Stephan H.
von Reuss
*a and
Frank C.
Schroeder
*b
aMax Planck Institute for Chemical Ecology, Department of Bioorganic Chemistry, Jena, Germany. E-mail: svonreuss@ice.mpg.de
bBoyce Thompson Institute and Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA. E-mail: Schroeder@cornell.edu
First published on 10th June 2015
Covering: up to 2015
The nematode Caenorhabditis elegans was the first animal to have its genome fully sequenced and has become an important model organism for biomedical research. However, like many other animal model systems, its metabolome remained largely uncharacterized, until recent investigations demonstrated the importance of small molecule-based signalling cascades for virtually every aspect of nematode biology. These studies have revealed that nematodes are amazingly skilled chemists: using simple building blocks from conserved primary metabolism and a strategy of modular assembly, C. elegans and other nematode species create complex molecular architectures to regulate their development and behaviour. These nematode-derived modular metabolites (NDMMs) are based on the dideoxysugars ascarylose or paratose, which serve as scaffolds for attachment of moieties from lipid, amino acid, carbohydrate, citrate, and nucleoside metabolism. Mutant screens and comparative metabolomics based on NMR spectroscopy and MS have so-far revealed several 100 different ascarylose (“ascarosides”) and a few paratose (“paratosides”) derivatives, many of which represent potent signalling molecules that can be active at femtomolar levels, regulating development, behaviour, body shape, and many other life history traits. NDMM biosynthesis appears to be carefully regulated as assembly of different modules proceeds with very high specificity. Preliminary biosynthetic studies have confirmed the primary metabolism origin of some NDMM building blocks, whereas the mechanisms that underlie their highly specific assembly are not understood. Considering their functions and biosynthetic origin, NDMMs represent a new class of natural products that cannot easily be classified as “primary” or “secondary”. We believe that the identification of new variants of primary metabolism-derived structures that serve important signalling functions in C. elegans and other nematodes provides a strong incentive for a comprehensive re-analysis of metabolism in higher animals, including humans.
Surprisingly, nematode biochemistry and metabolism remained largely uncharacterized until recent investigations demonstrated the central importance of small molecule-based signalling cascades for nematode behaviour and development. Interorganismal chemical communication in nematodes was already suspected by Greet in 1964 who reported that males and females of the free living Panagrolaimus rigidus, when separated by a cellophane barrier, moved towards members of the opposite sex but not to members of the same sex, suggesting the release of gender-specific attractants, e.g. sex pheromones.6 Subsequent research demonstrated the presence of small molecule signals modulating gender-specific attraction in more than 40 representative nematodes including free living, entomopathogenic, plant-parasitic, and zoo-parasitic species.7–18 However, despite considerable efforts to isolate and identify nematode pheromones, molecular structures remained elusive for many decades.
Recent research focusing on the free-living Caenorhabditis elegans, the necromenic Pristionchus pacificus, and a few other species revealed that nematodes are amazingly skilled chemists: using simple building blocks from conserved primary metabolism and a strategy of modular assembly, they create complex molecular architectures, the nematode-derived modular metabolites (NDMMs) to regulate their development and behaviour. In this review, we will describe the discovery of this modular library, summarize current knowledge regarding the biosynthesis and perception of these components and outline possible implications for small-molecule signalling in higher organisms. Earlier reviews of this field focussed primarily on the biological activity of the initially discovered members of this family of signaling molecules.19,20
The first hint that small molecule signalling may play an important role in C. elegans life history emerged from studies of a peculiar state of developmental diapause called dauer, from the German word for “enduring”. Under unfavourable conditions such as lack of food, high temperature, or high population density, C. elegans larvae abort reproductive development and instead enter a specialized, non-feeding and highly stress resistant larval stage that is optimized for dispersal and long-term survival (Fig. 1a). When environmental conditions improve, these dauer larvae resume normal development to reproductive adults.27 Formation of dauer larvae is regulated by conserved signalling pathways, including insulin and TGF-β signalling as well as steroid hormone biosynthesis converging on the nuclear hormone receptor DAF-12, a liver-X and vitamin D receptor homologue.28–31 Given that dauer formation results in greatly extended lifespan, the mechanisms that regulate entry and exit from the dauer stage have become an important focus of aging research.28
Fig. 1 Initial identification of ascarosides in nematodes. (a) C. elegans life cycle. (b) Ascarosides with potent dauer inducing activity. (c) Lipid-like ascaroside from Ascaris spp. |
In 1982 Golden and Riddle reported that dauer formation is modulated by a worm-derived small molecule signal that is constitutively excreted by the worms and “may be a hydroxylated, short-chain fatty acid or a mixture of closely related compounds”.32,33 The nematode origin of this pheromone was proven unambiguously by showing that its production depends on a gene (of then unknown identity) named daf-22.34 However, the molecular structure of the dauer pheromone remained elusive for more than 20 years until several laboratories reported the identification of a family of dauer inducing glycosides derived from L-α-3,6-dideoxymannose, a sugar known as ascarylose, and a variety of hydroxylated short-chain fatty acids.35–38
Ascarylose-based glycosides were first described as an “unsaponifiable matter” that accounted for about 25% of the total lipid content of the human intestinal parasitic nematode Ascaris lumbricoides.39 At the time of this initial report (1912 !), the chemical structure of the A. lumbricoides-derived lipids could not be determined, and it was not until 1957 that the exact structures of the sugar and fatty acid parts of these ascarosides were determined (Fig. 1c).40 The A. lumbricoides-derived ascarosides feature very long aliphatic side chains (predominantly 29 and more carbons in length), are produced in copious amounts and are presumed to form part of a protective coating of Ascaris eggs.
In contrast, the dauer-inducing ascarosides identified from C. elegans incorporate short fatty acid-derived side chains of only 3–9 carbons (Fig. 1b) and are produced at low (nano- or micromolar) concentrations. Activity-guided fractionation of worm culture media with dauer-inducing activity resulted in the isolation of ascr#1 (daumone#1, C7),35† the methyl ketone ascr#2 (daumone#2, C6), ascr#3 (daumone#3, C9),36 and ascr#5 (C3),37 which were shown to induce dauer formation at near-physiological concentrations. However, even mixtures of synthetic samples of these compounds failed to fully reproduce the activity of unfractionated worm media, suggesting that there must be additional dauer-inducing components that had been missed by activity-guided fractionation. As described in the following section, the use of comparative metabolomics enabled the identification of important additional dauer pheromone components, including the so far most-potent dauer-inducing ascaroside, ascr#8 (Fig. 2).38 Ascr#8 represents the first example for an ascaroside that incorporates additional moieties beyond the ascarylose sugar and the fatty acid-derived side chain, and thus provided a first glimpse at the vast diversity of multi-modular ascarosides – the NDMMs – later identified from C. elegans, P. pacificus, and other nematodes.
Fig. 2 Modular ascarosides regulating development and mediating behavioural phenotypes in C. elegans, including building blocks derived from tryptophan (red) and folate (blue) metabolism. |
Soon after identification of the first dauer-inducing ascarosides, it became clear that ascarosides regulate many other aspects of nematode life history, including social behaviours and adult lifespan. For example, mixtures of ascr#2, its 2-glucoside ascr#4, ascr#3, and ascr#8 (Fig. 1b & 2) were found to act as potent male-specific attractants.38,41 These hermaphrodite-produced ascarosides strongly synergize in male attraction, that is, mixtures of these compounds were found to be much more attractive than when tested individually, even at elevated concentrations. Behavioural responses to these ascarosides were found to be highly sex-specific: whereas males are attracted by ascr#2 and ascr#3, these compounds act as deterrents for hermaphrodites. Generally, ascaroside concentrations required for behavioural responses (pico- to nanomolar) are much lower than those required for dauer induction.38
In addition, ascr#2 and ascr#3 (Fig. 1b) have been shown to regulate adult lifespan in a manner that is distinct from dauer induction. When exposed to nanomolar concentrations of these ascarosides after reaching adulthood, hermaphrodite worms live up to 25% longer than untreated controls.42 Other, yet unidentified ascarosides that are produced by male worms appear to shorten hermaphrodite lifespan.43
For the purpose of comparing wild-type and daf-22 metabolome samples, Differential analysis of 2D NMR spectra (DANS), a method that relies on comparison of high-resolution dqfCOSY spectra, proved particularly useful.38 DANS revealed a series of daf-22-dependent NMR signals that enabled the identification of several additional ascarosides, including ascr#8 (Fig. 2) which incorporates a p-aminobenzoic acid (PABA) moiety likely derived from folate metabolism.38 Ascr#8 was then shown to be highly active in promoting male attraction in synergism with ascr#2 and ascr#3 (Fig. 1b), in addition to strong dauer-inducing activity. Synthetic blends of ascr#2, ascr#3, and ascr#8 at near-physiological concentrations were found to fully reconstitute the behavioural activity of unfractionated exo-metabolome, demonstrating the utility of DANS in connecting biological activity and molecular structures.38
In addition, DANS revealed a group of daf-22 dependent indole carboxy ascarosides (e.g. icas#3 and icas#9), which carry an indole-3-carboxylate moiety at the 4-position of the ascarylose and were subsequently shown to act as highly potent hermaphrodite aggregation pheromones (Fig. 2).44 The first indole ascaroside, icas#9, had previously been identified via activity-guided fractionation as a minor component of the dauer pheromone;45 however, extensive bioassays revealed that icas#9 and icas#3 act as potent attractants and aggregation pheromones at concentrations significantly lower than those required for dauer formation.44 Prior to the identification of the indole ascarosides, the existence of an aggregation pheromone in C. elegans had not been suspected, demonstrating how comparative metabolomic analyses and subsequent identification of novel biogenic small molecules can lead to the discovery of new and unexpected phenotypes.
Fig. 3 (a) mbas#3 and hbas#3, two modular ascarosides including tigloyl and p-hydroxybenzoyl modules putatively derived from amino acid or short-chain fatty acid metabolism (red) that were identified via targeted metabolomics in mixed stage C. elegans cultures.46 (b) ascr#10, a male-produced hermaphrodite attractant, and osas#9, a compound produced abundantly by starved L1 larvae,47 incorporating succinyl (magenta) and octopamine (green) moieties.49 |
Furthermore, targeted ascaroside profiling using precursor ion screening enabled the identification of life-stage and sex-specific ascarosides. It should be noted that most of the original work on small-molecule signalling in C. elegans was based on the analysis and biological testing of extracts derived from large liquid cultures containing worms of all ages and life stages, including eggs and larvae. However, given that ascarosides serve a great variety of biological functions, it seemed likely that different life stages would produce different ascaroside blends. Comparative MS/MS analysis of metabolome extracts of samples of a few hundred hand-picked C. elegans males and hermaphrodites facilitated the identification of ascr#10 (Fig. 3) as a highly potent, specifically male-produced hermaphrodite attractant.47 In addition, HPLC-MS analysis of different larval stages showed that production of one of the main dauer-inducing ascarosides, ascr#2, greatly increases before entering the dauer stage, whereas dauer larvae themselves produce little or no ascarosides.48 MS/MS analysis of different developmental stages also resulted in the identification of new types of modular ascaroside derivatives. For example, osas#9 and osas#10, two ascarosides incorporating octopamine-N-succinyl moieties, were found to be produced specifically by L1 larvae and function as a dispersal signal in the absence of food (Fig. 3).49 In nematodes, octopamine serves as a neurotransmitter in a manner similar to serotonin in higher animals, and thus osas#9 and osas#10 appear to connect neurotransmitter signalling with ascaroside-based interorganismal communication.
Fig. 4 Attachment of a tryptophan-derived indole carboxy moiety to the deterrent ascr#3 results in the hermaphrodite attractant icas#3. In addition, ascr#3 is part of the male-attracting pheromone (see text).44 |
Attachment of additional building blocks at the carboxy terminus of the side chains can also modify biological function. As described above, ascr#8, which includes a 4-aminobenzoic acid moiety that most likely originates from bacterial folate metabolism38 is a potent male attractant (Fig. 2), whereas the corresponding unmodified ascaroside, ascr#7 (not shown), is inactive in this assay.38 Modification of the side chain carboxylic acid terminus may also be involved in the regulation of ascaroside secretion and storage. For example, a variety of β-glucosyl ester ascarosides (e.g. glas#3, Fig. 11) have been identified in C. elegans worm body extracts but could not be detected in exo-metabolomes samples.46 Overall, the observation that even seemingly minor structural changes in NDMMs can greatly affect biological activity strongly suggests that their biosynthesis must be precisely regulated.
Fig. 5 Structures of the side chain-hydroxylated dhas#18, a pheromone identified from the sour paste nematode P. redivivus,54 and easc#18, a component of the dauer pheromone of the entomopathogenic nematode H. bacteriophora.54 |
Given that the use of ascarosides as signalling molecules is highly conserved among nematodes, it seems possible that other organisms have evolved the capability to detect and respond to these nematode-specific molecular markers. Recent work showed that, in fact, ascarosides mediate cross-kingdom ecological interactions: in fungi that feed on nematodes, sensing of nematode-produced ascarosides was found to induce formation of specific mycelial structures that serve as “nematode traps”.58
Previous work had suggested that, like in C. elegans, dauer formation in P. pacificus depends on small molecules. Furthermore, it had been shown that in P. pacificus a unique dimorphism of mouth development is likely controlled by inter-organismal small-molecule signalling. Under conditions of high population density, worms develop a wider mouth (“eurystomatous”) equipped with a tooth-like structure that enables a predatory lifestyle consuming other nematodes, whereas under low population density conditions worms develop a narrow mouth (“stenostomatous”) optimized for a bacterial diet.60,61 Since none of the ascarosides previously identified from C. elegans elicited dauer formation or mouth dimorphism in P. pacificus, it appeared that other, species-specific components must be responsible. Extensive analysis of active P. pacificus exo-metabolome fractions via NMR spectroscopy and MS/MS revealed a fascinatingly diverse array of multimodular NDMMs, incorporating building blocks from all major primary metabolic pathways including carbohydrate, lipid, amino acid, nucleoside, and neurotransmitter metabolism (Fig. 6).
Fig. 6 Ascaroside and paratoside-derived metabolites in P. pacificus. Major components of the P. pacificus exo-metabolome derived from assembly of building blocks from carbohydrate, lipid, amino acid (red), and nucleoside (blue) metabolism, as well as TCA cycle-derived succinate (magenta). Also shown is the highly conserved tRNA nucleoside, N6-threonylcarbamoyladenosine (t6A), a putative precursor of paratosides npar#1–3.62 |
Many of the building blocks in the P. pacificus NDMMs featured unexpected structural modifications. For example, whereas all known C. elegans NDMMs include L-ascarylose as the central scaffold, the P. pacificus metabolites are based on two different di-deoxysugars, L-ascarylose and the related L-paratose, a new sugar, the D-enantiomer of which having previously been reported from bacteria.63 Additional structural and functional diversity of the P. pacificus NDMMs derives from dimerization of ascarosides. For example, dasc#1, formally derived from dimerization of ascr#1, strongly induces the eurystomatous mouth form, whereas the monomeric ascr#1 is inactive (Fig. 7).62,64 Another dimeric ascaroside, ubas#1, which is formally derived from dimerization of two different ascarosides, the (ω-1)-side chain-functionalized ascr#9 and (ω)-functionalized oscr#9, contributes to dauer formation in some P. pacificus strains (Fig. 6). The structures of ubas#1 further includes an unusual ureido isobutyrate moiety that likely originates from metabolism of the nucleobase thymine. Nucleoside metabolism further contributes to dauer signalling in P. pacificus via a family of paratose-based NDMMs, npar#1–3, which incorporate a L-threonylcarbamoyl adenosine moiety that most likely originates from canonical threonylcarbamoyl adenosine (t6A), a highly conserved nucleoside found adjacent to the anticodon triplet of a subset of tRNAs (Fig. 6).62 Most surprisingly, however, a xylopyranoside residue was observed in npar#1 instead of the canonical ribofuranoside variant of t6A found in tRNA.62 Physiological concentrations of npar#1 strongly induced dauer formation in P. pacificus, whereas the corresponding unmodified paratoside part#9 was much less active62 Two additional derivatives have been described. npar#2 represents the N-deglycosylated analogue of npar#1, whereas in npar#3 the adenosine moiety of N-deglycosylated npar#1 is replaced by the corresponding 8-hydroxy derivative.65 Hydroxylated adenosine derivatives have been shown to be produced as a result of oxidative damage to nucleic acids,66 suggesting that npar#3 functions as an indicator of oxidative stress.
Fig. 7 The dimeric ascaroside dasc#1 promotes development of the wide (eurystomatous) mouth form that enables a predatory lifestyle consuming other nematodes. |
P. pacificus also produces a family of NDMMs that, similar to osas#9 in C. elegans, incorporate putatively neurotransmitter-derived moieties, e.g. pasc#9 (Fig. 6). Analogous to the incorporation of N-succinyl-octopamine in osas#9 (Fig. 3), pasc#9, the most abundant NDMM in all P. pacificus strains analyzed so far, incorporates a N-succinyl-phenylethanolamine moiety, suggesting that N-succinylation plays a role in nematode neurotransmitter metabolism.49,62 In contrast to osas#9 in C. elegans (Fig. 3), the succinamide in pasc#9 is linked to the carboxy terminus of the side chain instead of the ascarylose.
Recently, a derivative of pasc#9 has been described in which an additional anthranilic acid likely derived from tryptophan is attached to the succinate unit. This compound, named pasa#9, represents the first NDMM composed of 5 different modules (Fig. 6).64 The structure of pasa#9 demonstrates that additional modules must not necessarily be incorporated via direct attachment to the ascaroside scaffold, but may also get linked to other modules, suggesting that NDMMs may be even more structurally diverse than previously suspected. Penta-modular pasa#9 and pasc#9 are accompanied by smaller amounts of the cyclic succinimide pasy#9 (not shown), which may represent a by-product of pasc#9 biosynthesis or could be derived from decomposition of pasa#9.62,64
Many of the new chemical structures proposed on the basis of spectroscopic analysis of the P. pacificus metabolome were structurally much more complex than the NDMMs previously identified from C. elegans and other nematodes (Fig. 6). As a result, confirmation of the proposed structures via total synthesis emerged as a major bottle neck in the characterization of P. pacificus NDMMs. For example, the synthesis of npar#1 was based on a sequence with 13 linear steps. Therefore, proposed structures were prioritized for synthesis based on their relative abundances and results from activity-guided fractionation; however, structures of a large number of additional NDMMs remain to be confirmed via synthesis which will then also facilitate their biological evaluation. As in the case of C. elegans, the identification of additional NDMMs in P. pacificus may lead to the discovery of new small-molecule mediated phenotypes.
Correspondingly, the biological activity of the identified NDMMs is strongly structure-dependent.62 A screen of six different natural P. pacificus strains has further shown that bioactivity of different NDMMs varies greatly between different natural isolates.64 These observations indicate that NDMM receptors evolve rapidly, as also suggested by the finding that laboratory strains of C. elegans are less sensitive to dauer pheromone than the parent wildtype strain due to rapid accumulation of mutations in receptor-coding genes.67 Comparison of the metabolomes of the six different natural P. pacificus strains further revealed marked differences in the relative abundances of NDMM in different strains.64 For example, the ascaroside pasc#9 varied more than six-fold between strains. Several NDMMs, for example ubas#1 and ubas#2, were completely absent in two of the six analysed strains. Furthermore, it was found that some P. pacificus strains respond strongly to NDMMs they do not produce themselves, which suggested that NDMMs may play a role in communication and/or competition between different strains.64,68 Similarly, analysis of several C. elegans wild type isolates indicated that the production52 and dauer inducing activity69 of ascr#2 and ascr#3 is strongly strain-specific, suggesting the possibility of “dishonest” signalling between competing C. elegans strains.69
L-Ascarylose (or L-paratose) are glycosidically connected to fatty acid-derived side chains of varying length and functionality that have been shown to originate from peroxisomal β-oxidation,45,46,56 producing homologous series of the glycolipid core structures. In some nematode species, the glycolipid profiles are dominated by one or two chain lengths, whereas other species have broader distributions.52 Although the origin of these fatty acid chains is not well understood, the general predominance of odd-numbered side chains indicates that these are derived from long-chain odd-numbered fatty acids via multiple steps of β-oxidation.
In most ascarosides/paratosides, the fatty acid residue is attached to the dideoxysugar at the (ω-1)-position and the corresponding chiral centre always carries the (R)-configuration. Less common are (ω)-linked side chains, as in ascr#5, a major component of the C. elegans dauer pheromone, which carries an (ω)-linked 3-hydroxypropanoic acid residue (Fig. 1b),37 and several other compounds based on longer chained (ω)-linked side chains identified from both C. elegans and P. pacificus.46,62 As described in the following section, targeted metabolomic analysis of peroxisomal β-oxidation mutants revealed complete series of longer chain homologues with (ω-1)- and (ω)-hydroxylated side chains ranging from 3 to 21 carbons which suggested that (ω-1)- and (ω)-hydroxylation occurs upstream of peroxisomal β-oxidation.46
Fig. 8 Side chain shortening of long-chain ascarosides via iterative four-step peroxisomal β-oxidation in C. elegans.46,71 |
Subsequent studies showed that C. elegans genes coding for homologues of enzymes catalyzing the remaining three enzymatic steps in peroxisomal β-oxidation also participate in ascaroside biosynthesis. This includes three acyl-CoA oxidases (acox-1,46,70acox-2 and acox-371), the enoyl-CoA hydratase maoc-1,46 and the 3-hydroxyacyl-CoA dehydrogenase dhs-28.45,46 The role of acox-1, maoc-1, dhs-28, and daf-22 in chain shortening of ascarosides via peroxisomal β-oxidation was investigated using the ascaroside-targeted MS/MS screen described above.46 Mutation of acox-1, maoc-1, or dhs-28 was shown to result in accumulation of shunt metabolites with structural features supportive of the enzymatic functions of these genes, which originally had been predicted solely based on homology. For example, production of short-chain ascarosides (<9 carbon side chain) and ascarosides with α,β-unsaturated side chains, which are highly abundant in C. elegans wild type, is attenuated in acox-1 mutant metabolomes. Instead, acox-1 mutant worms accumulate large amounts of saturated longer-chain ascarosides (especially C9, C11, and C13), indicating that acox-1 serves as acyl-CoA oxidase introducing a double bond in the first step of peroxisomal β-oxidation.46 Additional work revealed that homodimers of the acyl-CoA oxidase acox-1 specifically act on the CoA ester of ascarosides with nine carbon side chains, whereas heterodimers of acox-3 and acox-1 act on ascaroside CoA esters of shorter carbon chains. Furthermore, homodimers of acox-2 appear to act specifically on ascaroside CoA esters of (ω)-oxygenated side chains and thus may control the biosynthesis of the dauer pheromone component ascr#5.71
In contrast to acox-1, maoc-1, and dhs-28, targeted metabolomic analyses via HPLC-MS/MS did not reveal any shunt metabolites whose structures could serve to corroborate the predicted function of daf-22 as a 3-ketoacyl-CoA thiolase.46 This was not unexpected, given that β-ketoacids such as they would be expected to form from accumulating substrates of DAF-22 (Fig. 8) easily decarboxylate or may undergo other chemical transformations. To identify such daf-22-dependent shunt metabolites, the daf-22 metabolome was re-analyzed using 2D NMR-based comparative metabolomics with a focus on spectroscopic signatures present in the mutant metabolome, but absent in wildtype. For this purpose, multivariate differential analysis by 2D NMR spectroscopy (mvaDANS) was developed, which integrates automatic crosspeak identification and binning with statistical analysis via principal component analysis. mvaDANS then revealed a series of very long-chain ascaroside ethanolamides and other long-chain ascarosides as shunt metabolites in daf-22 mutants (Fig. 9).56 Subsequent re-analysis of the metabolomes of other peroxisomal β-oxidation mutants revealed similar shunt metabolites. Formation of these long-chain ethanolamides in response to defects in daf-22 and other components of peroxisomal β-oxidation was associated with a severe depletion of endocannabinoid pools, demonstrating an unexpected interaction between peroxisomal lipid β-oxidation and the biosynthesis of endocannabinoids (Fig. 9), which are major regulators of lifespan in C. elegans.56
Fig. 9 Interaction of endocannabinoid biosynthesis with peroxisomal β-oxidation. daf-22 mutation abolishes processing of long-chain ascaroside CoA esters, whose conversion into ascaroside ethanolamides is associated with reduced EPEA and anandamide production, likely due to depletion of phosphatidylethanolamine pools. Shown in red are shunt metabolites identified in daf-22 mutant endo-metabolomes via mvaDANS.56 |
Fig. 10 Model for the biosynthesis of the avoidance signal osas#9 in C. elegans. tbh-1 mutant worms produce large quantities of the shunt metabolite tsas#9 due to accumulation of the octopamine precursor tyramine.49 |
Finally, comparative analysis of exo- and endo-metabolome (worm body extracts) samples revealed that C. elegans and P. pacificus exhibit significant control over ascaroside release.46,62,64 For example, ascaroside glucosyl esters (e.g. glas#3, Fig. 11) have been found only in the C. elegans endo-metabolome (worm body extract), but not the exo-metabolome, suggesting that such glucosides may be involved in ascaroside storage or transport.46
Fig. 11 glas#3, an ascaroside glucosyl ester identified in the C. elegans endo-metabolome.46 |
However, so far only a relatively small number of G-protein coupled receptors involved in ascaroside perception have been identified,67,78,79 whereas the diversity of identified ascaroside functions suggests that many more are yet to be discovered. SRBC-64 and SRBC-66 are two heterotrimeric GPCRs expressed in the ASK chemosensory neurons that mediate dauer formation in response to ascr#1, ascr#2, and ascr#3 (Fig. 1b).79 Two members of the serpentine chemoreceptor class g family, srg-36 and srg-37, discovered through genetic analysis of pheromone resistant C. elegans strains obtained from high density cultivation for several years, encode redundant GPCRs for the dauer inducing activity of ascr#5, demonstrating that adaptation to specific environments can entail rapid remodelling of the chemoreceptor repertoire. Deletion of a srg gene paralogous to srg-36 and srg-37 has also been linked to resistance to dauer formation in a laboratory Caenorhabditis briggsae strain, indicating that parallel genetic changes can affect life-history traits across species.67 Finally, heterodimers of DAF-37 and DAF-38, two GPCRs identified using DAF-8 immunoprecipitation, cooperatively mediate ascaroside perception. daf-37 mutants are defective in ascr#2-mediated dauer-induction but respond normally to other ascarosides, whereas daf-38 mutants are partially defective to several different ascarosides. Furthermore, cell-specific overexpression revealed that ascr#2-mediated dauer formation depends on DAF-37 expression in ASI neurons, whereas DAF-37 expression in ASK neurons regulates behaviour.78 Using a photoaffinity-labeled ascr#2 probe and amplified luminescence assays (AlphaScreen), it was further demonstrated that ascr#2 binds directly to DAF-37.78
Perception of natural dauer pheromone samples, i.e. ascaroside-containing exo-metabolome, has been shown to act upstream of several conserved signalling pathways, including the insulin, TGF-β, serotonin, and guanylyl cyclase pathways.27,31 However, it is not known if and to what extent downstream pathways of individual dauer-inducing ascarosides differ. Nonetheless, it has been shown that different phenotypes induced by the same ascaroside can depend on different downstream signalling components. For example, ascarosides ascr#2 and ascr#3 extend lifespan in adult worms via sir-2.1, a homologue of the mammalian histone-deacetylase SIRT-1,42 and the same two compounds also induce dauer via the insulin signalling pathway.36 However, sir-2.1 is not required for dauer induction by ascr#2 and ascr#3,27 and neither is insulin signalling required for ascr#2 and ascr#3-mediated longevity.42
Further elucidation of the biosynthesis of ascaroside-based NDMMs will reveal how input from conserved primary metabolism is transduced to create signals that regulate development, behaviour, and many other aspects of the life history of nematode model organisms. Moreover, since ascaroside signalling is highly conserved among nematodes, a detailed understanding of their biosynthesis and regulation may enable new approaches for the treatment of human nematode infections or the control of parasitic nematodes in agriculture. Lastly, it should be noted that ascaroside-based NDMMs are likely just one of several families of small molecule signals. Mass spectrometric analyses of the C. elegans metabolome have revealed evidence for several 1000 yet unidentified compounds,46 and nematode genomes feature many genes that, based on homology, may encode small-molecule biosynthetic pathways, including one gene with clear homology to microbial hybrid PKS/NRPSs (polyketide synthase/non-ribosomal peptide synthetases),80 and whose functions remain to be elucidated.
Footnote |
† For ascaroside nomenclature see www.smid-db.org |
This journal is © The Royal Society of Chemistry 2015 |