Caenorhabditis elegans receptors related to mammalian vascular endothelial growth factor receptors are expressed in neural cells

Through a genomic survey of the Caenorhabditis elegans genome for genes encoding tyrosine kinase receptors (RTK) we identified a family of four RTKs which are structurally related to vascular endothelial growth factor receptors (VEGFRs). We named this family the ver gene family (for Vascular Endothelial growth factor receptor Related). It was intriguing to find this type of RTK in an animal devoid of a vascular system. The common sites of expression of the ver genes are specialized cells of neural origin: ver-1 (T17A3.1) is expressed in the support (glial) cells of amphid and phasmid neurons, ver-2 (T17A3.8) in ADL, a pair of chemosensorial neurons, and ver-3 (F59F3.1) in the ALA neuron. In mammals, the VEGFRs are associated with angiogenesis and neurogenesis. We provide here the first observation that these molecules may be primarily and solely involved in neurogenesis in a living organism.     

Caenorhabditis elegans has scores of hedgehog-related genes: sequence and expression analysis.

Previously, we have described novel families of genes, warthog (wrt) and groundhog (grd), in Caenorhabditis elegans. They are related to Hedgehog (Hh) through the carboxy-terminal autoprocessing domain (called Hog or Hint). A comprehensive survey revealed 10 genes with Hog/Hint modules in C. elegans. Five of these are associated with a Wart domain in wrt genes, and three with multiple copies of the Ground domain in grd genes. Both the Wart domain and the Ground domain occur also in genes encoding no Hog domain. Further, we define a new group of genes related to the grd genes, called ground-like (grl). Overall, C. elegans has more than 50 genes belonging to these gene families. Phylogenetic and sequence analysis shows that the wrt, grd, and grl genes are derived from each other. Further examination reveals a sequence motif with similarity to the core of the amino-terminal-signaling domain of Hh proteins. Our data suggest that the wrt, grd, grl, and hh genes are derived from a single ancestral gene. wrt, grd, and grl genes are also present in other nematodes, but so far not in any other phyla. Conversely, hh is not found presently in C. elegans nor other nematodes. Thus, the nematode genes could be the homologs of Hh molecules in other phyla. The membrane molecule Patched has been shown previously to be a receptor of Hh. Many Patched-related proteins are present in C. elegans, which may be targets of the hh-related genes. No Hedgehog-interacting protein (Hip) was found. We analyzed the expression patterns of eight wrt and eight grd genes. The results show that some closely related genes are expressed in the same tissues, but, overall, the expression patterns are diverse, comprising hypodermis, seam cells, the excretory cell, sheath and socket cells, and different types of neurons.     

Classification of transmembrane protein families in the Caenorhabditis elegans genome and identification of human orthologs

The complete genome sequence of the nematode Caenorhabditis elegans provides an excellent basis for studying the distribution and evolution of protein families in higher eukaryotes. Three fundamental questions are as follows: How many paralog clusters exist in one species, how many of these are shared with other species, and how many proteins can be assigned a functional counterpart in other species? We have addressed these questions in a detailed study of predicted membrane proteins in C. elegans and their mammalian homologs. All worm proteins predicted to contain at least two transmembrane segments were clustered on the basis of sequence similarity. This resulted in 189 groups with two or more sequences, containing, in total, 2647 worm proteins. Hidden Markov models (HMMs) were created for each family, and were used to retrieve mammalian homologs from the SWISSPROT, TREMBL, and VTS databases. About one-half of these clusters had mammalian homologs. Putative worm-mammalian orthologs were extracted by use of nine different phylogenetic methods and BLAST. Eight clusters initially thought to be worm-specific were assigned mammalian homologs after searching EST and genomic sequences. A compilation of 174 orthology assignments made with high confidence is presented.     

The large srh family of chemoreceptor genes in Caenorhabditis nematodes reveals processes of genome evolution involving large duplications and deletions and intron gains and losses

The srh family of chemoreceptors in the nematode Caenorhabditis elegans is very large, containing 214 genes and 90 pseudogenes. It is related to the str, stl, and srd families of seven-transmembrane or serpentine receptors. Like these three families, most srh genes are concentrated on chromosome V, and mapping of their chromosomal locations on a phylogenetic tree reveals 27 different movements of genes to other chromosomes. Mapping of intron gains and losses onto the phylogenetic tree reveals that the last common ancestral gene of the family had five introns, which are inferred to have been lost 70 times independently during evolution of the family. In addition, seven intron gains are revealed, three of which are fairly recent. Comparisons with 20 family members in the C. briggsae genome confirms these patterns, including two intron losses in C. briggsae since the species split. There are 14 clear C. elegans orthologs for these 20 genes, whose average amino acid divergence of 68% allows estimation of 85 gene duplications in the C. elegans lineage since the species split. The absence of six orthologs in C. elegans also indicates that gene loss occurs; consideration of all deletions and terminal truncations of srh pseudogenes reveals that large deletions are common. Together these observations provide insight into the evolutionary dynamics of this compact animal genome.     

Multigene families of immunoglobulin domain-containing innate immune receptors in zebrafish: Deciphering the differences

Five large multigene families encoding innate-type immune receptors that are comprised of immunoglobulin domains have been identified in bony fish, of which four do not possess definable mammalian orthologs. The members of some of the multigene families exhibit unusually extensive patterns of divergence and the individual family members demonstrate marked variation in interspecific comparisons. As a group, the gene families reveal striking differences in domain type and content, mechanisms of intracellular signaling, basic structural features, haplotype and allelic variation and ligand binding. The potential functional roles of these innate immune receptors, their relationships to immune genes in higher vertebrate species and the basis for their adaptive evolution are of broad interest. Ongoing investigations are expected to provide new insight into alternative mechanisms of immunity.     

Mimicry of a G Protein Mutation by Pertussis Toxin Expression in Transgenic Caenorhabditis elegans

Pathogens produce virulence factors that interact directly with host molecules, but in many cases the host targets are unknown. The genetic and molecular identification of these orphan targets is often not feasible with mammalian experimental models. However, a substantial number of known targets are molecules and pathways that are conserved among eukaryotes, and therefore the use of nonmammalian model hosts to identify orphan targets may prove useful. To demonstrate the feasibility of this approach, we transformed the nematode Caenorhabditis elegans with a gene encoding the catalytic subunit of pertussis toxin (PTX), which in mammals inactivates G(o/i)alpha proteins. Expression of PTX in C. elegans produced phenotypes almost identical to those of a null mutation in the nematode gene encoding G(o/i)alpha. Furthermore, PTX suppressed the phenotype of a constitutively active form of nematode G(o/i)alpha protein. These results indicate that PTX is functional in nematodes and acts specifically on the C. elegans homologue of the mammalian target.     

An unusual receptor tyrosine kinase of Schistosoma mansoni contains a Venus Flytrap module

Previous studies have suggested that successful development of the parasitic helminth Schistosoma mansoni must be dependent on an adaptative molecular dialogue with its hosts and on the existence of receptors for growth factors and hormones. Attempts to identify a homolog of the insulin receptor (IR) have led us to characterize a new receptor tyrosine kinase (RTK) molecule in S. mansoni. SmRTK-1 is an integral membrane protein with a single membrane-spanning sequence separating an extracellular ligand-binding domain and a cytoplasmic TK domain. Structural and phylogenetic analyses of the kinase domain of SmRTK-1 confirmed its similarity to IR catalytic domains. However, sequence analysis of the extracellular domain of SmRTK-1 revealed similarity with various proteins (such as drug receptors) that share a structure known as the Venus Flytrap (VFT) module. Alignment with other VFT modules for which the structure has been solved was used to generate a 3D model of the putative VFT module of SmRTK-1. Phylogenetic analysis indicated that the SmRTK-1 VFT module was closer to that of the GABA(B) receptor. Numerous RTK genes recently discovered in vertebrate and invertebrate species code for large families of modular proteins with diverse structures and ligand-binding specificities. SmRTK-1 probably represents a new class of RTK whose function remains to be determined. RTKs are present in all metazoans and associated with the control of metabolism, growth and development. The preferential localization of SmRTK-1 in sporocyst germinal cells and ovocytes could be in favor of its function in schistosome growth and differentiation.     

Cloning and characterization of genes encoding alpha and beta subunits of glutamate-gated chloride channel protein in Cylicocyclus nassatus

The invertebrate glutamate-gated chloride channels (GluCls) are receptor molecules and targets for the avermectin-milbemycin (AM) group of anthelmintics. Mutations in GluCls are associated with ivermectin resistance in the soil dwelling nematode Caenorhabditis elegans and the parasitic nematode Cooperia oncophora. In this study, full-length cDNAs encoding alpha and beta subunits of GluCl were cloned and sequenced in Cylicocyclus nassatus, a common and important cyathostomin nematode parasite of horses. Both genes possess the sequence characteristics typical of GluCls, and phylogenetic analysis confirms that these genes are evolutionarily closely related to GluCls of other nematodes and flies. Complete coding sequences of C. nassatus GluCl-alpha and GluCl-beta were subcloned into pTL1 mammalian expression vector, and proteins were expressed in COS-7 cells. Ivermectin-binding characteristics were determined by incubating COS-7 cell membranes expressing C. nassatus GluCl-alpha and GluCl-beta proteins with [(3)H]ivermectin. In competitive binding experiments, fitting the data to a one site competition model, C. nassatus GluCl-alpha was found to bind [(3)H]ivermectin with a high amount of displaceable binding (IC(50)=208 pM). Compared to the mock-transfected COS-7 cells, the means of [(3)H]ivermectin binding were significantly different for C. nassatus GluCl-alpha and the Haemonchus contortus GluCl (HcGluCla) (p=0.018 and 0.023, respectively) but not for C. nassatus GluCl-beta (p=0.370). This is the first report of orthologs of GluCl genes and in vitro expression of an ivermectin-binding protein in a cyathostomin species. These data suggest the likelihood of a similar mechanism of action of AM drugs in these parasites, and suggest that mechanisms of resistance may also be similar.     

Mechanistic insights and identification of two novel factors in the C. elegans NMD pathway

The nonsense-mediated mRNA decay (NMD) pathway selectively degrades mRNAs harboring premature termination codons (PTCs). Seven genes (smg-1-7, for suppressor with morphological effect on genitalia) that are essential for NMD were originally identified in the nematode Caenorhabditis elegans, and orthologs of these genes have been found in several species. Whereas in humans NMD is linked to splicing, PTC definition occurs independently of exon boundaries in Drosophila. Here, we have conducted an analysis of the cis-acting sequences and trans-acting factors that are required for NMD in C. elegans. We show that a PTC codon is defined independently of introns in C. elegans and, consequently, components of the exon junction complex (EJC) are dispensable for NMD. We also show a distance-dependent effect, whereby PTCs that are closer to the 3' end of the mRNA are less sensitive to NMD. We also provide evidence for the existence of previously unidentified components of the NMD pathway that, unlike known smg genes, are essential for viability in C. elegans. A genome-wide RNA interference (RNAi) screen resulted in the identification of two such novel NMD genes, which are essential for proper embryonic development, and as such represent a new class of essential NMD genes in C. elegans that we have termed smgl (for smg lethal). We show that the encoded proteins are conserved throughout evolution and are required for NMD in C. elegans and also in human cells.     

Gene discovery in the adenophorean nematode Trichinella spiralis: an analysis of transcription from three life cycle stages

Expressed sequence tags (ESTs) were produced from cDNA libraries for immature L1, mature muscle larva and adult stages of the adenophorean nematode Trichinella spiralis. 10,130 ESTs were grouped into 3454 gene clusters. The clusters represent a conservative estimate of 3262 unique genes. Interspecific comparisons of the predicted proteins support an ancient divergence of clade I nematodes from other nematodes in the phylum Nematoda. Furthermore, apparent clade I or Trichocephalida-specific proteins were identified, which may include molecular determinants important in the evolution of these species. Similarity matches identified 463 C. elegans genes homologs that confer phenotypes by RNA interference. Classification of predicted proteins suggested diverse cellular, metabolic and extracellular functions, significantly expanding the dataset of T. spiralis proteins with prospective, and potentially critical, functions. Several lines of evidence suggested stage-specific expression of certain genes beyond those previously identified. Evidence was obtained for the existence of large gene families encoding isoforms of known secreted proteins, such as p43 and TspE1. Unexpectedly, diverse isoforms of the muscle larva p43 gene appear to be expressed by immature L1. Proteinases, kinases, antioxidant proteins and enzymes involved in glycan synthesis are implicated in T. spiralis interactions with its hosts. Numerous genes were identified that encode predicted proteins in these categories. The genes discovered, when put into context of functional classification, stage of expression, and biology of the parasite, should substantially enhance experimental potential for research on this parasite.     

The role of mycelium production and a MAPK-mediated immune response in the C. elegans-Fusarium model system

Fusariosis is an emerging infectious complication of immune deficiency, but models to study this infection are lacking. The use of the soil nematode Caenorhabditis elegans as a model host to study the pathogenesis of Fusarium spp. was investigated. We observed that Fusarium conidia consumed by C. elegans can cause a lethal infection and result in more than 90% killing of the host within 120 hours, and the nematode had a significantly longer survival when challenged with Fusarium proliferatum compared to other species. Interestingly, mycelium production appears to be a major contributor in nematode killing in this model system, and C. elegans mutant strains with the immune response genes, tir-1 (encoding a protein containing a TIR domain that functions upstream of PMK-1) and pmk-1 (the homolog of the mammalian p38 MAPK) lived significantly shorter when challenged with Fusarium compared to the wild type strain. Furthermore, we used the C. elegans model to assess the efficacy and toxicity of various compounds against Fusarium. We demonstrated that amphotericin B, voriconazole, mancozeb, and phenyl mercury acetate significantly prolonged the survival of Fusarium-infected C. elegans, although mancozeb was toxic at higher concentrations. In conclusion, we describe a new model system for the study of Fusarium pathogenesis and evolutionarily preserved host responses to this important fungal pathogen.     

How do pathogens drive the evolution of paired receptors?

Paired receptors are families of membrane proteins characterized by similar extracellular regions but different transmembrane and cytoplasmic regions, meaning that some members can give inhibitory signals and others activating signals. Well‐characterized examples include the KIR, SIRP, Ly49, Nkpr, and Siglec families. The difference in the repertoire of these genes in mouse and man indicates that these families have evolved rapidly. For example, KIRs are found in humans and not mice, and Ly49 shows the converse. These genes are often very polymorphic, e.g. KIR and the number of genes can vary as shown for Ly49 in different mouse strains. Paired receptors are expressed mainly on NK and myeloid cells and their evolution is thought to be pathogen driven. In this article, we review various receptor families for which pathogen interactions are known and discuss the possible molecular mechanisms driving their evolution.     

Caenorhabditis elegans as a model host for Staphylococcus aureus pathogenesis

Staphylococcus aureus, an important pathogen of humans and other warm-blooded animals, is also capable of killing the nematode Caenorhabditis elegans. Here, we show that C. elegans organisms that are fed S. aureus die over the course of several days in a process that is correlated with the accumulation of bacteria within the nematode digestive tract. Several S. aureus virulence determinants known or speculated to be important in mammalian pathogenesis, including the quorum-sensing global virulence regulatory system agr and the global virulence regulator sarA, the alternative sigma factor sigma(B), alpha-hemolysin, and V8 serine protease, are required for full pathogenicity in nematodes. In addition, several defined C. elegans mutants were examined for susceptibility to S. aureus infection. Enhanced susceptibility to S. aureus killing was observed with loss-of-function mutations in the C. elegans genes esp-2/sek-1 and esp-8/nsy-1, which encode components of a conserved p38 MAP kinase signaling pathway involved in nematode defense against multiple pathogens. These results suggest that key aspects of S. aureus pathogenesis have been conserved, irrespective of the host, and that specific C. elegans host factors can alter susceptibility to this gram-positive human pathogen.     

The expression of the homologue of the Caenorhabditis elegans lin-45 raf is regulated in the motile stages of the plant parasitic nematode Meloidogyne artiellia

The Ras-MAPK signal transduction pathway controls multiple developmental events and is involved in the processing of olfactory information in the free living nematode Caenorhabditis elegans. We have studied the Ras-MAPK pathway in the plant parasitic nematode Meloidogyne artiellia. The genes Mt-let-60, Mt-lin-45, Mt-mek-2 and Mt-mpk-1 have been isolated and sequenced. Each of them shows a high level of sequence similarity to its presumed ortholog in C. elegans and key functional domains are structurally conserved. Furthermore, we show that the M. artiellia recombinant MEK-2 protein can phosphorylate and activate the M. artiellia recombinant MPK-1 and the recombinant MEK-2 itself can be phosphorylated and activated by immunoprecipitated mammalian Raf. Surprisingly, the Mt-lin-45 message is not detectable in freshly emerged juveniles or in male specimens, suggesting that it may be quickly degraded in these life stages.     

The activity and hydrogen peroxide sensitivity of the peroxiredoxins from the parasitic nematode Haemonchus contortus

The requirement of aerobic organisms to control damage caused by reactive oxygen species has led to the evolution of the antioxidant systems. Peroxiredoxins are a large family of peroxidases which detoxify hydrogen peroxide at the expense of thiols. The parasitic nematode Haemonchus contortus contains two peroxiredoxins, HcPrx1 a mitochondrial protein and HcPrx2 a cytoplasmic protein. Although both peroxiredoxins contain the conserved eukaryotic motifs 'GGLG' and 'YF', identified as critical for hydrogen peroxide instability, both were stable to high concentrations of hydrogen peroxide, demonstrating different functions to their mammalian counterparts. H. contortus also contains two thioredoxin reductases and five different thioredoxin-like proteins. The activity of both peroxiredoxins was specific for the thioredoxin system; however, both could also be regenerated by the glutathione system when coupled to the nematode specific thioredoxin HcTrx5. Analysis of homologous genes in Caenorhabditis elegans showed that only CePrx2, which is secreted, was sensitive to the external oxidant hydrogen peroxide. However, both peroxiredoxins KO C. elegans were sensitive to intracellular free radicals and both peroxiredoxins protected DNA from free radical attack. The results demonstrate that the hydrogen peroxide detoxification and the antioxidant activity of the peroxiredoxins are separate activities that are independent of the 'GGLG' and 'YF' motifs.     

The Haemonchus contortus UNC-49B subunit possesses the residues required for GABA sensitivity in homomeric and heteromeric channels

Hco-UNC-49 is a GABA receptor from the parasitic nematode Haemonchus contortus that has a relatively low overall sequence similarity to vertebrate GABA receptors but is very similar to the UNC-49 receptor found in the free living nematode Caenorhabditis elegans. While the nematode receptors do share >80% sequence similarity they exhibit different sensitivities to GABA. In addition, the UNC-49C subunit appears to be a positive modulator of GABA sensitivity in the H. contortus heteromeric channel, but is a negative modulator in the C. elegans heteromeric channel. The cause(s) of these differences is currently unknown since the structural elements essential for GABA sensitivity in nematode receptors have been largely unexplored. Thus, the overall aim of this study was to investigate the residues that are important for UNC-49 receptor sensitivity through the use of homology modeling, site-directed mutagenesis, and two-electrode voltage clamp. This study revealed that Met(170) in Loop B of the GABA binding-site may partially account for the observed differences in GABA receptor sensitivity between the nematode species. Residues in Loops A-D that have been reported to form the GABA binding pocket in mammalian receptors, including those forming the conserved 'aromatic box', also appear to play analogous roles in Hco-UNC-49. In addition, the two mutations that produced the most significant reduction in GABA sensitivity were R66S and Y166S. Homology modeling indicates that these two residues share a hydrogen bond and are positioned close to the carboxyl end of the GABA molecule. However, of residues examined in this study, only those on the Hco-UNC-49B subunit and not its subunit partner, Hco-UNC-49C, appear important for GABA sensitivity. Overall, results from this study suggest that the binding site of the UNC-49 heteromeric GABA receptor exhibits some differences compared to classical vertebrate GABA(A) receptors.     

Systemic immunoregulatory and pathogenic functions of homeostatic chemokine receptors

IL-17 is a potent proinflammatory cytokine produced by activated memory T cells. The large-scale sequencing of the human and other vertebrate genomes has revealed the presence of additional genes encoding proteins clearly related to IL-17, thus defining a new family of cytokines. There are at least six members of the IL-17 family in humans and in mice. Initial characterization suggests that like IL-17, several of these newly identified molecules have the ability to modulate immune function. Neither the IL-17 family nor the cognate receptors that have been identified for these molecules bear obvious sequence similarity to other known families of proteins. Thus, they represent a distinct signaling system that appears to have been highly conserved across vertebrate evolution. The potent inflammatory actions that have been identified for several of these factors and the emerging associations with major human diseases suggest that these proteins may have significant roles in inflammatory processes.     

Specificity of the innate immune system and diversity of C-type lectin domain (CTLD) proteins in the nematode Caenorhabditis elegans

The nematode Caenorhabditis elegans has become an important model for the study of innate immunity. Its immune system is based on several signaling cascades, including a Toll-like receptor, three mitogen-activated protein kinases (MAPK), one transforming growth factor-beta (TGF-beta), the insulin-like receptor (ILR), and the programmed cell death (PCD) pathway. Furthermore, it also involves C-type lectin domain- (CTLD) containing proteins as well as several classes of antimicrobial effectors such as lysozymes. Almost all components of the nematode immune system have homologs in other organisms, including humans, and are therefore likely of ancient evolutionary origin. At the same time, most of them are part of a general stress response, suggesting that they only provide unspecific defense. In the current article, we re-evaluate this suggestion and explore the level of specificity in C. elegans innate immunity, i.e. the nematode's ability to mount a distinct defense response towards different pathogens. We draw particular attention to the CTLD proteins, which are abundant in the nematode genome (278 genes) and many of which show a pathogen-specific response during infection. Specificity may also be achieved through the differential activation of antimicrobial genes, distinct functions of the immunity signaling cascades as well as signal integration across pathways. Taken together, our evaluation reveals high potential for immune specificity in C. elegans that may enhance the nematode's ability to fight off pathogens.