Abstract
In humans, defects in leucine catabolism cause a variety of inborn
errors in metabolism. Here, we use Caenorhabditis elegans to
investigate the impact of mutations in mccc-1, an enzyme that functions
in leucine breakdown. Through untargeted metabolomic and transcriptomic
analyses we find extensive metabolic rewiring that helps to detoxify
leucine breakdown intermediates via conversion into previously
undescribed metabolites and to synthesize mevalonate, an essential
metabolite. We also find that the leucine breakdown product
3,3-hydroxymethylbutyrate (HMB), commonly used as a human
muscle-building supplement, is toxic to C. elegans and that bacteria
modulate this toxicity. Unbiased genetic screens revealed interactions
between the host and microbe, where components of bacterial pyrimidine
biosynthesis mitigate HMB toxicity. Finally, upregulated ketone body
metabolism genes in mccc-1 mutants provide an alternative route for
biosynthesis of the mevalonate precursor
3-hydroxy-3-methylglutaryl-CoA. Our work demonstrates that a complex
host–bacteria interplay rewires metabolism to allow host survival when
leucine catabolism is perturbed.
__________________________________________________________________
In animals and humans, roughly 80% of dietary leucine is used for
protein synthesis and the remainder is broken down by a dedicated
metabolic pathway^[48]1 ([49]Fig. 1a). A key enzyme complex in the
leucine breakdown pathway, 3-methylcrotonyl-CoA carboxylase (3-MCC;
encoded by MCCC1 and MCCC2), catalyses the biotin-dependent
carboxylation of 3-methylcrotonyl-CoA (3MC-CoA) to form
3-methylglutaconyl-CoA, ultimately leading to the production of
acetyl-CoA, an essential metabolite for lipogenesis and energy
production and mevalonate, a key intermediate for the production of
isoprenoids ([50]Fig. 1a)^[51]2–[52]4. Leucine breakdown and MCCC1 have
been shown to play a role in early adipocyte differentiation, and
genetic variants of both MCCC1 and MCCC2 are associated with 3-MCC
deficiency, characterized by elevated leucine catabolites but showing
highly variable clinical presentation, suggesting that additional
factors influence the aetiology of 3-MCC deficiency^[53]5–[54]7.
Fig. 1 |. C. elegans mccc-1 is an orthologue of human methylcrotonyl-CoA
carboxylase MCCC1.
Fig. 1 |
[55]Open in a new tab
a, Leucine degradation pathway. The location of mccc-1, the major
enzyme studied here, is shown in bold. C. elegans genes are indicated
in colour (blue, leucine breakdown; yellow, branched-chain fatty acid
synthesis; pink, mevalonate synthesis; human genes are indicated in
black). b,c, Quantification of HMB (b) and HMB-carnitine (c) from exo-
and endo-metabolome extracts of control and mccc-1(ww4) mutant animals.
Each bar represents the mean value of three biologically independent
experiments, indicated by a white dot, with error bar showing the mean
± s.d. Statistical significance was determined using a two-sided
unpaired Student’s t-test.
In addition to its cellular metabolic function, leucine and its
breakdown product, 3,3-hydroxymethylbutyrate (HMB), are frequently used
as dietary supplements to increase muscle building and exercise
endurance and performance, as well as in elderly patients with
sarcopenia or type 2 diabetes^[56]8–[57]10. In humans, under normal
physiological conditions, approximately 5% of leucine is metabolized
into HMB^[58]11. While both metabolites are generally well tolerated,
their effects on physiology have not been comprehensively
studied^[59]12.
The disruption of metabolic enzyme function often leads to a
hard-to-predict rewiring of metabolism, which can be influenced by
environmental factors such as diet^[60]13. Further, the bacteria that
inhabit the gut, known as the intestinal microbiota, greatly influence
animal physiology, in part via production of specific metabolites that
enter host metabolism^[61]14–[62]16. However, the influence of gut
microbiota on central metabolism of the host and on the effects of
dietary supplements remains poorly understood. The complex interplay
between diet, microbiota, metabolic insults and resulting rewiring is
difficult to study in humans.
We, and others, have shown that the nematode Caenorhabditis elegans
provides a genetically tractable model system with which to study the
consequences of metabolic perturbations^[63]17–[64]20. C. elegans
metabolism is highly conserved with humans and, because it is a
bacterivore, its diet can be modulated to study bacterial influence on
cellular processes^[65]21–[66]25. Therefore, C. elegans and its
bacterial diet can be used as a model to study metabolic perturbations
and how these are modulated by bacterial influences as models for
dietary or microbiota effects.
Here, we used C. elegans to study the physiological and molecular
effects of altered leucine catabolism. First, we demonstrate that C.
elegans mccc-1 is a functional orthologue of human MCCC1, and that
mccc-1 mutant animals exhibit extensive metabolic rewiring, as shown
via comparative metabolomics and transcriptomics. Like human patients
with 3-MCC deficiency, C. elegans mccc-1 mutant animals accumulate the
leucine breakdown products HMB and HMB-carnitine. In addition, we
discovered multiple shunt metabolites derived from leucine breakdown,
including N-acyl amino acid (AA) conjugates and modular glucosides
(MOGLs), whose production may prevent the buildup of toxic leucine
catabolites. Notably, while HMB and other leucine catabolites are
tolerated by the animal when fed a Comamonas aquatica DA1877 (hereafter
referred to as Comamonas) diet, these compounds are toxic in animals
fed Escherichia coli OP50. We performed an unbiased bacterial genetic
screen that revealed that components of Comamonas pyrimidine
biosynthesis are required for the protective effect. We further found
that mccc-1 mutant animals upregulate the expression of ketone body
metabolism genes as an alternate way to provide precursors for
mevalonate synthesis. Altogether, our findings yield insights into the
multifaceted metabolic rewiring in animals with mccc-1 deficiency.
Results
C. elegans mccc-1 is an orthologue of human MCCC1
Previously, we isolated four alleles of the mccc-1 gene in a forward
genetic screen for mutations that prevent the transcriptional
repression of the acyl-CoA dehydrogenase acdh-1 when animals are fed
Comamonas^[67]26 ([68]Extended Data Fig. 1). C. elegans MCCC-1 is 53%
identical to human MCCC1 and the two proteins give reciprocal best
BlastP values, suggesting that they are orthologues ([69]Extended Data
Fig. 1). We focused on the mccc-1(ww4) mutant strain that has a G150R
missense mutation within the biotin carboxylase domain. We compared the
metabolome of mccc-1(ww4) mutants to control animals using
high-performance liquid chromatography (HPLC) coupled to high
resolution mass spectrometry (HRMS). We separately analysed metabolites
retained in the animal (endo-metabolome) and metabolites excreted into
the medium (exo-metabolome) and found that two metabolites that
accumulate in the urine and serum of humans with 3-MCC deficiency, HMB
and HMB-carnitine, accumulate in both the endo- and exo-metabolome of
mccc-1(ww4) mutant animals^[70]27 ([71]Fig. 1b,[72]c and
[73]Supplementary Table 1). HMB levels were higher in the
exo-metabolome relative to the endo-metabolome, indicating that this
metabolite is predominantly excreted, whereas HMB-carnitine was more
abundant in the endo-metabolome of mccc-1(ww4) mutant animals. Together
with the high similarity in protein sequence, these results indicate
that mccc-1 is a functional C. elegans orthologue of human MCCC1.
Conjugation reactions redirect leucine catabolic flux
Next, we investigated the biochemical fate of leucine in mccc-1(ww4)
mutant animals by stable isotope labelling with ^13C[6]-leucine
([74]Extended Data Fig. 2a). We focused on metabolites that were
isotopically labelled and at least fourfold enriched in mccc-1(ww4)
mutants relative to control animals (summarized in [75]Supplementary
Table 1). These metabolites were further distinguished as
^13C[6]-metabolites that carry the complete carbon skeleton of leucine
or as ^13C[5]-metabolites that are generated after decarboxylation by
the BCKDH complex in the second step of leucine catabolism ([76]Fig.
2a).
Fig. 2 |. Novel metabolites accumulate in mccc-1(ww4) mutant animals.
Fig. 2 |
[77]Open in a new tab
a, Schematic of ^13C[6]-leucine tracing. Each orange dot represents a
stable ^13C isotope. b, Quantification of 3-methylcrotonyl
(3MC)-carnitine from exo- and endo-metabolome extracts of control and
mccc-1(ww4) mutant animals. c, Structure of MOGLs with positions of the
conjugated R or X groups indicated. d, Quantification of mecglu#1. e,
Extracted ion chromatograms (EICs) for m/z 285.0944, corresponding to
mecglu#1 in extracts of control and mcccc-1 animals, or synthetic
mecglu#1 co-injected with extract, as indicated. f, Quantification of
2-ketoisocaproate (2KIC) and 2-hydroxyisocaproate (2HIC). g,
Quantification of N-acyl 2HIC-AA conjugates. h, EICs for m/z 244.1554,
corresponding to 2HIC-Ile and -Leu in control, mccc-1(ww4) mutant
animals and synthetic samples, as indicated. i, C. elegans leucine
degradation pathway, with shunt metabolites highlighted in red and
^13C-labelled moieties in MOGL marked in blue. Each bar represents the
mean value of three biologically independent experiments, indicated by
a white dot, with error bar showing the mean ± s.d. Statistical
significance was determined using a two-sided unpaired Student’s
t-test.
^13C[5]-metabolites that were enriched in mccc-1(ww4) animals include
HMB and HMB-carnitine, as well as 3MC-carnitine, which was not detected
in control animals ([78]Figs. 1b,[79]c and [80]2b). In contrast,
tiglyl-carnitine, the isoleucine-derived isomer of leucine-derived
3MC-carnitine, was not enriched and was instead decreased in
mccc-1(ww4) mutant animals, demonstrating the specific accumulation of
leucine-derived metabolites in the mutant ([81]Extended Data Fig. 2b).
Additionally, ^13C[5]-medium-chain acylcarnitine esters, such as
5-methylhexanoyl-l-carnitine (C7ISO) and 7-methyloctanoyl-l-carnitine
(C9ISO) were significantly enriched in the exo-metabolome of
mccc-1(ww4) mutant animals ([82]Extended Data Fig. 2c). Increased
levels of branched-chain fatty acids are likely the result of shunt
metabolites derived from isovaleryl-CoA, which can be diverted during
fatty acid elongation by conjugation to carnitine and excretion from
the cell ([83]Extended Data Fig. 2c and [84]Supplementary Table
1)^[85]28. Other ^13C[5]-labelled compounds include a putative
HMB-choline conjugate and S-acyl pantetheine conjugates, the latter
likely represent decomposed coenzyme A during sample preparation
([86]Extended Data Fig. 2c and [87]Supplementary Table 1)^[88]29.
The metabolomic analysis of the exo- and endo-metabolomes of
mccc-1(ww4) mutant animals additionally revealed accumulation of a
series of previously undescribed metabolites whose MS/MS spectra
suggested that they represent MOGLs that incorporate 3MC and/or HMB
([89]Fig. 2c). MOGLs comprise a recently discovered family of
metabolites built around a core glucose that can be acylated with
diverse moieties representing AA, nucleic acid and neurotransmitter
metabolism ([90]Fig. 2c)^[91]30,[92]31. More than 20 different MOGLs
were ^13C[5]-labelled following ^13C[6]-Leu supplementation, some of
which may incorporate multiple 3MC and/or HMB moieties, based on
analysis of MS/MS spectra ([93]Extended Data Fig. 2d and
[94]Supplementary Table 1). Given that the putative 3MC- and
HMB-derived MOGLs were strongly enriched in mccc-1(ww4) mutant animals
([95]Fig. 2d and [96]Supplementary Table 1), we synthesized an
authentic standard of one of the proposed MOGLs, with 3MC attached to
the anomeric position of glucose (which we named mecglu#1). The
synthetic compound exhibited identical chromatographic retention time
and MS/MS fragmentation to the natural compound, confirming our
assignment ([97]Fig. 2e and [98]Extended Data Fig. 2e). In contrast to
most previously described MOGLs^[99]30,[100]31, mecglu#1 and many of
the other 3MC- and HMB-derived MOGLs were more abundant in the exo-
than in the endo-metabolome of mccc-1(ww4) mutant animals, suggesting
that production of 3MC- and HMB-containing MOGLs may represent a
mechanism to reduce accumulation of these leucine catabolites in the
animal ([101]Supplementary Table 1).
^13C[6]-labelled metabolites enriched in mccc-1(ww4) mutant animals
include the known leucine breakdown products 2-ketoisocaproate (2KIC)
and 2-hydroxyisocaproate (2HIC) ([102]Fig. 2f)^[103]32. Additionally,
we detected a series of previously undescribed ^13C[6]-labelled
metabolites highly enriched in the endo-metabolome of mccc-1(ww4)
mutant animals, whose MS/MS spectra suggested that they represent AA
conjugates of 2HIC ([104]Fig. 2g). We first synthesized standards by
conjugating either l-Leu or l-Ile with racemic (R/S)-2HIC to
demonstrate that stereoisomers of these compounds can be
chromatographically separated ([105]Fig. 2h). Subsequent synthetic
conjugation of l-Leu to (S)-2HIC yielded a single stereoisomer with
identical chromatographic retention time and MS/MS fragmentation as the
naturally occurring metabolite ([106]Extended Data Fig. 2f), indicating
that this series of metabolites likely represents AAs conjugated to
(S)-2HIC. Additionally, these data indicate that stereospecific
reduction of 2KIC to (S)-2HIC in C. elegans proceeds with the same
stereospecificity as is observed in mammals^[107]33 ([108]Fig. 2h).
Notably, 2HIC was conjugated to Leu, Val, Ile, Phe, Met and Arg, an
overlapping subset of hydrophobic AAs previously found to be conjugated
to 3-hydroxypropionate (3HP) and lactate^[109]34–[110]36 ([111]Fig. 2g
and [112]Supplementary Table 1). Whereas 3HP-AAs were more abundant in
the exo-metabolome^[113]35, the 2HIC-AAs instead primarily accumulated
in the endo-metabolome ([114]Fig. 2g). We did not detect AA conjugates
of 2KIC, nor did we detect AA conjugates of 3MC or HMB, which were
preferentially incorporated into MOGLs and excreted. Notably, the
abundance of several other families of Leu-derived metabolites did not
differ between control and mccc-1(ww4) mutant animals. For example,
N-acetyl- and N-propionyl-Leu conjugates were not significantly
enriched in mccc-1(ww4) mutant animals and abundances of branched-chain
fatty ethanolamine derivatives were similar between the two strains as
well ([115]Extended Data Fig. 2g and [116]Supplementary Table 1).
Collectively, our metabolomic analyses and characterization of several
series of shunt metabolites reveals a complex biochemical network of
discrete and specific conjugation reactions that redirect leucine
catabolic flux in mccc-1(ww4) mutant animals ([117]Fig. 2i).
Genotype and diet reduce leucine breakdown product toxicity
The detection of multiple leucine-derived shunt metabolites in
mccc-1(ww4) mutant animals suggests that the accumulation of leucine
breakdown products is toxic. To test this hypothesis, we directly
supplemented the leucine breakdown products 2KIC, 2HIC, isovalerate,
3MC or HMB, and examined the effect on the development of control and
mccc-1(ww4) mutant animals. We found that HMB, 3MC and isovalerate
elicit toxicity in control animals fed Comamonas and that isovalerate
and 3MC are much more toxic than HMB ([118]Fig. 3a and [119]Extended
Data Fig. 3a). Further, these leucine breakdown products elicited
stronger toxic effects in the mutants ([120]Fig. 3b). Finally, we found
that HMB was also toxic to the three other mccc-1 mutant strains found
in our initial screen but did not observe toxicity in either of two
mccc-2 mutant strains ([121]Extended Data Fig. 3b).
Fig. 3 |. Leucine breakdown products are toxic and toxicity is modulated by
C. elegans genotype and bacterial diet.
Fig. 3 |
[122]Open in a new tab
a,b, Toxicity of leucine breakdown products as measured by the
proportion of animals reaching the L4 stage in control animals (a) and
mccc-1(ww4) mutant animals (b) fed Comamonas. c,d, Toxicity of leucine
breakdown products using the same criteria in control animals (c) and
mccc-1(ww4) mutant animals (d) fed E. coli OP50. Each bar represents
the mean value of three biologically independent experiments, indicated
by a white dot, with error bar showing the mean ± s.d. Statistical
significance was determined using a one-sided unpaired Student’s
t-test.
We previously found that the toxic effects of metabolites or
therapeutic drugs in C. elegans can be modulated by bacterial
diet^[123]23,[124]37,[125]38. We initially performed the experiments
described above in animals fed the Comamonas diet, because that diet
was used to identify the mccc-1 mutants^[126]26. Remarkably, we found
that control animals fed E. coli are much more sensitive to 2KIC, 2HIC
and HMB, relative to animals fed Comamonas, whereas 3MC and isovalerate
were comparatively well tolerated ([127]Fig. 3c). However, mccc-1(ww4)
mutant animals were more sensitive to all five compounds when fed E.
coli OP50 ([128]Fig. 3d). Finally, while 2KIC and 2HIC are more toxic
to animals fed E. coli relative to those fed Comamonas, their toxicity
was not altered in mccc-1(ww4) mutants ([129]Fig. 3c,[130]d).
HMB has been deemed non-toxic in mammals^[131]39–[132]41. However, we
observed different levels of HMB-elicited toxicity in C. elegans
depending on genotype and bacterial diet. Therefore, we determined the
metabolic fate of HMB by supplementing control and mccc-1(ww4) mutant
animals fed Comamonas with a non-lethal dose (10 mM) of
^13C[3]-labelled HMB and analysing labelled HMB-derived metabolites. In
both control and mccc-1(ww4) mutant animals, we detected a putative
MOGL containing ^13C[3]-HMB, as well as mono-methyl-branched-chain
fatty acids, derived from isovaleryl-CoA, which were further
metabolized to sphinganine and lysophosphatidylcholine (LPC) ([133]Fig.
4a–[134]e and [135]Supplementary Table 1). We did not, however, detect
labelled 2KIC or 2HIC. These results imply that supplemented HMB is
converted into isovaleryl-CoA via the intermediate 3MC-CoA, that is,
that the reactions that generate HMB are reversible ([136]Fig. 4f). As
isovalerate and 3MC, which can be generated from these metabolites when
they lose the CoA, are more toxic than HMB ([137]Extended Data Fig.
3a), and because HMB cannot be degraded via the 3-MCC-dependent route
in mccc-1(ww4) mutants, these results may help to explain HMB toxicity.
These results also support our finding that 2KIC and 2HIC are equally
toxic to control and mccc-1(ww4) animals (fed either bacterial diet)
because these two metabolites are synthesized from a branch of the
leucine catabolic pathway that does not directly involve mccc-1. In
fact, these metabolites are generated upstream of isovalerate and are
separated from the reverse reactions by the irreversible metabolic
reaction catalysed by the branched-chain ketoacid dehydrogenase (BCKDH)
complex ([138]Fig. 2a).
Fig. 4 |. Metabolic fate of HMB.
Fig. 4 |
[139]Open in a new tab
a, Schematic of ^13C[3]-HMB tracing. Orange dot indicates labelled
^13C. b, HPLC–HRMS analysis of the endo-metabolomes of control and
mccc-1(ww4) mutant animals supplemented with 10 mM ^13C[3]-HMB showing
uptake of HMB by C. elegans. Extracted ion chromatograms EICs for m/z
117.0557 and 120.0655 (negative ion mode), corresponding to HMB and
^13C[3]-HMB. The abundance of naturally occurring HMB in control
animals (light blue trace) was low compared with mccc-1(ww4) mutants
(yellow trace), whereas both control and mccc-1(ww4) mutant animals
exhibited similar levels of ^13C[3]-HMB (dark blue and orange,
respectively). c, EICs for m/z 303.1050 and 306.1151, corresponding to
sodium adducts of the MOGL HMB-glucoside and ^13C[3]-HMB-glucoside
(positive ion mode). Only ^13C[3]-HMB-glucoside was observed in control
animals (dark blue trace), whereas naturally occurring HMB-glucoside
was also observed and more abundant in mccc-1(ww4) mutant animals
(yellow trace). d,e, HPLC–HRMS analysis of control animals supplemented
with ^13C[3]-HMB or HMB, as indicated. Animals fed ^13C[3]-HMB
exhibited ^13C[3] isotopic enrichment in iso-branched lipids, such as
sphinganine (d) and lysophosphatidylcholine (LPC) bearing a C17 acyl
group (e), indicating that exogenous HMB can be reduced to
isovaleryl-CoA that can feed back into branched-chain fatty acid
biosynthesis. ^13C[1] and ^13C[2] isotopes represent natural abundance
of ^13C. Similar levels of ^13C[3]-enrichment in iso-branched lipid
derivatives were observed in mccc-1(ww4) mutants supplemented with
^13C[3]-HMB. f, Metabolism of supplemented ^13C[3]-HMB in leucine
degradation pathway in mccc-1(ww4) mutant animals is indicated in
green.
As MCCC-1 is a mitochondrial enzyme, we wanted to determine whether HMB
buildup leads to a disruption in mitochondrial membrane potential. We
used tetramethylrhodamine ethyl ester (TMRE) staining to examine the
mitochondrial membrane potential of control and mccc-1(ww4) mutant
animals. We observed a reduction in mitochondrial membrane potential
following exposure to 60 mM HMB in both control and mccc-1(ww4) mutant
animals fed Comamonas, as indicated by TMRE staining ([140]Extended
Data Fig. 4). This suggests a negative impact of HMB on mitochondrial
function^[141]42.
Taken together, the two leucine breakdown products directly upstream of
the reaction catalysed by 3-MCC are specifically toxic in mccc-1(ww4)
mutant animals fed either bacterial diet and toxicity of all five
metabolites is enhanced on an E. coli OP50 diet. These results suggest
that there are different mechanisms of detoxification associated with
these metabolites, which correlates with the observation of the
different novel shunt metabolites described above.
Comamonas pyrimidine synthesis suppresses HMB toxicity
Next, we focused on the bacterial mechanisms that modulate HMB
toxicity. One noteworthy difference between E. coli and Comamonas is
that Comamonas produces vitamin B12 but E. coli does not. As vitamin
B12 has substantial effects on C. elegans
metabolism^[142]18,[143]25,[144]37,[145]43, we asked whether
supplementation of E. coli with vitamin B12 or disrupting Comamonas
vitamin B12 production would modulate HMB toxicity. However, neither
supplementation with vitamin B12 in animals fed E. coli ([146]Fig. 5a)
nor disruption of its biosynthesis in Comamonas affected HMB toxicity
([147]Fig. 5b).
Fig. 5 |. Vitamin B12 does not attenuate HMB toxicity.
Fig. 5 |
[148]Open in a new tab
a, Dose–response curves of animals reaching the L4 stage and fed
Comamonas, E. coli OP50 or E. coli OP50 supplemented with increasing
concentrations of HMB and with or without supplementation of 64 nM
vitamin B12. Data represent five Comamonas, six E. coli OP50 and nine
E. coli OP50 with vitamin B12 supplemented biologically independent
experiments and error bars indicate mean ± s.d. b, Toxicity of titrated
HMB in animals fed E. coli OP50, wild-type Comamonas, or two Comamonas
mutants cbiA^− and cbiB^−, which cannot synthesize vitamin B12. Each
bar represents the mean value of three biologically independent
experiments, indicated by a white dot, with error bar showing the mean
± s.d.
There are several mechanisms by which bacteria can affect the host
response to supplemented compounds such as metabolites and therapeutic
drugs, including differential uptake by the bacteria, which may
modulate drug delivery to C. elegans, direct modification of the
compound or by providing bacterial metabolites to the animal that
result in modulation of compound toxicity^[149]44,[150]45. To
discriminate between these possibilities, we first asked whether active
bacterial metabolism is required to either enhance (in E. coli) or
mitigate (in Comamonas) HMB toxicity in C. elegans. We found that HMB
was more toxic when animals were fed dead bacteria compared with
animals fed live bacteria of either species ([151]Fig. 6a–[152]c). This
result indicates that active bacterial metabolism is required to
mitigate HMB toxicity and that active Comamonas metabolism is most
protective.
Fig. 6 |. Live bacteria are required to mitigate HMB toxicity.
Fig. 6 |
[153]Open in a new tab
a, HMB toxicity on live versus dead (kanamycin-killed) bacteria as
measured by the proportion of animals reaching the L4 stage. Each bar
represents the mean value of three biologically independent
experiments, indicated by a white dot, with error bar showing the mean
± s.d. b, Images of C. elegans grown with HMB supplementation and fed
live or kanamycin-killed bacteria. Figures represent one of three
biologically independent experiments. Scale bar, 1 mm. c, Percentage of
animals reaching beyond the L1 stage (percentage L1+) under the
condition of 60 mM HMB supplementation, fed E. coli OP50, wild-type
Comamonas or their powders. Each bar represents the mean value of three
biologically independent experiments, indicated by a white dot, with
error bar showing the mean ± s.d.
To identify Comamonas genes involved in the modulation of HMB toxicity
in C. elegans we screened a collection of ~5,700 Comamonas transposon
mutant strains^[154]37 and found five mutant strains that enhanced HMB
toxicity in control animals ([155]Extended Data Fig. 5a and
[156]Extended Data Table 1). Two of these mutant strains harbour
transposons in genes in the Comamonas pyrimidine biosynthesis pathway
(pyrC and pyrE; [157]Fig. 7a). Both bacterial mutant strains exhibited
reduced growth compared with wild-type bacteria and, while pyrC^−
mutant growth could be rescued by supplementation with either orotate
or uracil, pyrE^− mutants could only be rescued by uracil, which
corroborates the predicted biosynthetic functions of these enzymes
([158]Extended Data Fig. 5b). We further found that supplementation
with either orotate or uracil rescued HMB toxicity in C. elegans fed
pyrC^− bacteria and likewise, supplementation with uracil rescued HMB
toxicity in C. elegans fed Comamonas pyrE^− mutants ([159]Fig. 7b and
[160]Extended Data Fig. 5c).
Fig. 7 |. Toxicity of HMB is mitigated by Comamonas pyrimidine metabolism.
Fig. 7 |
[161]Open in a new tab
a, Bacterial pyrimidine biosynthesis pathway. The two mutants found in
the screen are indicated in blue. b, C. elegans grown with and without
60 mM HMB and fed E. coli OP50, wild-type or either of the two
Comamonas mutants found in the screen that were supplemented with
uracil or orotate during bacterial culture. Outlined areas emphasize
the rescue of the pyrC and pyrE mutant E. coli by uracil and orotate.
Figures represent one of three biologically independent experiments.
Scale bar, 1 mm. c, HMB toxicity exhibited by control or mccc-1(ww4) C.
elegans strains with combinations of UTP glucose-1-phosphate
uridylyltransferases mutations or knockdown and fed either wild-type or
ΔgalU mutant Comamonas. Each bar represents the mean value of four
biologically independent experiments, indicated by a white dot, with
error bar showing the mean ± s.d. Statistical significance was
determined using a one-sided unpaired Student’s t-test. NS, not
significant. d, Model of UDP-glucose mediated detoxification. C.
elegans and Comamonas genes are indicated in blue. Metabolic processes
occurring separately in C. elegans and the bacterium Comamonas are
distinguished by green dashed lines.
Notably, uracil supplementation did not alleviate HMB toxicity in
animals fed E. coli OP50, even though this bacterial strain is an
uracil auxotroph ([162]Fig. 7b and [163]Extended Data Fig. 5c). We
tested two additional E. coli strains, BW25113 and HT115 and found that
C. elegans fed these strains were less sensitive to HMB than animals
reared on OP50 and that toxicity was also not suppressed by uracil
supplementation in these strains. Further, we found that feeding E.
coli pyrE^− mutant bacteria did not impact HMB toxicity in the host,
even upon uracil supplementation ([164]Extended Data Fig. 6).
Collectively, these data show that Comamonas pyrimidine synthesis is
required for the protective effect of these bacteria to HMB toxicity in
C. elegans. Further, these data indicate that the modest protective
effect of live E. coli is independent of pyrimidine metabolism.
Therefore, bacteria can protect C. elegans from metabolite toxicity by
distinct mechanisms.
Transcriptional metabolic rewiring in mccc-1(ww4) animals
So far, our metabolomic data indicate that there is extensive metabolic
rewiring in mccc-1(ww4) mutant animals, that this rewiring functions to
detoxify leucine breakdown products, and that bacterial metabolism
aides in this detoxification. As metabolism can be rewired by changing
the expression of metabolic enzymes and transporters and because
metabolic genes are extensively transcriptionally regulated in C.
elegans^[165]46–[166]48, we compared the transcriptomes of mccc-1(ww4)
mutant and control animals by RNA-seq. Using a threshold of 1.5-fold
change and P < 0.05, we identified 156 up- and 939 downregulated genes
in the mutant ([167]Supplementary Table 2). While only 99 (11%) of the
downregulated genes encode annotated metabolic enzymes, 50 (32%)
upregulated genes are associated with metabolism, suggesting that the
transcriptional activation of metabolic genes contributes to the
metabolic rewiring seen in the mutant animals ([168]Extended Data Fig.
7a).
Upregulated genes in mccc-1(ww4) mutant animals include acdh-1, which
agrees with the screen in which the mutant allele was originally
identified^[169]26. Among the upregulated genes in mccc-1(ww4) mutant
animals are four genes encoding predicted UDP-glycosyltransferases
(UGTs), enzymes that attach sugars to other molecules for excretion and
detoxification^[170]49. The C. elegans genome encodes 67 UGT enzymes,
most of which are completely uncharacterized^[171]50. The two most
highly induced ugt genes in mccc-1(ww4) mutant animals, ugt-53 and
ugt-32, showed ~11-fold and eightfold greater expression relative to
control animals, respectively ([172]Extended Data Fig. 7b). We
generated single mutant ugt-53(ww60) and ugt-32(ww58) animals by
CRISPR/Cas9 genome editing ([173]Extended Data Fig. 7c), crossed these
animals with mccc-1(ww4) mutants and tested all resulting single and
double mutants for HMB sensitivity. While we did not observe a change
in HMB toxicity in the ugt mutants, we found that ugt-32(ww58);
mccc-1(ww4) double mutant animals were more sensitive to HMB than
mccc-1(ww4) alone ([174]Extended Data Fig. 7d,[175]e). Remarkably,
deletion of ugt-53 may slightly decrease HMB toxicity ([176]Extended
Data Fig. 7e). The sensitivity to HMB was further increased by feeding
ugt-32(ww58); mccc-1(ww4) double mutant animals the Comamonas pyrE^−
mutant ([177]Extended Data Fig. 7d). These results suggests that C.
elegans ugt-32 may be upregulated in mccc-1(ww4) mutant animals to
detoxify leucine breakdown products. As HMB is toxic at high doses, we
were not able to assess MOGL levels in the mccc-1(ww4);ugt mutant
animals. Therefore, we measured the levels of excreted 3MC glucosides
in the mutant combinations without HMB supplementation. We did not
detect a change in any of the 3MC glucosides in any of the mutant
combinations ([178]Extended Data Fig. 7f and [179]Supplementary Table
3). These results suggest that other genes are involved as well, likely
because detoxification is multifaceted involving different metabolic
conjugation reactions.
Comamonas pyrimidine biosynthesis genes pyrC and pyrE are required to
mitigate HMB toxicity in C. elegans and, in mccc-1(ww4) mutant animals,
HMB is conjugated to glucose to generate MOGLs that are excreted. As C.
elegans ugt-32 is mildly involved in alleviating HMB toxicity, and
because these genes encode enzymes that conjugate sugars to toxic
molecules, we hypothesized that Comamonas pyrimidine biosynthesis may
be used to generate UDP-glucose that is then used by the animal to
produce MOGLs. Therefore, we tested whether the production of
UDP-glucose is necessary for the protection exhibited by Comamonas.
UDP-glucose is synthesized by the enzyme UTP glucose-1-phosphate
uridylyltransferase. The Comamonas genome has one such transferase
(galU), whereas the C. elegans genome encodes two biochemically
verified uridylyltransferases (rml-1 and D1005.2)^[180]50–[181]52. We
aimed to generate a Comamonas ΔgalU mutant and a C. elegans D1005.2
mutant by CRISPR/Cas9 genome editing ([182]Extended Data Fig.
7g,[183]h). However, while we succeeded in generating a Comamonas ΔgalU
mutant strain, we were not able to establish a C. elegans D1005.2
mutant line because the deletions we generated were homozygous lethal.
Therefore, we used RNAi to knockdown D1005.2 expression in subsequent
experiments. In addition, we used a deletion mutant of the essential
gene rml-1 that likely is a partial loss-of-function allele. We found
that HMB toxicity increased when we fed animals Comamonas ΔgalU mutant
bacteria and that this effect was stronger in mccc-1(ww4) mutant
animals ([184]Fig. 7c). We did not observe a further significant change
in HMB toxicity upon either additional deletion of rml-1(ok233) or
knockdown of D1005.2 ([185]Fig. 7c). However, when we depleted D1005.2
in the mccc-1(ww4); rml-1(ok233) mutant background and fed animals
Comamonas ΔgalU mutant bacteria, we observed a slight increase in HMB
toxicity. This increase is only moderate likely because we used a
partial loss-of-function allele of rml-1 and because we only have
achieved partial knockdown of the essential gene, D1005.2, by
performing RNAi in animals fed E. coli HT115 before transferring them
to the Comamonas diets.
Taken together, these results suggest that Comamonas pyrimidine
metabolism may be important to mitigate HMB toxicity in C. elegans by
providing the animal with UDP-glucose, which provides the glucose
moiety to synthesize MOGLs ([186]Fig. 7d). However, as the effect of
the Comamonas ΔgalU mutant is weaker than that of mutants in bacterial
pyrimidine metabolism, the latter pathway must be involved in HMB
detoxification by other mechanisms as well.
Leucine breakdown supports C. elegans mevalonate synthesis
Our RNA-seq data also revealed significantly altered expression of
ketone body metabolism genes in mccc-1(ww4) mutant animals ([187]Fig.
8a and [188]Supplementary Table 2). In contrast with valine and
isoleucine, which are propiogenic, leucine is a ketogenic AA, and its
catabolites are intermediates for other biosynthetic pathways,
including 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA), which is a
precursor of mevalonate synthesis ([189]Fig. 8b). Mevalonate is the key
intermediate for the endogenous production of isoprenoids, for example,
the co-factor ubiquinone, the geranyl and farnesyl moieties serving as
protein membrane anchors or isopentenyladenosine, an essential
component of tRNA^[190]3,[191]4. HMG-CoA can be derived from leucine
but can also be synthesized de novo from condensation of
acetoacetyl-CoA with acetyl-CoA by HMG-CoA synthase (HMGS-1), followed
by reduction to mevalonate by HMG-CoA reductase (HMGR-1) ([192]Fig.
8b). As the production of HMG-CoA from leucine is impaired in
mccc-1(ww4) mutants, we hypothesized that upregulation of ketone body
metabolism genes may support mevalonate synthesis. Consistent with this
idea, we found that both sur-5 and C05C10.3, which are predicted to
produce acetoacetyl-CoA, are more highly expressed in mccc-1(ww4)
mutant animals ([193]Fig. 8a). Therefore, we reasoned that, if
mevalonate synthesis relied on both leucine catabolism, which is
dependent on MCCC-1 activity, as well as ketone body metabolism,
simultaneous depletion of both pathways should generate a stronger
phenotype than depletion of either pathway alone ([194]Fig. 8c).
Previous reports have shown that animals carrying deletions in hmgs-1
or hmgr-1 are not viable, while knockdown of hmgs-1 reduces adult
viability and depletion of hmgr-1 does not show a detectable
phenotype^[195]53–[196]55. Viability of the hmgr-1 deletion mutant
animals can be rescued with low concentrations of mevalonate; however,
these animals show reduced reproductive potential. When we knocked down
hmgs-1 we also observed reduced adult viability, and this was enhanced
in mccc-1(ww4) mutant animals ([197]Fig. 8d). Moreover, the
reproductive potential of hmgr-1 knockdown animals was reduced in
mccc-1(ww4) mutant animals ([198]Fig. 8e). Further, supplementation
with mevalonate, but not the ketone body 3-hydroxybutyrate rescued all
of the phenotypes ([199]Fig. 8b,[200]d,[201]e). The increased
sensitivity to hmgr-1 RNAi was consistently observed across mccc-1
mutant alleles, but not with mccc-2 mutant alleles ([202]Extended Data
Fig. 8). Taken together, C. elegans leucine catabolism is important for
mevalonate synthesis and its perturbation is compensated by an increase
in ketone body metabolism genes.
Fig. 8 |. Mevalonate biosynthesis is rewired via ketone bodies when leucine
breakdown is impaired.
Fig. 8 |
[203]Open in a new tab
a, Fold change in mRNA levels of differentially expressed C. elegans
ketone body metabolism genes in mccc-1(ww4) mutant animals measured by
RNA-seq. b, Cartoon depicting leucine breakdown dependent and
independent HMG-CoA synthesis facilitating the generation of
mevalonate. In the absence of leucine breakdown (mccc-1(ww4) animals),
HMG-CoA synthesis depends on precursors derived from ketone body
production such as acetoacetate. The dashed line shows that acetyl-CoA
generated by HMG-CoA breakdown can be recycled back to form HMG-CoA but
both acetyl-CoA and acetoacetyl-CoA can additionally be generated by
other cellular pathways, such as tyrosine metabolism. Enzymes active in
the absence of MCCC-1 activity are shown in red. Enzymes with elevated
expression in mccc-1(ww4) animals are bold. c, Cartoon of changes in C.
elegans mevalonate levels between control and mccc-1(ww4) mutant
animals (grey) in the presence (black) or absence (red) of hmgs-1 or
hmgr-1. d, Bar graphs showing viability of P0 animals exposed to
mevalonate biosynthesis gene RNAi and supplemented with 50 mM or 20 mM
for 3-hydroxybutyrate (3HB) or mevalonate (Mev), respectively. Outlined
areas emphasize the reduced reproductive potential of
hmgr-1;mccc-1(ww4) mutant animals. NS, not significant. Each bar
represents the mean value of five biologically independent experiments,
indicated by a white dot, with error bar showing the mean ± s.d.
Statistical significance was determined using a one-sided unpaired
Student’s t-test. e, Offspring of P0 animals exposed to mevalonate
biosynthesis gene RNAi from P0 animals and supplemented with 50 mM or
20 mM for 3HB or Mev, respectively. Figures represent one of five
biologically independent experiments. Scale bar, 1 mm. f, Model of
rewired leucine degradation pathway in mccc-1(ww4) mutant animals and a
proposed metabolic connection to Comamonas pyrimidine metabolism.
Orange arrows indicate rewired metabolic processes in mccc-1(ww4)
mutant animals. C. elegans genes are shown in blue.
Discussion
Combining metabolomics, transcriptomics and de novo structure
elucidation revealed extensive metabolic rewiring in a C. elegans
mutant with perturbed leucine catabolism. This rewiring serves two
purposes: to detoxify toxic leucine breakdown intermediates and to
support mevalonate biosynthesis by an alternate metabolic route
([204]Fig. 8f).
Our data indicate that the detoxification of leucine breakdown products
is multifaceted and relies on several distinct biochemical
transformations, resulting in the production of diverse, previously
undescribed shunt metabolites, including N-acylated AAs,
2-hydroxyisocapoate AA conjugates, methyl-branched acylcarnitines and
MOGLs. Previous studies in humans found that AA conjugations to lactate
or fatty acids are catalysed by cytosolic non-specific dipeptidase 2
(CNDP2) or peptidase M20 domain containing 1 enzyme (PM20D1)
activities^[205]34,[206]36,[207]56. We therefore speculate that
2HIC-AAs production may involve similar peptidases. However, we did not
find any change in expression in putative C. elegans peptidases in our
mccc-1(ww4) mutant RNA-seq analysis. Of note, 2HIC toxicity is also
modulated by bacterial diet with greater toxicity observed when
supplemented animals are grown on E. coli OP50 versus Comamonas,
similar to HMB, indicating that metabolic factors produced by the
bacteria could also aide in mitigating 2HIC toxicity in the animal. We
found that HMB can undergo reverse conversion reactions that lead to
the production of potentially toxic intermediates such as 3MC. Future
studies will be required to identify the enzymes catalysing these
reactions.
Notably, we found that many leucine catabolites, including HMB, are
toxic to C. elegans and that this toxicity is modulated by bacterial
diet. As active bacterial metabolism is required to mitigate HMB
toxicity, it is unlikely that the bacteria modulate delivery to the
animal by differential HMB uptake. Instead, our findings suggest that
the production of bacterial metabolites such as UDP-glucose may help to
mitigate HMB toxicity in the animal. However, because mutants in
pyrimidine biosynthesis affect HMB toxicity more severely than mutants
required for the bacterial synthesis of UDP-glucose, bacterial
pyrimidine metabolism must be involved in additional detoxification
mechanisms. Our data further suggest that within the animal, C. elegans
UGT enzymes function in the detoxification of leucine breakdown
products. We speculate that UGT enzymatic activity would add glucose,
potentially from the UDP-glucose generated in bacteria, to toxic
leucine catabolites such as HMB, thereby generating MOGLs that are
excreted from the animal. However, this model is currently supported by
weak genetic interactions, potentially due to the large UGT family in
C. elegans, several of which are upregulated in mccc-1 mutant animals
and may have compensatory function. Therefore, further work is needed
to determine whether bacterial pyrimidine biosynthesis or UDP-glucose
and C. elegans UGT enzymes directly modify HMB through the proposed
mechanism. It is also possible that Comamonas directly metabolizes HMB
in supplementation experiments. However, if so, it is not likely to be
a contributing factor to the detoxification of HMB produced by C.
elegans because it would have to be significantly taken up from the
animal by living bacteria in the gut, which is perhaps not likely to
occur. Finally, because vitamin B12 does not affect HMB detoxification,
it is clear that Comamonas affects C. elegans metabolism and physiology
by both vitamin B12-dependent and independent mechanisms, consistent
with our previous findings of drug toxicity^[208]23,[209]38.
Another finding is that perturbation of C. elegans leucine catabolism
rewires mevalonate biosynthesis. Because mccc-1 is required for HMG-CoA
synthesis from leucine, our data suggest that mevalonate biosynthesis
in mccc-1(ww4) mutants is bolstered by increased transcription of
ketone body genes providing input to HMGS-1, an alternative route to
produce HMG-CoA. While it is well known that leucine catabolism
produces HMG-CoA, it has also been proposed that HMB can be directly
converted to HMG-CoA via an unknown enzyme^[210]57. If that were true,
mccc-1 loss-of-function would not be expected to affect mevalonate
metabolism. However, our data indicate that mevalonate biosynthesis in
mccc-1 mutant animals is compensated by an alternate route using ketone
bodies, even though these animals produce ample HMB.
Taken together, our work shows that defective leucine breakdown results
in activation of a previously uncharted biochemical network of
conjugation reactions and the transcriptional compensation for
biosynthetic processes. Future studies will determine whether these
processes are conserved in mammals, including humans or whether diverse
organisms have evolved different solutions to handle the toxic effect
of metabolic intermediates such as HMB.
Methods
C. elegans strains
C. elegans strains were cultured using standard procedures at 20 °C
(ref. [211]58). N2 (Bristol) was used as the wild-type strain. The
VL749 wwIs24(Pacdh-1::GFP + unc-119(+)) strain is the background
control strains for the mccc-1(ww2), mccc-1(ww4), mccc-1(ww20) and
mccc-1(ww38) alleles (strain VL907, VL1080, VL1520 and VL1521,
respectively) and other strains generated herein^[212]17,[213]26. The
strains FX22463 mccc-2(tm12463) and FX25273 mccc-2(tm15274) were
obtained from National BioResource Project, Japan. FX22463 and FX25273
were backcrossed three times with N2 and crossed to VL749 to generate
VL1528 mccc-2(tm12463); wwIs24(Pacdh-1::GFP + unc-119(+)) and VL1529
mccc-2(tm15274); wwIs24(Pacdh-1::GFP + unc-119(+)), respectively. The
strains VL1369 ugt-32(ww58), VL1384 ugt-53(ww60), VL1524
D1005.2(ww65)/+ heterozygote and VL1525 D1005.2(ww66)/+ heterozygote
were generated by CRISPR-Cas9 genome editing and backcrossed three
times with N2 (refs. [214]59,[215]60). The VL1369 and VL1384 strains
were crossed to VL749 to generate VL1410 ugt-32(ww58);
wwIs24(Pacdh-1::GFP + unc-119(+)) and VL1411 ugt-53(ww60);
wwIs24(Pacdh-1::GFP + unc-119(+)), respectively. VL1410 and VL1411 were
further crossed to mccc-1(ww4) to create strains VL1412 mccc-1(ww4);
ugt-32(ww58); wwIs24(Pacdh-1::GFP + unc-119(+)) and VL1413 mccc-1(ww4);
ugt-53(ww60); wwIs24(Pacdh-1::GFP + unc-119(+)), respectively. The
strains MG278 rml-1(ok233) was retrieved from the C. elegans Gene
Knockout Consortium. MG278 rml-1(ok233) was backcrossed threes time
with N2 and crossed to VL749 or VL1080 to generate VL1451 rml-1(ok233);
wwIs24(Pacdh-1::GFP + unc-119(+)) or VL1452 rml-1(ok233); mccc-1(ww4);
wwIs24(Pacdh-1::GFP + unc-119(+)), respectively.
Bacterial strains
E. coli OP50, E. coli HT115(DE3), E. coli BW25113 and Comamonas
aquatica DA1877 (referred to as Comamonas) were obtained from the
Caenorhabditis Genetics Center. The E. coli pyrE^− mutant was retrieved
from the E. coli Keio collection^[216]61. The Comamonas cbiA^−, cbiB^−,
valS^−, recB^−, pyrC^− and pyrE^− mutants were retrieved from the
Comamonas mutant collection^[217]37. The Comamonas ΔgalU mutant was
generated using a modified CRISPR-Cas9 genome editing system (see
below). Bacterial cultures were prepared in lysogeny broth (LB) from a
single-colony inoculation and incubated at 37 °C for 24 h with 200 rpm
orbital shaking.
Sequence alignment of C. elegans MCCC-1 to human MCCC1
Protein sequences of human MCCC1 (A0A0S2Z693) and C. elegans MCCC-1
([218]O45430) were obtained from Uniprot ([219]www.uniprot.org).
Sequences were aligned using the online tool Benchling
([220]www.benchling.com).
Culturing C. elegans for metabolomics
First larval stage (L1) animals were cultured on nematode growth medium
(NGM) agar seeded with Comamonas approximately 70 h at 20 °C until they
reached the gravid adult stage. Eggs were obtained by subjecting gravid
adults to buffered bleached solution (20% NaOCl, Fisher, SS290–1), 10%
10 N NaOH and 70% H[2]O). Eggs were hatched and synchronized in M9
buffer (22 mM KH[2]PO[4], 42 mM Na[2]HPO[4], 86 mM NaCl, 1 mM MgSO[4]
and 1 L H[2]O). Approximately 50,000 L1 animals were placed in a 25-ml
Erlenmeyer flask filled with 10 ml S medium (1 l S basal (5.85 g NaCl,
1 g K[2] HPO[4], 6 g KH[2]PO[4], 1 ml cholesterol (5 mg ml^−1 in
ethanol) in 1 l H[2]O), 10 ml 1 M potassium citrate, 10 ml trace metal
solution, 3 ml 1 M CaCl[2] and 3 ml 1 M MgSO[4]) with a concentrated
Comamonas bacterial pellet from a 300-ml overnight LB culture. Animals
were incubated at 20 °C with 180 rpm orbital shaking for 60 h for
checking the developmental stage and/or adding 10 mM pH 6.0-adjusted
leucine or HMB. Animals were further incubated for 12 h until they
reached young adult stage. Animals and conditioned medium were
separated by 264g centrifugation for 2 min and extracted separately.
For analysis of animal bodies (endo-metabolome), animal pellets were
washed three times with M9 buffer followed by one wash with H[2]O.
Conditioned medium (exo-metabolome) was centrifuged at 10,000g for 10
min to remove residual Comamonas bacteria. Approximately 7 ml of
clarified exo-metabolome was transferred to a new conical tube. The
endo- and exo-metabolome samples were snap frozen in liquid nitrogen
and stored at −80 °C.
Sample preparation for HPLC–HRMS
Animal bodies (endo-metabolome) and conditioned medium (exo-metabolome)
were frozen and processed separately, as described above. For
preparation of endo-metabolome extracts, samples were lyophilized for
18–24 h using a VirTis BenchTop 4K Freeze Dryer. After the addition of
1 ml methanol directly to the conical tube in which animals were
frozen, samples were sonicated for 5 min (2 s on–off pulse cycle at 90
A) using a Qsonica Q700 Ultrasonic Processor with a water bath cup horn
adaptor (Qsonica 431C2). Following sonication, an additional 4–9 ml of
methanol was added, depending on sample size, and the extract rocked
overnight at room temperature (22 °C). The conical tubes were
centrifuged (3,000g, 22 °C, 5 min) and the resulting clarified
supernatant transferred to a clean 8- or 20-ml glass vial which was
concentrated to dryness in an SC250EXP Speedvac Concentrator coupled to
an RVT5105 Refrigerated Vapor Trap (Thermo Scientific). The resulting
powder was suspended in 100–250 μl of methanol, depending on sample
size, followed by vigorous vortex and brief sonication. This solution
was transferred to a clean microfuge tube and subjected to
centrifugation (20,000g, 22 °C, 5 min) in an Eppendorf 5417 R
centrifuge to remove precipitate. The resulting supernatant was
transferred to an HPLC vial and analysed by HPLC–HRMS.
For preparation of exo-metabolome extracts, samples were lyophilized
for ~48 h using a VirTis BenchTop 4K Freeze Dryer. Dried material was
extracted in 5–15 ml methanol, depending on the sample size and rocked
overnight at room temperature. The conical tubes were centrifuged
(3,000g, 22 °C, 5 min) and the resulting clarified supernatant
transferred to clean 8- or 20-ml glass vials which were concentrated in
vacuo and suspended in methanol as described for endo-metabolome
samples.
HPLC–HRMS analysis
Reversed-phase chromatography was performed using a Vanquish HPLC
system controlled by Chromeleon Software (Thermo Fisher Scientific) and
coupled to an Orbitrap Q-Exactive HF mass spectrometer controlled by
Xcalibur software (Thermo Fisher Scientific) or by a Dionex Ultimate
3000 HPLC system coupled to an Oribtrap Q-Exactive mass spectrometer
controlled by the same software. Extracts prepared as described above
were separated on a Thermo Scientific Hypersil Gold column (150 × 2.1
mm, particle size 1.9 μm, part no. 25002–152130) maintained at 40 °C
with a flow rate of 0.5 ml min^−1. Solvent A: 0.1% formic acid (Fisher
Chemical Optima LC–MS grade; A11750) in water (Fisher Chemical Optima
LC–MS grade; W6–4); solvent B: 0.1% formic acid in acetonitrile (Fisher
Chemical Optima LC–MS grade; A955–4). A/B gradient started at 1% B for
3 min after injection and increased linearly to 98% B at 20 min,
followed by 5 min at 98% B, then back to 1% B over 0.1 min and finally
held at 1% B for an additional 2.9 min.
The mass spectrometer parameters were spray voltage of −3.0 kV/+3.5 kV;
capillary temperature of 380 °C; probe heater temperature of 400 °C;
sheath, auxiliary and sweep gas at 60, 20 and 2 a.u., respectively;
S-Lens RF level of 50; resolution of 60,000 or 120,000 at m/z 200; and
AGC target of 3E6. Each sample was analysed in negative (ESI−) and
positive (ESI+) electrospray ionization modes with m/z range 117–1,000.
Parameters for MS/MS (dd-MS2): MS1 resolution of 60,000; AGC target of
1E6. MS2 resolution, 30,000; and AGC target of 2E5. The maximum
injection time was 60 ms; isolation window was 1.0 m/z; stepped
normalized collision energy 10, 30; dynamic exclusion was 1.5 s; and
the top eight masses were selected for MS/MS per scan.
HPLC–HRMS RAW data were converted into mzXML file format using
MSConvert (v.3.0, ProteoWizard) and were analysed using Metaboseek
software v.0.9.9 and normalized to the abundance of ascr#3 (an
ascaroside featuring a nine-carbon α,β-unsaturated carboxylic acid side
chain) in negative ionization mode or normalized to the abundance of
ascr#2 (an ascaroside featuring a six-carbon side chain bearing a
ketone functionality) in positive ionization mode as an approximate
measure of sample size for replicates from the same genotype. To
account for variation between genotypes, metabolites were normalized as
a ratio to ascr#3 or ascr#2 and the resulting quotient multiplied by
the genotype average (performed independently for endo- and
exo-metabolome samples), thereby removing the effect of variation
between genotypes. Quantification was performed with Metaboseek
software v.0.9.9 ([221]Metaboseek.com) or via integration using
Xcalibur QualBrowser v.4.1.31.9 (Thermo Fisher Scientific) using a
5-ppm window around the m/z of interest. Statistical analysis for
metabolomics was performed with Metaboseek software v.0.9.9 and with
GraphPad Prism v.9.5.0.
General synthetic procedures
Unless stated otherwise, all reactions were carried out under argon
(Ar) atmosphere in flame-dried glassware. All commercially available
reagents were used as purchased unless otherwise stated. All solvents
were dried over activated 3 Å molecular sieves for a minimum of 24 h
unless used in reactions containing aqueous reagents. Solutions and
solvents sensitive to moisture and oxygen were transferred via standard
syringe and cannula techniques. Reactions were cooled with iced water
or dry ice–acetone baths or heated with mineral oil baths depending on
reaction temperature. Unless stated otherwise, all chemicals and
reagents were purchased from Sigma-Aldrich. l-isoleucine tert-butyl
ester hydrochloride, l-leucine tert-butyl ester hydrochloride and
trifluoroacetic acid (TFA) were purchased from TCI. The
4-dimethylaminopyridine (DMAP) was purchased from Fluka.
Dichloromethane (DCM), ethyl acetate, hexanes and methanol were
purchased from Fisher Scientific. Thin-layer chromatography was
performed using J. T. Baker Silica Gel IB2F plates. Flash
chromatography was performed using Teledyne Isco CombiFlash systems and
Teledyne Isco RediSep Rf silica columns. All deuterated solvents were
purchased from Cambridge Isotopes. Nuclear Magnetic Resonance (NMR)
spectra were recorded on a Bruker AV 500 (500 MHz) spectrometer at
Cornell University’s NMR facility. ^1H NMR chemical shifts are reported
in ppm (δ) relative to residual solvent peaks (7.26 ppm for CDCl[3] and
3.31 ppm for CD[3]OD). ^1H NMR chemical shifts are reported as follows:
chemical shift, multiplicity (s, singlet; d, doublet; t, triplet; m,
multiplet), coupling constants (Hz) and integration. ^13C NMR chemical
shifts are reported in ppm (δ) relative to residual solvent peaks
(77.16 ppm for CDCl[3] and 49.00 ppm for CD[3]OD). All NMR data
processing was conducted using Mnova v.14.2.3
([222]https://mestrelab.com/).
Chemical syntheses
graphic file with name nihms-2043324-f0001.jpg
(2S,3R,4S,5S,6R)-3,4,5-Trihydroxy-6-(hydroxymethyl) tetrahydro-2H-pyran-2-yl
3-methylbut-2-enoate (1).
Based on a previously reported procedure^[223]62, we added
N,N-dimethylformamide (5 ml) to a flask of α-d-glucose (551 mg, 3.06
mmol, 1.0 eq.), 3,3-dimethylacrylic acid (398.0 mg, 3.98 mmol, 1.3 eq.)
and triphenylphosphine (1.2 g, 4.59 mmol, 1.5 eq.) under argon and
cooled to 0 °C, before adding diisopropyl azodicarboxylate (0.9 ml,
4.59 mmol, 1.5 eq.) to the flask. The reaction mixture was stirred at 0
°C for 1 h and warmed up to room temperature. After 17 h, the mixture
was concentrated in vacuo. Flash-column chromatography on silica using
a gradient of 0–50% methanol in DCM afforded mecglu#1 (1) as a
colourless oil (86.0 mg, 11%).
^1H NMR (500 MHz, methanol-d[4]).
δ (ppm) 5.73 (m, 1H), 5.45 (d, J = 8.1 Hz, 1H), 3.80 (dd, J = 1.9, 12.0
Hz, 1H), 3.68–3.61 (m, 2H), 3.39 (dt, J = 8.7 Hz, 1H), 3.36–3.32 (m,
2H), 2.15 (d, J = 0.9 Hz, 3H), 1.90 (d, J = 1.0 Hz, 3H).
^13C NMR (125 MHz, methanol-d[4]).
δ (ppm) 166.4, 161.0, 116.2, 95.1, 78.7, 78.0, 73.9, 71.1, 62.3, 27.5,
20.5.
graphic file with name nihms-2043324-f0002.jpg
N-(2-Hydroxyisocaproyl)-l-isoleucine tert-butyl ester (2).
We added 4 ml dichloromethane to a vial containing 2-hydroxyisocaproic
acid (45 mg, 0.34 mmol, 1.0 equiv.), l-isoleucine tert-butyl ester
hydrochloride (98 mg, 0.44 mmol, 1.3 equiv.),
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) hydrochloride (224
mg, 1.17 mmol, 3.4 equiv.) and DMAP (83 mg, 0.68 mmol, 2.0 equiv.). The
resulting solution was stirred at room temperature overnight and
concentrated in vacuo. Flash-column chromatography on silica using a
gradient of 0–100% ethyl acetate in n-hexane afforded 2 (86 mg, 85%).
^1H NMR (500 MHz, chloroform-d).
δ 4.51 (ddd, J = 8.8, 4.6, 1.9 Hz, 1H), 4.19 (ddd, J = 9.7, 3.3, 2.3
Hz, 1H), 1.96–1.84 (m, 2H), 1.68–1.53 (m, 2H), 1.49 (s, 9H), 1.30–1.17
(m, 2H), 0.99–0.93 (m, 12H).
^13C NMR (126 MHz, chloroform-d).
δ 174.2, 174.0, 171.1, 170.9, 82.2, 82.2, 70.9, 70.6, 56.5, 56.4, 44.0,
43.9, 38.2, 38.2, 28.1, 25.3, 25.2, 24.6, 24.6, 23.5, 23.5, 21.4, 15.4,
15.4, 11.7.
graphic file with name nihms-2043324-f0003.jpg
N-(2-Hydroxyisocaproyl)-l-isoleucine (3).
We added trifluoroacetic acid (TFA) (0.52 ml, 6.8 mmol, 100 equiv.) to
a solution of 2 (20 mg, 0.068 mmol, 1 equiv.) in 1 ml of DCM. The
resulting solution was stirred for 1 h and then concentrated to dryness
in vacuo, yielding 2HIC-l-Ile (3, 21 mg, 127%).
^1H NMR (500 MHz, methanol-d[4]).
δ 4.43 (t, J = 5.6 Hz, 1H), 4.10 (td, J = 9.8, 3.7 Hz, 1H), 2.00–1.92
(m, 1H), 1.92–1.83 (m, 1H), 1.61–1.47 (m, 3H), 1.31–1.21 (m, 1H),
0.99–0.95 (m, 12H).
^13C NMR (126 MHz, methanol-d[4]).
δ 176.2, 176.1, 173.0, 173.0, 70.0, 69.9, 56.0, 55.9, 43.5, 43.2, 37.4,
37.3, 24.8, 24.7, 24.2, 24.1, 22.5, 22.5, 20.4, 20.3, 14.6, 14.6, 10.5.
graphic file with name nihms-2043324-f0004.jpg
N-((S)-2-hydroxyisocaproyl)-l-leucine tert-butyl ester (4).
We added 4 ml dichloromethane to a vial containing
(S)-2-hydroxyisocaproic acid (46 mg, 0.35 mmol, 1.0 equiv.), l-leucine
tert-butyl ester hydrochloride (106 mg, 0.47 mmol, 1.3 equiv.), EDC
hydrochloride (229 mg, 1.20 mmol, 3.4 equiv.) and DMAP (89 mg, 0.73
mmol, 2.0 equiv.). The resulting solution was stirred at room
temperature overnight and concentrated in vacuo. Flash-column
chromatography on silica using a gradient of 0–100% ethyl acetate in
n-hexane afforded 4 (72 mg, 68%).
^1H NMR (500 MHz, methanol-d[4]).
δ 4.39 (t, J = 7.3 Hz, 1H), 4.08 (dd, J = 9.6, 3.7 Hz, 1H), 1.92–1.83
(m, 1H), 1.73–1.66 (m, 1H), 1.65–1.62 (m, 2H), 1.59–1.50 (m, 2H), 1.49
(s, 9H), 0.99–0.94 (m, 12H).
^13C NMR (126 MHz, methanol-d[4]).
δ 176.2, 171.9, 81.5, 69.9, 51.0, 43.5, 40.6, 26.8, 24.7, 24.1, 22.5,
21.8, 20.6, 20.4.
graphic file with name nihms-2043324-f0005.jpg
N-((S)-2-Hydroxyisocaproyl)-l-leucine (5).
We added TFA (0.53 ml, 6.9 mmol, 100 equiv.) to a solution of 4 (21 mg,
0.069 mmol, 1 equiv.) in 1 ml of dichloromethane. The resulting
solution was stirred for 1 h and then concentrated to dryness in vacuo.
Flash-column chromatography on silica using a gradient of 0–100%
methanol in DCM afforded (S)-2HIC-L-Leu (5, 19 mg, 113%).
^1H NMR (500 MHz, methanol-d[4]).
δ 4.39 (t, J = 7.1 Hz, 1H), 3.98 (dd, J = 9.6, 3.7 Hz, 1H), 1.80–1.82
(m, 1H), 1.60–1.56 (m, 3H), 1.48–1.35 (m, 2H), 0.88–0.82 (m, 12H).
^13C NMR (126 MHz, methanol-d[4]).
δ 176.3, 174.4, 70.0, 50.1, 43.4, 40.6, 24.7, 24.1, 22.5, 22.0, 20.5,
20.4.
Developmental rate assay
For the developmental rate assay, overnight bacterial cultures were
concentrated by resuspending the bacteria in H[2]O after centrifuge at
2,500g for 30 min. E. coli was concentrated fivefold, Comamonas,
cbiA^−, and cbiB^− were concentrated tenfold, and Comamonas pyrC^− and
pyrE^− mutants were concentrated 20-fold. Then, 0.2 ml concentrated
bacteria was seeded and dried onto 35-mm NGM plates.
Animals were fed a diet of E. coli or Comamonas for three generations
before assay. Approximately 100 L1 animals were added to their
respective bacteria-seeded 35-mm NGM plates containing various
concentrations of pH 6.0-adjusted HMB, 3-methylcrotonate, or
isovalerate. L1 animals were incubated for 70 h at 20 °C and post-L4
stage animals were counted. The percentage of post-L4 developed animals
was calculated compared with the non-supplement condition of same
bacterial diet. For the vitamin B12 supplement condition, we used 64 nM
adenosyl cobalamin (Sigma-Aldrich, C0884). We obtained dose–response
curves using the following equation that was fit to the biological
replicate datasets:
[MATH:
y=Bottom+(Top
-Bottom)/
(1+10((logLD50-X)×HillSlope)) :MATH]
For starved and kanamycin treated dead bacterial diet, L1 animals were
incubated for 70 h at 20 °C and post-L4 stage animals were counted to
obtain the percentage animals that developed beyond this stage.
TMRE staining
L1 animals were cultured on NGM agar seeded with Comamonas
approximately 30 h at 20 °C until they reached the third larval (L3)
stage. Animals were treated with 10 μM TMRE in S Basal buffer (44 mM
KH[2]PO[4], 5 mM K[2]HPO[4] and 0.1 M NaCl in H[2]O) and incubated at
room temperature with gentle rocking for 45 min, covered in foil.
Animal pellets were washed two times by 161g centrifugation for 1 min
in S Basal buffer. After washing, animals were placed in NGM plates
without TMRE for 30 min. Animals were mounted on a 2% agar pad to
assess mitochondrial morphology and measure TMRE intensity of the
intestinal mitochondria.
Bacterial killing
E. coli OP50 and Comamonas were cultured in 50 ml LB medium in a 250-ml
flask at 37 °C for 24 h with 200 rpm shaking. The bacterial culture was
transferred to 50-ml tube. Transferred bacteria was pelleted by 3,000g
centrifugation for 30 min. Supernatant was discarded and bacterial
pellet was resuspended in 100 ml H[2]O containing 250 μg ml^−1
kanamycin. Resuspended bacteria were evenly separated in two 250-ml
flasks and incubated for 24 h with 200 rpm shaking. For checking
bacterial survival, 1 ml of 100 ml culture was aliquoted and washed
three times by 1 ml H[2]O. Washed bacterial culture was plated on LB
agar and incubated at 37 °C for 24 h. When no viable colonies were
detected, the killed bacteria were stored at 4 °C. Before seeding
killed bacteria on NGM agar, they were washed three times with 100 ml
H[2]O.
Bacterial powder from E. coli OP50 or Comamonas was produced as
described previously^[224]38. In brief, a single bacterial colony was
cultured overnight and subsequently diluted 1:100 in LB medium without
antibiotics to reach the log phase (OD[600nm] = 0.8 to 1.0). The
bacterial cultures were collected and washed three times with sterile
water. Each pellet was weighed and resuspended in sterile water at a
concentration of 1 gram of wet weight per 25 ml. The bacteria were then
disrupted using a Microfluidics M-110P lab homogenizer set to 22,000
psi for ten cycles. The disrupted cells were flash-frozen and
lyophilized using a Labconco FreeZone 2.5-l benchtop freeze
dryer/lyophilizer until completely dry. The dried bacterial powder was
then dissolved in sterile water at a concentration of 50 mg ml^−1. The
prepared powder solution was plated on LB agar to ensure no bacterial
growth.
Microscopy
For TMRE stained mitochondria, 3–4 animals in the third larval (L3)
stage were mounted onto a single slide and imaged together within 5 min
using a ZEISS LSM800 confocal microscope at ×63 magnification to
minimize time spent for each slide as the prolonged scoping time can
lead to mitochondrial fission and loss of the network.
For Nomarksi pictures, synchronized L1 animals were grown in NGM agar
plates with each metabolite supplemented for 72 h for measuring the
number of post-L4 animals at P0 and 144 h for observing F1 embryonic
lethality and larval development at 20 °C. Brightfield images of each
plate were taken using a Nikon Eclipse Ci (microscope) and Nikon DS-Ri2
(Camera) at ×20 magnification.
Bacterial mutant screens
Primary screens were performed in 96-well plates containing modified
NGM agar (substitute K[2]HPO[4] buffer (pH 6.0) to PBS (Gibco,
10010023) containing 60 mM HMB and 10 μg ml^−1 gentamycin). Bacterial
mutants were cultured from frozen glycerol stocks in 1 ml LB medium
with gentamycin and incubated at 37 °C for 24 h with 200 rpm shaking.
Then, 50 μl of bacterial mutant overnight culture was seeded in each
well of a 96-well plate and dried in an aseptic hood. Approximately 20
L1 animals were seeded in each well and incubated for 70 h at 20 °C.
Comamonas mutants that did not support C. elegans development in the
presence of HMB were considered hits. The mutant collection was
screened twice.
All hits were retested using approximately 50 L1 animals grown on 35 mm
NGM agar plates plus the corresponding antibiotics and containing 60 mM
HMB. Comamonas hits that retested were sequenced to identify the
location of the transposon insertion as described previously^[225]37.
For C. elegans developmental rate assays (see above) with the Comamonas
mutants, bacteria were cultured without supplement, with 10 mM NaOH
(solvent control), 1 mM uracil or 1 mM orotic acid, and incubated at 37
°C for 24 h with 200 rpm shaking. Cultured bacteria were centrifuged
for 30 min at 2,054g. Supernatant was decanted and bacterial cells were
resuspended in H[2]O. Larval development was captured after 20 °C
incubation for 70 h on 35-mm NGM agar plates containing 0 or 60 mM HMB
by using Nikon DS-Ri camera in Nikon Eclipse Binocular Ergonomic
microscope at ×20 magnification.
Bacterial growth rate
Bacterial growth rate was measured in flat-bottom 96-well plates. For
different nucleobase supplementation, 5 μl diluted bacterial cultures
(OD[600nm] = 0.4) were inoculated to 0.2 ml LB medium containing 1 mM
of uracil, adenine, thymine, guanine, cytosine or orotate dissolved in
10 mM NaOH. Plates were incubated at 37 °C for 24 h with 200 rpm
orbital shaking. Bacterial growth was monitored every 30 min by
measuring OD[600nm] in an EON microplate spectrophotometer.
RNA extraction, RNA-seq and data analysis
L1 animals were cultured on NGM agar seeded with Comamonas
approximately 70 h at 20 °C until they reached the gravid adult stage.
Gravid adults were bleached and synchronized eggs were obtained as
described above. Approximately 500 L1 animals were incubated at 20 °C
until they reached at young adult stage. Then, 250 young adults were
picked and transferred to 0.5 ml TRIzol (Thermo Fisher, 15596018).
Animals were immediately frozen in liquid nitrogen and stored at −80 °C
for RNA extraction. Animals in TRIzol were freeze-cracked in liquid
nitrogen and warmed to 37 °C three times. RNA was separated from TRIzol
by adding 50 μl of 1-bromo-3-chloropropane (MRC, BP 151) and purified
by following manufacturer’s protocol of Directzol RNA miniprep kit
(Zymo research, R2050). Extracted RNA was sent to BGI genomics for
sequencing, read mapping and analysis. Differentially expressed genes
were identified with thresholds of 1.5-fold change with PPEE < 0.05 of
statistical significance, where PPEE indicates the post probability of
equally expressed^[226]63 genes. In addition, metabolic differentially
expressed genes were annotated and categorized according to the
annotations used in the iCEL1314 genome-scale metabolic network
model^[227]21,[228]48,[229]64 and pathway enrichment analysis using
WormPaths was used to detect significantly induced or repressed
metabolic pathways^[230]65.
CRISPR/Cas9 genome editing
To delete ugt-32 and ugt-53 in C. elegans, we modified a previously
described CRISPR-Cas9 genome editing protocol^[231]59. In brief, to
delete each gene, we injected two sgRNAs targeting both 5′ and 3′ ends
of each ugt coding region. dpy-10 sgRNA was co-injected as a co-CRISPR
marker with dpy-10(cn64) ssDNA as a repair template^[232]66. After
injecting 100 animals, F2 rollers were screened and genomic DNA PCR was
performed to each roller animal to detect knockout alleles of ugt
genes. Each ugt deletion mutant was outcrossed three times with N2.
To delete D1005.2 in C. elegans, we designed to induce gene deletion
through non-homologous end-joining repair of double strand breakdown
that was described in a previously reported CRISPR-Cas9 genome editing
protocol^[233]67.
To delete the galU genomic region in Comamonas, we modified a
Pseudomonas CRISPR-Cas9 genome editing method^[234]68. We found a
single UTP glucose-1-phosphate uridylyltransferase (galU) orthologue in
the Comamonas genome^[235]37. sgRNA was designed to target 5′ end of
galU open reading frame and it was incorporated to pACRISPR plasmid by
Golden gate assembly^[236]68. We predicted that the Comamonas genome
does not encode the DNA end binding protein, KU or a DNA ligase, and
therefore, we predict that it does not use non-homologous end-joining
DNA repair machinery^[237]37,[238]69. Therefore, to enable homologous
recombination, adjacent DNA sequences of both the 5′ and 3′ end of the
galU genomic region were provided as a DNA repair template by Gibson
assembly. For Gibson assembly, the vector was linearized by PCR from a
circular plasmid followed by a DpnI restriction enzyme digest (NEB,
R0176) to remove the methylated circular plasmid DNA template.
We found that Comamonas is resistant to β-lactam antibiotics.
Therefore, we replaced the antibiotic selection marker, amp^R in the
pACRISPR plasmid with a gentamycin resistant aacC1 marker which
originates from a Pseudomonas transposon, Tn1696 by Gibson
assembly^[239]70,[240]71. This Amp^R replaced plasmid, pACRISPR-gent
was validated by plating E. coli DH5α having pACRISPR-gent plasmid on
LB agar plates containing different antibiotics. Cas9 nuclease coding
sequence was encoded in pCasPA plasmid with λ-Red recombination system
proteins that increased efficiency of homologous recombination^[241]68.
To prepare Comamonas for electroporation, a single colony was
inoculated in 5 ml LB medium and incubated at 37 °C for 24 h with 200
rpm orbital shaking. Overnight culture was diluted 50-fold in fresh 5
ml LB medium and incubated until OD[600nm] = 0.6. This diluted
secondary culture (1.5 ml) was washed three times with H[2]O at room
temperature. After a third wash, bacteria were resuspended in 0.1 ml
H[2]O and mixed with 0.5 μg plasmid DNA. Prepared bacteria and plasmid
DNA were electroporated at 15 kV cm^−1 in a Gene Pulser Cuvette
(Bio-Rad). After electroporation, bacteria were transferred in a 15-ml
round-bottom tube and immediately 1 ml prewarmed (37 °C) super optimal
broth with catabolite repression (SOC) medium was added. Cells were
incubated at 37 °C for 2 h with 200 rpm orbital shaking and seeded on
LB agar plates containing antibiotics (10 μg ml^−1 tetracycline for
pCasPA; 10 μg ml^−1 gentamycin for pACRISPR-gent) and incubated at 37
°C for 2 days with parafilm sealing. The presence of plasmid was
validated by antibiotic selection, colony PCR and plasmid PCR.
For expression of Cas9 nuclease and λ-Red recombination system
proteins, Comamonas was inoculated in 5 ml LB medium containing
antibiotics (10 μg ml^−1 tetracycline for pCasPA and 10 μg ml^−1
gentamycin for pACRISPR-gent) and incubated at 37 °C for 24 h with 200
rpm orbital shaking. Overnight culture was 1:20 diluted in fresh 20 ml
LB medium with antibiotics and incubated until OD[600nm] = 0.6 in 125
ml flask for aeration. Then, a 20-ml culture was washed three times
with H[2]O at room temperature. After the third wash, the bacterial
pellet was resuspended in 20 ml bacterial M9 plus amino acids,
l-arabinose and isopropyl β-d-1-thiogalactopyranoside (IPTG) (1× M9
salt, 0.4% glucose, 1 mM MgSO[4], 1 mM CaCl[2], 0.002% histidine,
0.006% leucine, 0.003% lysine, 0.002% methionine, 0.5% adenine sulfate,
20 mg ml^−1 l-arabinose and 2 mM IPTG). Resuspended bacteria were
incubated at 37 °C for 48 h with 200 rpm orbital shaking. Then, 50 μl
of the bacterial culture was seeded on LB agar plates containing
antibiotics and incubated at 37 °C for 2 days with parafilm sealing.
Each single colony was validated by colony PCR and sequencing of
isolated genomic DNA.
To remove the pCasPA and pACRISPR-gent plasmids from Comamonas ΔgalU
mutants, a single colony was inoculated in 5 ml LB medium without any
antibiotics and incubated at 37 °C for 24 h with 200 rpm orbital
shaking. The overnight culture was diluted to 1:10,000 in LB medium and
100 μl of diluted culture was seeded on LB agar containing no
antibiotics or selective antibiotics and incubated overnight at 37 °C.
Five colonies from non-antibiotic-containing LB agar were tested to
verify the absence of the plasmids by plating each colony on LB agar
containing tetracycline or gentamycin for antibiotic selection.
RNA interference
Animals were fed a diet of E. coli HT115 for three generations before
assay. RNAi clones were cultured in LB containing 50 μg ml^−1
ampicillin at 37 °C for 20 h and seeded on NGM agar with 2 mM IPTG
(Fisher Scientific). Plates were dried and incubated at room
temperature for 48 h. After 2 days, approximately 100 synchronized L1
animals were plated, followed by incubation at 20 °C for 72 h for
counting P0 adult viability and continuously incubated further 72 h to
examine F1 embryo viability. For the D1005.2 RNAi experiment, animals
were exposed to vector or D1005.2 RNAi for more than two generations
before being fed with Comamonas or Comamonas ΔgalU mutant diets.
Statistic and reproducibility
Statistical analysis was performed using a Student’s t-test. The data
were analysed using the Data Analysis Toolpak in Microsoft Excel to
compare the means and s.d. between groups. Data distribution was
assumed to be normal, but this was not formally tested. Individual data
points are shown in the figures. No data were excluded from the
analyses. No statistical methods were used to pre-determine sample
sizes. For HPLC–HRMS we used 50,000 animals, which has been shown to
provide enough material to give a detectable reading for the assay. For
developmental/lethality we used ~100 worms, which has been shown to
provide a sample size which will produce statistically significant
results^[242]37. For the Comamonas mutant screen we used 10–20 worms
per well to prevent the bacteria from being consumed before the end of
the experiment. For the RNA-seq we used 250 worms per condition, which
provides the minimum amount of total RNA to generate a statistically
significant number of reads. Multiple biological replicates for each
experiment were conducted as mentioned in the figure legends. For all
experiments, batches of C. elegans for the specified genotype under
each condition were randomly selected from experimental plates and
assayed as appropriate. Data collection and analysis were not performed
blind to the conditions of the experiments as blinding is not relevant
to our study; knowing the genotype or condition did not bias the study.
Extended Data
Extended Data Fig. 1 |. Alignment of human (H.s.) MCCC1 and C. elegans (C.e.)
MCCC-1 protein sequences.
Extended Data Fig. 1 |
[243]Open in a new tab
a, Alignment of protein sequences, asterisks indicate reported amino
acid alterations in human 3-MCC deficiency patients. Four C. elegans
mccc-1 mutant alleles are in bold, and their altered amino acids are
marked below C. elegans MCCC-1. Biotin carboxylase and biotinyl-binding
domains are marked as black and grey underbars, respectively. b,
Cartoon of the C. elegans MCCC-1 protein showing the mutant alleles.
Extended Data Fig. 2 |. Conjugation products of leucine and its catabolites.
Extended Data Fig. 2 |
[244]Open in a new tab
a, A schematic of Leu supplements provided to animals during C. elegans
culture in liquid medium used to generate exo- and endo-metabolome
samples. b, c, Bar graphs showing tiglyl-carnitine (b),
isovaleryl-carnitine, 5-methylhexanoyl(C7ISO)-carnitine,
7-methyloctanoyl(C9ISO)-carnitine, HMB-choline, and HMB-pantetheine (c)
levels in control and mccc-1(ww4) mutant animals. d, e, f, HPLC–HRMS
analysis of mecglu#72: 3-methylcrotonyl, HMB and mecglu#:
3-methylcrotonyl (2x) (d), mecglu#1 (e), 2HIC-Leu (f). g, Bar graphs
showing N-acetyl-Leu and N-propionyl-Leu levels in control and
mccc-1(ww4) mutant animals. Each bar represents the mean value of three
biologically independent experiments, indicated by a white dot, with
error bar showing the mean ± standard deviation. Statistical
significance was determined using a two-sided unpaired Student’s
t-test.
Extended Data Fig. 3 |. HMB, isovalerate, and 3MC toxicity.
Extended Data Fig. 3 |
[245]Open in a new tab
a, Dose–response curves of control animals fed Comamonas supplemented
with HMB, isovalerate or 3MC. Data represent five HMB, and three each
for IV and 3MC supplemented biologically independent experiments and
error bars indicate mean ± s.d. b, Toxicity of 60 mM HMB in C. elegans
control, mccc-1(ww2), mccc-1(ww4), mccc-1(ww20), mccc-1(ww38),
mccc-2(tm12463) or mccc-2(tm15274) mutant animals fed Comamonas. Each
bar represents the mean value of three biologically independent
experiments, indicated by a white dot, with error bar showing the mean
± standard deviation. Statistical significance was determined using a
one-sided unpaired Student’s t-test.
Extended Data Fig. 4 |. Mitochondrial membrane potential decreases in C.
elegans fed Comamonas supplemented with HMB.
Extended Data Fig. 4 |
[246]Open in a new tab
a, Box plot representing the intensity of TMRE staining in C. elegans
control or mccc-1(ww4) mutant mitochondria with or without HMB
supplementation and fed a Comamonas diet. The vertical lines (whiskers)
extend from the minimum to the maximum scores, excluding outliers,
which are represented by circles above the maximum value. The box
itself delineates the interquartile range (IQR), capturing the middle
50% of scores, with the bottom and top edges indicating the 25th and
75th percentiles, respectively. A horizontal line within the box marks
the median value, providing a clear indication of the distribution’s
centre. Outliers, indicating data points significantly above the
maximum score, underscore variations beyond the typical range of
observations. Data represents control (n = 53), mccc-1(ww4) mutants (n
= 50), control (n = 54) and mccc-1(ww4) mutants (n = 54) with 60 mM HMB
supplementation from three biologically independent experiments. n.s
indicates statistically not significant p-value. Statistical
significance was determined using a two-sided unpaired Student’s
t-test. b, Representative images of TMRE stained mitochondria. Figures
represent one of three biologically independent experiments. Scale bar,
10 μm.
Extended Data Fig. 5 |. Comamonas pyrimidine biosynthesis is required to
mitigate HMB toxicity.
Extended Data Fig. 5 |
[247]Open in a new tab
a, Schematic of Comamonas transposon mutant screen with 60 mM HMB. b,
Bacterial growth of wild-type and mutant strains and supplemented with
nucleobases as indicated. c, Bar graphs of C. elegans grown with and
without 60 mM HMB and fed wild-type or either of the two Comamonas
mutants supplemented with uracil or orotate during bacterial culture.
Each bar represents the mean value of three biologically independent
experiments,indicated by a white dot, with error bar showing the mean ±
standard deviation.
Extended Data Fig. 6 |. Supplementation with uracil does not alleviate HMB
toxicity in C. elegans fed an E. coli diet.
Extended Data Fig. 6 |
[248]Open in a new tab
a, Toxicity of titrated HMB in animals fed E. coli OP50, HT115, BW25113
or Comamonas supplemented with 1 mM uracil during bacterial culture. b,
Toxicity of titrated HMB in animals fed E. coli OP50, or Comamonas
pyrE^− mutants supplemented with uracil during bacterial culture. c,
Toxicity of 40 mM HMB in animals fed E. coli BW25113, E. coli pyrE^−
mutant, Comamonas, or Comamonas pyrE^− mutants supplemented with uracil
during bacterial culture. Each bar represents the mean value of three
biologically independent experiments, indicated by a white dot, with
error bar showing the mean ± standard deviation. Statistical
significance was determined using a one-sided unpaired Student’s
t-test.
Extended Data Fig. 7 |. UTP glucose-1-phosphate uridylyltransferase gene
expression is increased in mccc-1(ww4) mutant animals and partially protects
against HMB toxicity.
Extended Data Fig. 7 |
[249]Open in a new tab
a, RNA-seq data of differentially expressed metabolic genes in
mccc-1(ww4) mutants fed Comamonas. b, Fold change in mRNA levels of
upregulated C. elegans ugt genes in mccc-1(ww4) mutants measured by
RNA-seq. c, Cartoon of ugt-32 and ugt-53 deletion alleles created by
CRISPR/Cas9 genome editing. d, e, Toxicity of 20 mM (d) or 40 mM (e)
HMB toxicity in six different C. elegans strains fed wild-type or pyrE
mutant Comamonas as measured by the proportion of animals reaching the
L4 stage. Each bar represents the mean value of three biologically
independent experiments, indicated by a white dot, with error bar
showing the mean ± standard deviation. Statistical significance was
determined using a one-sided unpaired Student’s t-test. n.s indicates
statistically not significant p-value. f, Bar graphs showing
exometabolomic mecglu# levels in six different C. elegans strains fed
Comamonas. Each bar represents the mean value of three biologically
independent experiments, indicated by a white dot, with error bar
showing the mean ± standard deviation. Statistical significance was
determined using a two-sided unpaired Student’s t-test. g, h, Cartoon
of Comamonas galU deletion (g) and C. elegans D1005.2 deletions (h)
generated by CRISPR/Cas9 genome editing.
Extended Data Fig. 8 |. mccc-1 mutants, but not mccc-2 mutants are vulnerable
to hmgr-1 RNAi knockdown.
Extended Data Fig. 8 |
[250]Open in a new tab
Offspring of P0 animals exposed to hmgr-1 RNAi with or without
mevalonate supplementation. Figures represent one of three biologically
independent experiments. Scale bar, 1 mm.
Extended Data Table 1 |.
Comamonas mutant strains that modify HMB toxicity in C. elegans
Comamonas mutant Disrupted gene product by transposon insertion
Homologous gene in E. coli K-12MG1655
Plate number/ well location in library
14A7 Valyl tRNA synthase ValS
39D7 Exodeoxyribonuclease V beta recB
41H7 Orotate phosphoribosyltransferase pyrE
42H11 Dihydroorotase pyrC
51D3 Intergenic region Not applicable
[251]Open in a new tab
Supplementary Material
Supplementary Information
[252]NIHMS2043324-supplement-Supplementary_Information.pdf^ (2MB, pdf)
Supplementary Tables 1-4
[253]NIHMS2043324-supplement-Supplementary_Tables_1-4.xlsx^ (158.4KB,
xlsx)
Acknowledgements