facilitate discrimination of a variety of repellents by these polymodal sensory neurons.

A thermostable sugar-binding protein from the Archaeon Pyrococcus horikoshii as a probe for the development of a stable fluorescence biosensor for diabetic patients.

In this work is presented the first attempt to develop an innovative ultrastable protein-based biosensor for blood glucose detections. The gene of a putative thermostable sugar-binding protein has been cloned and expressed in E. coli. The recombinant protein has been purified to homogeneity by thermoprecipitation and affinity chromatography steps. The recombinant protein is a monomer with an apparent molecular weight of 55,000 as judged by gel filtration and sodium dodecyl sulfate polyacrylamide gel eletrophoresis. Circular dichroism experiments showed that the protein possesses a secondary structure content rich in alpha-helices and beta-structures and that the protein is highly stable as investigated in the range of temperature between 20 and 95 degrees C. Fluorescence spectroscopy experiments demonstrated that the recombinant protein binds glucose with a dissociation constant of about 10 mM, a concentration of sugar very close to the concentration of glucose present in the human blood. A docking simulation on the modeled structure of the protein confirms its ability to bind glucose and proposes possible modifications to improve the affinity for glucose and/or its detection. The obtained results suggest the use of the protein as a probe for a stable glucose biosensor.

Worms taste bitter: ASH neurons, QUI-1, GPA-3 and ODR-3 mediate quinine avoidance in Caenorhabditis elegans.

An animal's ability to detect and avoid toxic compounds in the environment is crucial for survival. We show that the nematode Caenorhabditis elegans avoids many water-soluble substances that are toxic and that taste bitter to humans. We have used laser ablation and a genetic cell rescue strategy to identify sensory neurons involved in the avoidance of the bitter substance quinine, and found that ASH, a polymodal nociceptive neuron that senses many aversive stimuli, is the principal player in this response. Two G protein alpha subunits GPA-3 and ODR-3, expressed in ASH and in different, nonoverlapping sets of sensory neurons, are necessary for the response to quinine, although the effect of odr-3 can only be appreciated in the absence of gpa-3. We identified and cloned a new gene, qui-1, necessary for quinine and SDS avoidance. qui-1 codes for a novel protein with WD-40 domains and which is expressed in the avoidance sensory neurons ASH and ADL.

C. elegans responds to chemical repellents by integrating sensory inputs from the head and the tail.

The phasmids are bilateral sensory organs located in the tail of Caenorhabditis elegans and other nematodes. The similar structures of the phasmids and the amphid chemosensory organs in the head have long suggested a chemosensory function for the phasmids. However, the PHA and PHB phasmid neurons are not required for chemotaxis or for dauer formation, and no direct proof of a chemosensory function of the phasmids has been obtained. C. elegans avoids toxic chemicals by reversing its movement, and this behavior is mediated by sensory neurons of the amphid, particularly, the ASH neurons. Here we show that the PHA and PHB phasmid neurons function as chemosensory cells that negatively modulate reversals to repellents. The antagonistic activity of head and tail sensory neurons is integrated to generate appropriate escape behaviors: detection of a repellent by head neurons mediates reversals, which are suppressed by antagonistic inputs from tail neurons. Our results suggest that C. elegans senses repellents by defining a head-to-tail spatial map of the chemical environment.

feh-1 and apl-1, the Caenorhabditis elegans orthologues of mammalian Fe65 and beta-amyloid precursor protein genes, are involved in the same pathway that controls nematode pharyngeal pumping.

The multigenic family of mammalian Fe65s encodes three highly similar proteins with the same modular organisation: a WW domain and two phosphotyrosine-binding domains. The PTB2 domain of these proteins binds to the cytosolic domains of the Alzheimer's beta-amyloid precursor protein APP and related proteins APLP1 and APLP2, generating a highly redundant system that is hard to dissect by reverse genetics. By searching potential Fe65-like genes in the nematode Caenorhabditis elegans, we identified a single gene, feh-1 (Fe65 homolog-1), encoding a protein with a high sequence similarity to mammalian Fe65s. FEH-1 is also functionally related to mammalian orthologues; in fact its PTB2 domain binds to APL-1, the product of the C. elegans orthologue of APP. Staining with specific antibodies show that the neuromuscular structures of the pharynx are the sites in which FEH-1 is present at highest levels. Expression studies with reporters indicate that the feh-1 gene is also expressed by a subset of the worm neurons. We generated and isolated a deletion allele of feh-1, and the corresponding homozygous mutants arrest as late embryos or as L1 larvae, demonstrating for the first time an essential role for a Fe65-like gene in vivo. The pharynx of homozygous larvae does not contract and the worms cannot feed. Analysis of pharyngeal pumping in heterozygous worms and in feh-1 RNA-interfered worms indicates that dosage of feh-1 function affects the rate of pharyngeal contraction in C. elegans. Interference with apl-1 double-stranded RNA showed a similar effect on pharyngeal pumping, suggesting that FEH-1 and APL-1 are involved in the same pathway. The non-redundant system of the nematode will prove useful for studying the basic biology of the Fe65-APP interaction and the molecular events regulated by this evolutionarily conserved system of interacting proteins.

The Kallmann syndrome gene homolog in C. elegans is involved in epidermal morphogenesis and neurite branching.

Kallmann syndrome is an inherited disorder defined by the association of anosmia and hypogonadism, owing to impaired targeting and migration of olfactory axons and gonadotropin-releasing hormone secreting neurons. The gene responsible for the X-linked form of Kallmann syndrome, KAL-1, encodes a secreted protein of still elusive function. It has been proposed that KAL-1 might be involved in some aspects of olfactory axon guidance. However, the unavailability of a mouse model, and the difficulties in studying cellular and axonal migration in vertebrates have hampered an understanding of its function. We have identified the C. elegans homolog, kal-1, and document its function in vivo. We show that kal-1 is part of a mechanism by which neurons influence migration and adhesion of epidermal cells undergoing morphogenesis during ventral enclosure and male tail formation. We also show that kal-1 affects neurite outgrowth in vivo by modulating branching. Finally, we find that human KAL-1 cDNA can compensate for the loss of worm kal-1 and that overexpression of worm or human KAL-1 cDNAs in the nematode results in the same phenotypes. These data indicate functional conservation between the human and nematode proteins and establish C. elegans as a powerful animal in which to investigate KAL function in vivo. Our findings add a new player to the set of molecules, which appear to underlie both morphogenesis and axonal/neuronal navigation in vertebrates and invertebrates.

Roles for Caenorhabditis elegans rad-51 in meiosis and in resistance to ionizing radiation during development.

We have investigated the role of Caenorhabditis elegans RAD-51 during meiotic prophase and embryogenesis, making use of the silencing effect of RNA interference (RNAi). rad-51 RNAi leads to severe defects in chromosome morphology in diakinesis oocytes. We have explored the effect of rad-51 RNAi in mutants lacking fundamental components of the recombination machinery. If double-strand breaks are prevented by spo-11 mutation, rad-51 RNAi does not affect chromosome appearance. This is consistent with a role for RAD-51 downstream of the initiation of recombination. In the absence of MRE-11, as in the absence of SPO-11, RAD-51 depletion has no effect on the chromosomes, which appear intact, thus indicating a role for MRE-11 in DSB induction. Intriguingly, rad-51 silencing in oocytes that lack MSH-5 leads to chromosome fragmentation, a novel trait that is distinct from that seen in msh-5 mutants and in rad-51 RNAi oocytes, suggesting new potential roles for the msh-5 gene. Silencing of the rad-51 gene also causes a reduction in fecundity, which is suppressed by mutation in the DNA damage checkpoint gene rad-5, but not in the cell death effector gene ced-3. Finally, RAD-51 depletion is also seen to affect the soma, resulting in hypersensitivity to ionizing radiation in late embryogenesis.

Perturbation of Conformational Dynamics of ASCUT-1 from Ascaris lumbricoides by Temperature and Sodium Dodecyl Sulfate.

ASCUT-1 is a protein found in cuticlin, the insoluble residue of the cuticles of the nematode Ascaris lumbricoides. It contains the CUT-1-like domain which is shared by members of a novel family of components of extracellular matrices. The monomeric form of ASCUT-1 contains a single tryptophan residue. An understanding of the structure-function relationship of the protein under different chemical-physical conditions is of fundamental importance for an understanding of its structure and function in cuticles. In this paper we report the effect of the temperature and sodium dodecyl sulfate on the structural stability of this protein. The structure of the protein was studied in the temperature range 25-85°C in the absence and in the presence of sodium dodecyl sulfate by frequency-domain measurements of the intrinsic fluorescence intensity and anisotropy decays. The time-resolved fluorescence data in the absence of SDS indicated that the tryptophanyl emission decays were well described by a bimodal lifetime distribution, and that the temperature increases resulted in the sharpening and in the shortening of the tryptophanyl lifetime distribution. In the presence of SDS an unimodal fluorescence lifetime distribution as well as a marked decrease in the anisotropy decay values were observed.