Complex small-molecule architectures regulate phenotypic plasticity in a nematode

Article abstract of DOI:10.1002/anie.201206797

Chemistry the worm's way: The nematode Pristionchus pacificus constructs elaborate small molecules from modified building blocks of primary metabolism, including an unusual xylopyranose-based nucleoside (see scheme). These compounds act as signaling molec

Full text of DOI:10.1002/anie.201206797

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DOI: 10.1002/anie.201206797
Metabolomics
Complex Small-Molecule Architectures Regulate
Phenotypic Plasticity in a Nematode**
Neelanjan Bose, Akira Ogawa, Stephan H. von Reuss, Joshua J. Yim,
Erik J. Ragsdale, Ralf J. Sommer,* and Frank C. Schroeder*
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Small molecules serve a wide variety of essential biological
functions.[1] They facilitate inter- and intra-species chemical
communication, are used for chemical defense and predation,
function as hormones and second messengers in animals and
plants, and serve as building blocks for biological macro-
molecules. In contrast to many groups of microorganisms and
plants, whose genomes encode a great variety of “secondary”
small-molecule biosynthetic pathways (for example, for
polyketides and non-ribosomal peptides),[2] most animals
are not known to have dedicated biosynthetic pathways to
generate structurally complex small molecules. Correspond-
ingly, many unusual metabolites that have been isolated from
basal animals (e.g., sponges and bryozoans, among others)
and arthropods have turned out to be of microbial origin or
acquired through their diet.[3] Recent studies of the model
organism Caenorhabditis elegans show that this nematode
produces a family of small-molecule signals, the ascarosides,
for example, ascr#1–3 and hbas#3 (Figure 1a), which control
multiple aspects of the life history of C. elegans, including
larval development, mating, and social behaviors.[4] These
signaling molecules are derived from combination of the
dideoxysugar ascarylose with a variety of lipid- and amino
acid-metabolism-derived moieties,[4e] suggesting that nemat-
odes, and perhaps other animals, harbor yet unrecognized
biosynthetic capabilities.
As part of a broad 2D NMR-spectroscopic screen of
nematode metabolomes, we analyzed the exometabolome
(the entirety of all secreted and excreted metabolites) of the
necromenic roundworm Pristionchus pacificus. Like C. ele-
gans, P. pacificus is a free-living nematode that is used as
a model organism for the study of developmental and
evolutionary biology.[5] P. pacificus forms a necromenic asso-
ciation with beetles, which may represent a pre-adaptation to
the evolution of true parasitism.[6] P. pacificus exhibits two
types of phenotypic plasticity that are key to its survival in the
wild. Like in many other nematode species, harsh environ-
mental conditions such as food shortage trigger developmen-
tal arrest as dauer worms (a highly stress-resistant alternate
Figure 1. a) Chemical formulas of previously identified small molecules
that regulate development and behavior in C. elegans. b) P. pacificus
exometabolome samples induce dauer arrest and affect mouth-form
dimorphism, promoting eurystomatous mouth development. Activity-
guided fractionation and 2D NMR-spectroscopic profiling were used to
identify the active components.
larval stage).[7] P. pacificus further exhibits a unique dimor-
phism in mouth development, an example of phenotypic
plasticity of morphology in an adult metazoan (Figure 1b).
Adult worms can have either a narrow (stenostomatous) or
a wide and more complex (eurystomatous) mouth opening,
the latter developing in response to conditions of low food
availability. The two different mouth forms are associated
with different feeding habits: stenostomatous worms are
considered to feed primarily on bacteria, whereas the
eurystomatous form is suited for predatory behavior toward
other nematodes. Previous studies of mixed-stage P. pacificus
cultures showed that both dauer formation and mouth
dimorphism are regulated by population density,[8] suggesting
that these two phenotypes are driven by inter-organismal
small-molecule signaling as part of a population density
monitoring system.[9]
2D NMR-based metabolomics of the P. pacificus exome-
tabolome fractions with dauer-inducing activity (Supporting
Information, Section 1.4) revealed a striking diversity of
signals suggestive of primary metabolites, for example, xylose,
threonine, adenosine, short-chain fatty acids, succinate, phe-
nylethanolamine, and several dideoxysugar derivatives (Sup-
porting Information, Figure S1); however, the NMR data
[*] N. Bose,[+] Dr. S. H. von Reuss, J. J. Yim, Prof. F. C. Schroeder
Boyce Thompson Institute and Department of Chemistry and
Department of Chemical Biology, Cornell University
Ithaca, NY (USA)
E-mail: schroeder@cornell.edu
Homepage: http://www.bti.cornell.edu/schroeder/
Dr. A. Ogawa,[+] Dr. E. J. Ragsdale, Prof. R. J. Sommer
Department for Evolutionary Biology
Max-Planck-Institute for Developmental Biology
Tꢀbingen (Germany)
E-mail: ralf.sommer@tuebingen.mpg.de
[+] These authors contributed equally to this work.
[**] We thank Parag Mahanti and Alex Artyukhin for helpful discussions
and Maciej Kukula for assistance with high resolution mass
spectrometry. This work was supported in part by the National
Institutes of Health (GM088290 and GM085285), the Cornell/
Rockefeller/Sloan-Kettering Training Program in Chemical Biology
and the Max-Planck Society.
Supporting information for this article (experimental details) is
available on the WWW under http://dx.doi.org/10.1002/anie.
201206797.
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indicated that most of these moieties are part of larger
assemblies. The dideoxysugar derivatives fell into two chemi-
cally different subgroups. One group was based on ascarylose,
a sugar that is widely produced by nematodes,[10] whereas
a second group of compounds appeared to include a related
sugar, paratose, which previously had been reported only in
bacteria (Figure S1).[11] Analyzing the P. pacificus exometa-
bolome by HPLC-MS/MS for the presence of any of the
approximately 150 ascarosides recently identified from C. ele-
gans,[4e] we detected ascr#1, ascr#9, and ascr#12, as previously
reported (Figure 2a).[10] However, these three compounds
accounted only for a small portion of the structures detected
by 2D NMR spectroscopy.
Combining the results from NMR and high resolution
HPLC-MS/MS analyses (Figure 2a, Figure S2, Table S1), we
proposed structures for the major unknown components of
the P. pacificus exometabolome, in some cases after addi-
tional fractionation to increase the content of minor compo-
nents (Figure 2b). The most abundant ascaroside derivative,
named pasc#9 (see Supporting Information, Section 1.1 and
Ref. [23]) was proposed as an N-succinyl-1-phenylethanol-
amide linked to ascarylose by way of a 4-hydroxypentanoic
acid chain (Figure 2b). This compound was accompanied by
two dimeric ascaroside derivatives, a dimer (dasc#1) of the
known ascr#1 in which one ascr#1 unit is attached to carbon 4
of the other ascr#1 unit, and a second dimer (ubas#1)
consisting of ascr#9 to which the (w)-oxygenated ascaroside
oscr#9 is attached at position 2 (Figure 2b). This second
dimer, ubas#1, also has a 3-ureido isobutyrate moiety
attached to carbon 4. To our knowledge, neither dimeric
ascarosides nor ureido isobutyrate-substituted metabolites
have previously been reported from nature.
These ascarosides were accompanied by two abundant
metabolites that included paratose instead of ascarylose,
npar#1 and part#9 (Figure 2b). In npar#1, the paratose
moiety was linked to a short lipid side chain, which in turn
was connected to threonine. This amino acid was connected
further by way of a carbamoyl group to a derivative of the
nucleoside adenosine (Figure 2b). Strikingly, this adenosine
was found to include a xylopyranose, and not ribofuranose or
deoxyribofuranose as in DNA, RNA, and known nucleoside-
based signaling molecules. The accompanying part#9 repre-
sents the paratose and side-chain portions of npar#1 (Fig-
ure 2b).
To confirm these structural assignments, determine the
stereochemistry, and explore their biological functions we
developed total syntheses for each of the proposed structures,
taking advantage of their modular nature (Figure 2c, Sup-
porting Information). Comparison of the NMR spectra of the
synthetic and natural samples established the configuration of
the N-succinyl-1-phenylethanolamide moiety in natural
pasc#9 as R (Figure S3). Similarly, comparison of the NMR
spectra and HPLC retention times allowed us to assign the
stereochemistry of the dimeric ascarosides, dasc#1 and ubas#1
(Figure S4, S5). The R-configuration of the 3-ureido isobuty-
rate moiety in ubas#1 is consistent with its likely origin from
Figure 2. a) High-resolution HPLC-MS analysis reveals molecules whose molecular formulae provide additional constraints for structures
proposed based on NMR-spectroscopic analysis of the P. pacificus exometabolome. b) Major components of the P. pacificus exometabolome
derived from assembly of building blocks from fatty acid (blue), carbohydrate (black), amino acid (green), and nucleoside (red) metabolism, as
well as TCA cycle-derived succinate (magenta). Also shown is the related tRNA nucleoside, N6-threonylcarbamoyladenosine (t6A). c) Abbreviated
syntheses for metabolites identified from P. pacificus and several non-natural stereoisomers (see Supporting Information).
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thymine catabolism.[12] Finally, we synthesized samples of the
paratoside part#9 and the unusual xyloadenosine derivative,
npar#1. By comparing the HPLC-retention times of natural
part#9 and several synthetic part#9 diastereomers, we found
that natural part#9 must be either d-paratosyl-4S-hydroxy-
pentanoic acid or l-paratosyl-4R-hydroxypentanoic acid (Fig-
ure S6). We initially assumed that part#9 and npar#1 con-
tained d-paratose, which had previously been described from
bacteria whereas l-paratose had not been found in nature.
Furthermore, d-paratose is a putative intermediate in the
biosynthesis of l-ascarylose,[11] on which all ascarosides in
nematodes are based.[4e,10,13] However, the HPLC retention
time and NMR spectra of a synthetic npar#1 diastereomer
including d-paratosyl-4S-hydroxypentanoic acid did not
match the data obtained for natural npar#1 (Figure S7a).
Therefore, we concluded that npar#1 must be based on l-
paratosyl-4R-hydroxypentanoic acid, which was confirmed by
synthesizing this diastereomer and comparing its spectro-
scopic data with those of natural npar#1 (Figure S7b,c). Using
chiral derivatization agents (Mosherꢀs acid chlorides),[14] we
further showed that part#9 is also based on l-paratose
(Figure S8). The sugar l-paratose has not previously been
found in nature; however, its occurrence in nematodes might
result from epimerization of l-ascarylose at position 2.
To exclude the possibility that the identified compounds
are bacterial metabolites, we additionally analyzed the
metabolome of the E. coli OP50 bacteria used as food for
P. pacificus. None of the identified compounds were found to
be present in the bacterial metabolome (Figure S9). Further-
more, we showed that all identified compounds are also
produced in P. pacificus cultures fed with Pseudomonas sp.
instead of E. coli as well as in axenic (bacteria-free) cul-
tures[4d,15] (Figure S10).
Next we asked whether assembly of the identified small
molecules from sugar, amino acid, lipid, and nucleoside-
derived building blocks is selective. To address this, we
carefully re-analyzed the entire P. pacificus exometabolome
by high-resolution HPLC-MS/MS, quantified the identified
compounds using synthetic standards (Table S1), and
screened for homologues or alternative combinations of the
primary metabolism-derived building blocks in our structures.
We found that pasc#9 is accompanied by trace amounts of two
homologues including six- and seven-carbon side chains,
which were also detected by NMR spectroscopy (Figure S11).
In addition, we detected a small amount of a homologue of
ubas#1 as well as a derivative of npar#1 whose MS data
indicated loss of the xylose (ubas#2 and npar#2 in Figure 2a,
Table S1). Importantly, we did not observe any non-specific or
seemingly random combinations of building blocks that
would suggest a non-enzymatic genesis of the identified
compounds.
physiologically observed (20 mm, Figure S12). In contrast,
physiological concentrations of the nucleoside derivative
npar#1 strongly induced dauer formation and appear to
account for most of the reported dauer-inducing activity in the
non-fractionated exometabolome (Figure 3a,c).[9] Addition-
ally, we observed weaker dauer induction with part#9 than
Figure 3. Regulation of mouth dimorphism and dauer induction by
synthetic samples of identified P. pacificus metabolites. All experiments
were performed in triplicate for each treatment. a,b) Compounds were
assayed at 1 mm concentration (*=p<0.01, **=p<0.001). c,d) Com-
pounds with significant activity at 1 mm (p<0.01) were subsequently
tested at a range of concentrations.
with npar#1, whereas all other compounds tested were
inactive in this assay. Testing our synthetic compounds in
the mouth dimorphism assay, we found that the dimeric
compound dasc#1, which was inactive in the dauer-formation
assay, strongly induces the eurystomatous mouth form (Fig-
ure 3b,d). In addition, weaker induction of the eurystomatous
mouth form was observed for high concentrations of pasc#9,
ascr#1, and npar#1, whereas ascr#9 and part#9 as well as the
dimeric ubas#1 were inactive at physiological concentrations
in the wild-type strain tested (Figure 3b, Table S1).
These results show that adult phenotypic plasticity and
larval development in P. pacificus are controlled by distinct
yet partially overlapping sets of signaling molecules. Whereas
mouth-form dimorphism is primarily regulated by dasc#1, the
product of highly specific ascaroside dimerization, dauer
formation is controlled by a molecule combining a paratoside
with an unusual nucleoside. Previous work showed that the
signaling molecules controlling phenotypic plasticity in
P. pacificus act upstream of evolutionarily conserved tran-
scription factors, including DAF-16/FOXO and the nuclear
hormone receptor DAF-12 (Figure 4),[18] whereby daf-12 is
required for both dauer induction and mouth-form dimor-
phism, whereas daf-16, is required for dauer induction but
dispensable for regulation of mouth-form dimorphism.[18a]
Therefore, the different subsets of small molecules regulating
dauer formation and mouth-form dimorphism appear to
target different downstream effectors. Based on the recent
We then tested synthetic samples of the identified
compounds for their activity in the P. pacificus dauer- and
mouth-form-dimorphism assays. As expected from previous
studies that showed that C. elegans exometabolome samples
are not active in the P. pacificus mouth-form dimorphism and
dauer assays,[16] we found that ascr#1, a compound abundantly
excreted by C. elegans,[4e,17] has no dauer-inducing activity in
P. pacificus, even at concentrations higher than what is
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conserved pathways or depends on dedicated enzymes
specific to P. pacificus (and perhaps other nematodes) is not
known. However, the finding that the P. pacificus compounds
are derived from assembly of modified primary metabolites
suggests that their biosynthesis is largely based on conserved
biochemical pathways (Figure 4). Notably, the P. pacificus
genome contains more than 25000 predicted genes with many
specific gene duplication events among genes encoding
primary metabolic enzymes.[21] Supporting the involvement
of primary metabolism in the biosynthesis of nematode
signaling molecules, recent investigations of ascaroside bio-
genesis in C. elegans showed that the lipid-like ascaroside side
chains are derived from conserved peroxisomal-b-oxida-
tion.[4e,22] Known signaling molecules and co-factors in
higher animals, for example, S-adenosyl methionine or
phosphatidylinositols, often rely on the combination of
building blocks derived from one or two different primary
metabolic pathways. Our results demonstrate that metazoans
may extend such strategies to produce signaling molecules of
much greater structural complexity, suggesting that detailed
spectroscopic re-analysis of metabolomes from higher ani-
mals, including mammals, may also reveal novel types of
modular small-molecule signals.
Figure 4. Small-molecule signaling in nematodes occupies a central
position connecting primary metabolism to evolutionarily conserved
transcription factors, including DAF-16/FOXO and the nuclear hor-
mone receptor (NHR) DAF-12, a vitamin D and liver X receptor
homologue.
Received: August 22, 2012
Published online: November 19, 2012
identification of several G-protein coupled receptors
(GPCRs) of the C. elegans dauer pheromone,[19] it is probable
that GPCRs expressed in specific chemosensory neurons
connect npar#1 and dasc#1 with their respective downstream
pathways in P. pacificus. Our results further demonstrate
species-specific co-option of small molecule biosynthetic
pathways for regulating different aspects of development, as
ascr#1 contributes to dauer in C. elegans,[17] whereas its dimer
dasc#1 regulates adult morphology in P. pacificus. Similar to
the multifunctional signaling properties of ascarosides in
C. elegans,[4] it is possible that dasc#1, npar#1, and the other
identified compounds serve additional signaling functions in
P. pacificus, for example, in mediating social behaviors and
sex-specific attraction.
The identified small-molecules appear to integrate spe-
cific inputs from the major primary metabolic pathways: fatty
acid, carbohydrate, amino acid, and nucleoside metabolism
(Figure 4). Specific assembly of building blocks from these
pathways using ester and amide linkages generates the
modular molecules identified, which are further distinguished
from previously known animal metabolites by the inclusion of
l-paratose and xylopyranose-based adenosine. This unprece-
dented nucleoside is likely derived from metabolism of
canonical (ribo)threonylcarbamoyl adenosine (t6A, Fig-
ure 2b), a highly conserved nucleoside found directly adja-
cent to the anticodon triplet of a subset of tRNAs.[20] t6A plays
an important role in maintaining translational fidelity; how-
ever, it usually accounts for only a very small fraction of
tRNAs, and the production of large quantities of xylopyr-
anose derivative in P. pacificus is surprising.
Keywords: metabolomics · model organisms ·
.
natural products · pheromones · small-molecule signaling
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