Abstract
Protection against pathogens is a major function of the gut microbiota.
Although bacterial natural products have emerged as crucial components
of host-microbiota interactions, their exact role in
microbiota-mediated protection is largely unexplored. We addressed this
knowledge gap with the nematode Caenorhabditis elegans and its
microbiota isolate Pseudomonas fluorescens MYb115 that is known to
protect against Bacillus thuringiensis (Bt) infection. We find that
MYb115-mediated protection depends on sphingolipids (SLs) that are
derived from an iterative type I polyketide synthase (PKS) cluster
PfSgaAB, thereby revealing a non-canonical pathway for the production
of bacterial SLs as secondary metabolites. SL production is common in
eukaryotes but was thought to be limited to a few bacterial phyla that
encode the serine palmitoyltransferase (SPT) enzyme, which catalyses
the initial step in SL synthesis. We demonstrate that PfSgaB encodes a
pyridoxal 5’-phosphate-dependent alpha-oxoamine synthase with SPT
activity, and find homologous putative PKS clusters present across
host-associated bacteria that are so far unknown SL producers.
Moreover, we provide evidence that MYb115-derived SLs affect C. elegans
defence against Bt infection by altering SL metabolism in the nematode
host. This work establishes SLs as structural outputs of bacterial PKS
and highlights the role of microbiota-derived SLs in host protection
against pathogens.
Subject terms: Symbiosis, Natural products, Bacterial host response,
Innate immunity
__________________________________________________________________
Here, Peters et al. report that Pseudomonas fluorescens protects
Caenorhabditis elegans against pathogen infection via polyketide
synthase-derived sphingolipids, uncovering a non-canonical bacterial
sphingolipid synthesis pathway that modulates host metabolism.
Introduction
A major function of the gut microbiota is its contribution to host
protection against pathogens^[62]1. The protective mechanisms conferred
by the gut microbiota are complex and include direct competitive or
antagonistic microbe–microbe interactions and indirect microbe-host
interactions, which are mediated by the stimulation of the host immune
response, promotion of mucus production, and maintenance of epithelial
barrier integrity^[63]2. Microbiota-derived metabolites are known to
play an important role in the crosstalk between the gut microbiota and
the immune system^[64]3–[65]5. Of these metabolites, bacterial natural
products have emerged as crucial components of host-microbiota
interactions^[66]6–[67]8.
Bacterial natural products (also called secondary or specialised
metabolites) are chemically distinct, often bioactive compounds that
are not required for viability, but mediate microbial and environmental
interactions^[68]9. Some of the most studied natural products include
polyketides, which are derived from polyketide synthase (PKS). PKS are
found in many bacteria, fungi, and plants, and produce structurally
diverse compounds by using an assembly line mechanism similar to fatty
acid synthases^[69]10. Many PKS-derived natural products show potent
antibiotic (e.g., erythromycin and tetracycline), antifungal (e.g.,
amphotericin and griseofulvin) or immunosuppressant (e.g., rapamycin)
activities^[70]11 and have thus long played a central role in advancing
therapeutic treatments for a wide range of medical conditions. The
majority of characterised polyketides were isolated from free-living
microbes, while only a few are known to be gut
microbiota-derived^[71]8. Most well-studied examples of PKS-derived
products from the microbiota are virulence factors associated with
pathogenicity^[72]12. Few PKS-encoded natural products were reported to
play a role in microbiota-mediated protection against pathogens both
directly and indirectly. For example, the antifungal polyketide
lagriamide supports direct symbiont-mediated defence of eggs against
fungal infection in the beetle Lagria vilossa^[73]13. A PKS cluster of
the rodent gut symbiont Limosilactobacillus reuteri is required for
activating the mammalian aryl hydrocarbon receptor, which is involved
in mucosal immunity^[74]14. Additionally, L. reuteri PKS was recently
demonstrated to exhibit antimicrobial activity and to drive
intraspecies antagonism^[75]15. Yet, the vast majority of
microbiota-encoded PKS are of unknown function and mechanistic studies
linking specific microbial natural products to host phenotypes are
scarce.
The Pseudomonas fluorescens isolate MYb115 belongs to the natural gut
microbiota of the model organism Caenorhabditis elegans^[76]16. It was
previously found that MYb115 protects C. elegans against the harmful
effects of infection with Bacillus thuringiensis (Bt) without directly
inhibiting pathogen growth, likely through an indirect, host-dependent
mechanism^[77]17,[78]18. The nature of the microbiota-derived
protective molecule and the involved host processes were unknown. Here,
we identify a biosynthetic gene cluster (BGC) in MYb115 encoding an
iterative type I PKS that is required for MYb115-mediated protection
and produces sphingolipids (SLs). Thus, we discovered a non-canonical
pathway for the production of bacterial SLs, which relies on a BGC, the
P. fluorescens PKS cluster PfSgaAB. Hence SLs are produced as secondary
metabolites. We additionally demonstrate that MYb115-derived SLs affect
C. elegans SL metabolism and establish the importance of C. elegans SL
metabolism for survival after Bt infection.
Results
P. fluorescens MYb115 PKS cluster is required for C. elegans protection
against Bt infection
The natural microbiota isolate P. fluorescens MYb115 protects C.
elegans against infection with the Gram-positive pathogenic B.
thuringiensis strain Bt247 likely through a host-dependent
mechanism^[79]18, but the nature of the microbiota-derived protective
molecule was unknown. We performed an antiSMASH analysis^[80]19 of the
MYb115 genome to identify natural product BGCs. We found three BGCs in
the MYb115 genome, encoding a non-ribosomal peptide synthetase (NRPS),
an iterative type I PKS cluster, and an arylpolyene pathway.
We modified the PKS and NRPS clusters of MYb115 by inserting the
inducible arabinose P[BAD] promoter. Thus, while induction of BGC
expression requires arabinose supplementation, no expression should be
observed in the absence of arabinose supplementation, mimicking a
deletion phenotype^[81]20. We assessed the ability of MYb115 P[BAD]sga
(MYb115 PKS cluster, Fig. [82]1A) and MYb115 P[BAD]nrpA (MYb115 NRPS
cluster, Fig. [83]1B) in an induced (+ arabinose) and non-induced
(− arabinose) state to protect C. elegans against Bt247 infection. We
found that infected C. elegans exposed to induced MYb115 P[BAD]sga
showed significantly increased survival when compared to infected worms
on MYb115 P[BAD]sga in a non-induced state (Fig. [84]1A, C and
Supplementary Data [85]1). Arabinose supplementation had no effect on
resistance of C. elegans to Bt infection on its standard laboratory
food Escherichia coli OP50 (Supplementary Data [86]1). While the PKS
gene cluster affects MYb115-mediated protection, we did not observe
significant differences in worm survival with or without arabinose
supplementation on the MYb115 P[BAD]nrpA strain (Fig. [87]1B and
Supplementary Data [88]1). We therefore focused on the P. fluorescens
MYb115 PKS gene cluster (hereafter PfSgaAB) in our subsequent analyses.
Fig. 1. MYb115 PKS cluster-derived SLs mediate protection against B.
thuringiensis infection.
[89]Fig. 1
[90]Open in a new tab
A, B Survival proportion of C. elegans N2 on P. fluorescens MYb115
P[BAD]sga (A) or MYb115 P[BAD]nrpA (B) induced with arabinose (solid
line) or in a non-induced state without arabinose supplementation
(dashed line) 24 h post infection with B. thuringiensis Bt247. Bt407
was used as a non-pathogenic control. The data shown is representative
of three independent runs with four replicates each (see Supplementary
Data [91]1). C LC-MS chromatogram of MYb115 P[BAD]sga extracts from
cultures with (solid line) and without (dashed line) arabinose
supplementation. Upon induction with arabinose, three compounds (1–3)
are produced. D Schematic representation of the MYb115 PKS gene cluster
and its modifications. Polyketide synthase (PKS) SgaA, alpha-oxoamine
synthase (AOS) SgaB and inducible arabinose promoter (P[BAD]). E
Survival proportion of N2 on E. coli OP50, MYb115, or MYb115 knockout
mutants. C. elegans on both tested mutants MYb115 ΔsgaA, and MYb115
ΔsgaB were significantly more susceptible (p = 1.17E-09 or
p = 2.00E-16, respectively) to infection with Bt247 than worms on
wildtype MYb115. Means ± standard deviation (SD) of n = 4, are shown in
survival assays (A, B, E), n = 3 in (F). Statistical analyses were
carried out using the generalized linear model (GLM) framework with a
binomial distribution. All tests were two-sided, and p-values were
adjusted for multiple comparisons using the Bonferroni correction.
Significance is indicated as ***p < 0.001. F Survival proportion of N2
on MYb115 ΔsgaA/P[vanCC]sgaAB, which expresses SgaAB under the vanillic
acid-inducible P[vanCC] promoter on the pSEVA631 plasmid. Survival was
assessed 24 h post-infection with Bt247, comparing vanillic
acid-induced (solid line) and non-induced (dashed line) conditions
(p = 3.53E-11). G LC-MS chromatogram of MYb115 wt, ΔsgaA and ΔsgaB. H
Correlation of area under the C. elegans survival curve (AUC) and peak
intensity, representing bacterial SL abundance. Each facet represents
the correlation for a specific bacterial SL compound (1–6), with
different bacterial treatments indicated by colour. Correlations were
calculated using the two-sided Spearman method, correlation
coefficients are shown with 95% confidence intervals. Source data and
additional survival runs are provided in Supplementary Data [92]1.
We then deleted either, the PKS SgaA (MYb115 ΔsgaA), or the
alpha-oxoamine synthase (AOS) SgaB (MYb115 ΔsgaB) (Fig. [93]1D) to
confirm the requirement of PfSgaAB in MYb115-mediated protection. While
MYb115 provided significant protection against infection in C. elegans
compared to worms on E. coli OP50 (Fig. [94]1E^[95]18), protection of
worms on both MYb115 mutants was lost (Fig. [96]1E). Protection in
MYb115 ΔsgaA was restored upon expression of SgaAB from the vanillic
acid-inducible P[vanCC] promoter on a plasmid (pSEVA631) (Fig. [97]1F).
These results clearly demonstrate a role of PfSgaAB in MYb115-mediated
protection.
P. fluorescens MYb115 PKS cluster (PfSgaAB) produces long chain sphinganines
and phosphoglycerol SLs
MYb115-mediated protection against Bt247 infection depends on the PKS
cluster PfSgaAB. We next asked which natural product is produced by
PfSgaAB. Using LC-MS, we identified three compounds that are produced
in MYb115 P[BAD]sga upon induction with arabinose (Fig. [98]1C). We
subsequently established that the compounds 1–3 are also produced, but
less abundant, in MYb115, and that both MYb115 deletion mutants (MYb115
ΔsgaA, and MYb115 ΔsgaB) are not able to produce compounds 1–3
(Fig. [99]1G). MS^2 experiments revealed that compounds 1–3 show
structural similarities to commercially available long chain
sphinganines (Fig. [100]S1). We determined the molecular composition
through isotopic labelling experiments and confirmed that compounds 1–3
are very long chain sphinganines (C24, C26, C28, Fig. [101]S2A–C).
Moreover, we performed lipidomic analysis of MYb115 using
high-resolution Liquid Chromatography Tandem Mass Spectrometry
(HRES-LC-MS/MS) and found that in addition to the three sphinganines
1–3 MYb115 produces compounds 4–6, each with masses 154 Da heavier than
those of the three sphinganine derivatives (Fig. [102]S2D–F and
Supplementary Data [103]3). Since the masses of 4–6 did not match any
known lipids in the MS-DIAL LipidBlast (version 68) dataset, we used
the exact mass and different lipid headgroups to propose structures for
compounds 4–6. We conclude that compounds 4–6 are most likely
phosphoglycerol sphingolipids (PG-sphingolipids). Next, we analysed the
relative abundance of sphinganines 1–3, and PG-sphingolipids 4–6 in
MYb115 and MYb115 P[BAD]sga induced by arabinose or repressed by
glucose supplementation. While the sphinganines 1–3 were more abundant
in the induced MYb115 P[BAD]sga samples, the total abundance of
PG-sphingolipids 4 and 5 did not differ compared to MYb115 supplemented
with arabinose (Fig. [104]S2G). Thus, increase in sphinganine
production does not necessarily lead to increase in PG-sphingolipid
production.
Many BGCs are silent under typical laboratory conditions and activation
of secondary metabolic pathways can be a challenge^[105]21. Indeed, we
observed substantial variations in the protective effect of MYb115
under our standard laboratory conditions over the course of this
project. We hypothesized that the variations in the protective effect
are related to variations in SL production. To test this hypothesis, we
used MYb115 P[BAD]sga and MYb115 ΔsgaA/P[vanCC]sgaAB, in which SL
production can be activated by arabinose and vanillic acid
supplementation, respectively, and that produce SLs at much higher
levels than wildtype MYb115 (Fig. [106]S3). We harvested MYb115
P[BAD]sga and MYb115 ΔsgaA/P[vanCC]sgaAB pellets of the same bacterial
cultures, whose protective effect we then tested in survival analyses,
and visualized SL production using MALDI mass spectrometry spot
assays^[107]22. SL production indeed varied between different bacterial
cultures, even under conditions of targeted induction. Most
importantly, we found that abundances of sphinganines 1–3 and
PG-sphingolipid 4 correlate significantly with worm survival following
Bt247 infection (Fig. [108]1H and Supplementary Data [109]1), providing
further evidence that host protection is dependent on these SLs. Since
our results also demonstrate that SL production and the associated
protective effect is variable under the given laboratory conditions, we
always controlled for the protective effect in our experiments.
A proposed pathway for iT1PKS cluster-dependent SL biosynthesis
SL synthesis in bacteria and eukaryotes involves the condensation of an
amino acid (typically serine in mammals) and a fatty acid (typically
palmitate in mammals) via the serine palmitoyltransferase (SPT) enzyme
that uses pyridoxal phosphate (PLP) as cofactor for serine
decarboxylation and coupling to palmitoyl-CoA^[110]23. In the case of
MYb115, the protective SLs 1–3 and PG-sphingolipid 4, are produced by
the two-gene cluster PfsgaAB (Sphinganine biosynthesis A and B), in
which sgaA encodes a PKS and sgaB encodes an AOS with predicted
structural homology to SPTs deposited on the Protein Data Bank
([111]https://www.rcsb.org/ PDB: 2JG2; 2X8U; 3A2B; 8GUH, Fig. [112]S4).
Like its SPT homologues, PfSgaB is expected to condense fatty
acyl-thioesters with L-serine to give 3-ketodihydrosphinganine
(3-KDS)-like intermediates, which is the first committed step in SL
biosynthesis (Fig. [113]2A^[114]24,[115]25). To confirm this activity,
the gene encoding PfSgaB was codon-optimised, synthesised and cloned
into pET28a with an N-terminal poly-His tag for downstream purification
(Figs. [116]S5 and [117]S6). Recombinant expression in E. coli
BL21(DE3) resulted in yellow-tinged biomass, from which PfSgaB was
purified to homogeneity using tandem cobalt-IMAC and size-exclusion
chromatography (SEC, Fig. [118]S7). SEC analysis provided an estimated
molecular weight (MW) of 108 kDA, consistent with protein dimerization
(Fig. [119]S8). We first incubated recombinant, purified PfSgaB with
varying concentration of L-serine and observed PLP:L-serine external
aldimine formation by UV–vis spectroscopy (413 nm), with and estimated
K[d] = 1.30 ± 0.0256 mM (Figs. [120]2B and [121]S9). Following this, we
probed PfSgaB condensation activity using acyl-CoAs 7–9 as a surrogate
co-substrates (Fig. [122]2C), capturing the CoASH by-product using
5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB, Fig. [123]S10A). Using this
colorimetric assay, a clear response was obtained when PfSgaB was
incubated with both L-serine and 7, in contrast to all negative
controls (Fig. [124]S10B). Furthermore, PfSgaB shows virtually
exclusive preference for L-serine over deoxysphingolipid-forming amino
acids L-alanine and glycine (Fig. [125]2D). Similar condensation
activity with L-serine was also observed when 8 and 9 were used as
co-substrates (Fig. [126]S10C). We subsequently identified all
corresponding 3-KDS products 10–12 by LC/ESI-MS (m/z = 300.29, 328.33,
356.37, see Fig. [127]2E, F). Moreover, through isotopic labelling
experiments we could show that ^13C^15N-labelled serine is incorporated
during sphinganine biosynthesis in MYb115 in vivo (Supplementary
Data [128]2). Taken all together, this data confirms the functional
assignment of PfSgaB as a SPT and the key gateway into SL biosynthesis
in MYb115.
Fig. 2. Proposed biosynthesis of P. fluorescens MYb115 PKS cluster
PfSgaAB-derived SLs.
[129]Fig. 2
[130]Open in a new tab
A Biosynthesis scheme of MYb115-derived PG-sphingolipids 4–6. The
production of 3-ketodihydrosphinganines (KDSs) is catalysed by the
iterative PKS (iPKS) PfSgaA and PLP-dependent serine
palmitoyltransferase (SPT) PfSgaB. The reduction of KDSs to
dihydrosphinganines 1–3 is presumably catalysed by the KDS reductase
homologue PfSgaC. B PLP external aldimine formation following the
addition of up to 10 mM L-serine (L-ser), monitored by UV–vis
spectroscopy. External aldimine formation is signified by an increase
in absorbance at 413 nm. C Schematic representation of PfSgaB-catalysed
decarboxylative condensation between acyl-CoAs 7–9 and L-ser to give
3-ketodihydrosphinganines 10–12. D Relative activity of PfSgaB in the
presence of C[16]-CoA 7 and L-serine, L-alanine or glycine, determined
using the DTNB assay (412 nm). UV–vis measurements were recorded after
20 min of incubation. Error bars represent the standard deviation of
three technical replicates. All measurements were corrected for
non-specific background absorbance. E Extracted ion (EI) chromatograms
of PfSgaB-derived products 10–12, detected by LC/ESI-MS. F [M + H]^+
ions of PfSgaB-derived products 10–12, detected by LC/ESI-MS. The
theoretical m/z is shown for each product.
Furthermore, we identified a putative short chain
dehydrogenase/reductase (SDR) in the MYb115 SL BGC (locus ID:
KW062_RS19775), which shares homology with several eukaryotic
3-ketodihydrosphinganine reductases (KDSR, see Figs. [131]S11 and
[132]12). KDSR homologues are also found in fungal BGCs that produce
SL-like, PKS-derived mycotoxins such as fumonisin (FUM13, Uniprot:
[133]W7LL82)^[134]26 and sphingofungin (SphF, Uniprot:
[135]B0XZV2)^[136]27. KDSR catalyses the reduction of 3-KDS to
dihydrosphinganine (DHS)^[137]28,[138]29; whilst this step is
ubiquitous in eukaryotic SL biosynthesis, it is unusual in bacterial SL
pathways outside of Bacteroides and Prevotella species^[139]30,[140]31.
Taken together with gene context, we propose that this enzyme,
hereafter named PfSgaC, mediates 3-KDS reduction in MYb115. The
inclusion of this eukaryotic-like step further distinguishes this BGC
from canonical bacterial SL biosynthesis.
Homologous PKS clusters are present across diverse bacterial genera
Iterative PKS were originally found in fungi and only rarely in
bacteria^[141]10. However, a large number of bacterial iterative PKS
were identified more recently^[142]32. While only a few bacterial
iterative PKS and their products have been studied, our work is to our
knowledge the first example of a PKS cluster shown to be involved in SL
biosynthesis and also the first description of a P. fluorescens isolate
as SL producer. We explored the distribution of the two-gene PfsgaAB in
bacteria listed in the NR NCBI database and found 6,101 homologous
putative PKS clusters (Supplementary Data [143]4). Interestingly, the
homologous PKS clusters were present in bacteria that are known to be
closely associated with hosts, including human pathogens and
opportunistic pathogens (Fig. [144]3A). When we analysed the
distribution of the target BGC class at the genus level, we found that
the putative PKS cluster is dominantly distributed in Burkholderia
(Fig. [145]3B). Interestingly, Burkholderia pseudomallei K96243 has
previously been shown to produce sphingosine-1-phosphate
lyases^[146]33,[147]34, but like Pseudomonas, Burkholderia is not yet a
known SL producer. The fact that we found the potential PKS cluster
SgaAB in Burkholderia suggests that they may be able to produce
sphingosine-1-phosphate and not just degrade it.
Fig. 3. Distribution of P. fluorescens MYb115 PKS cluster SgaAB homologues in
bacteria.
[148]Fig. 3
[149]Open in a new tab
The monomodular PKS (KW062_RS19805) and the alpha-oxoamine synthase
(KW062_RS19800) in P. fluorescens MYb115 ([150]NZ_CP078138) were
searched against the NR NCBI database
([151]https://www.ncbi.nlm.nih.gov/) using cblaster (1.8.1)^[152]89. A
Five representative PKS cluster SgaAB homologs from various bacterial
genera aligned and visualised using clinker^[153]90. B Total
distribution of 6101 PKS cluster SgaAB homologs across different
bacterial genera. The width of each box represents the percentage of
all identified PKS cluster SgaAB homologs, found in each bacterial
genus are provided as source data in Supplementary Data [154]4.
MYb115-derived SLs modulate the expression of genes related to pathogen
defence and contribute to intestinal barrier protection
In a first step towards exploring the function of microbiota-derived
SLs in mediating the interaction with the host, we tested whether
MYb115-produced SLs affect the ability of MYb115 to colonise the host
or modulate host feeding behaviour. We did not observe a difference in
host colonisation between MYb115 and MYb115 ΔsgaA (Fig. [155]S13A and
Supplementary Data [156]5), nor did we see differences in C. elegans
feeding behaviour on MYb115 and MYb115 ΔsgaA (Fig. [157]S13B and
Supplementary Data [158]5).
MYb115 protects C. elegans against Bt infection without directly
inhibiting pathogen growth, likely through an indirect, host-dependent
mechanism^[159]18. When grown on MYb115 C. elegans is also protected
against infection with another Bt strain, Bt679, that produces distinct
pore-forming toxins (PFTs) (Fig. S[160]14^[161]17,[162]18) and this
protection also depends on bacterial SL production (Fig. [163]S14 and
Supplementary Data [164]6). We thus considered that activation of
general host defence mechanisms may contribute to MYb115-mediated
protection and performed gene expression profiling of 1-day adult worms
on either protective MYb115 or non-protective MYb115 ΔsgaA in the
absence and presence of pathogenic Bt247 (Fig. [165]4A and
Supplementary Data [166]7). We did not observe any genes differentially
regulated between worms on SL-producing MYb115 and worms on the MYb115
ΔsgaA mutant when using an adjusted p-value cutoff of 0.05. This may
indicate that MYb115-derived SLs do not strongly affect C. elegans on
the transcript level, but more strongly influence the host on the
proteome or metabolome level. Using a less stringent cutoff
(non-adjusted p-value < 0.01), we nevertheless identified 122
differentially expressed (DE) genes between the two treatments in the
absence of Bt247 (23 genes were down regulated and 99 genes upregulated
in worms on MYb115 ΔsgaA (Supplementary Data [167]7) and 48 DE genes in
the presence of Bt247 (22 genes were down regulated and 26 genes
upregulated in worms on MYb115 ΔsgaA (Supplementary Data [168]7). Genes
that are related to C. elegans pathogen defence were indeed enriched in
both gene sets, including targets of known pathogen defence pathways,
such as the p38 and JNK-like MAPK pathways (Fig. [169]4B, C and
Supplementary Data [170]7). However, we did not find any evidence of
the involvement of the p38 MAPK and the JNK MAPK KGB-1 in decreasing
MYb115-mediated protection against Bt247 (Fig. [171]4B, C and
Supplementary Data [172]8).
Fig. 4. MYb115-mediated protection is independent of known C. elegans
pathogen defense pathways.
[173]Fig. 4
[174]Open in a new tab
A Transcriptional response of C. elegans to MYb115-derived SLs.
Enrichment analysis of genes differentially regulated between worms
exposed to SL-producing MYb115 and worms exposed to non-SL producing
MYb115 ΔsgaA in the presence of pathogenic Bt247 (Supplementary
Data [175]7). B, C Survival of p38 and JNK MAPK pathway mutants.
Means ± standard deviation (SD) of n = 4 (p38 MAPK pathway (B)); n = 3
(kbg-1(ums3) survival (C)) are shown in all survival assays.
Statistical analyses were carried out using the generalized linear
model (GLM) framework with a binomial distribution. All tests were
two-sided, and p-values were adjusted for multiple comparisons using
the Bonferroni correction. Significance is indicated as, ***p < 0.001,
**p < 0.01, *p < 0.05. All p-values can be found in Supplementary
Data [176]8. nsy-1(ag3) and sek-1(km4) share the same N2 control since
the experiment was conducted in parallel, with statistical analysis
adjusted accordingly, as highlighted in Supplementary Data [177]8.
Source data are provided in Supplementary Data [178]8.
The damage caused by Bt PFTs leads to loss of intestinal barrier
function and we have previously shown that MYb115 limits Bt-induced
damage to the intestinal epithelium^[179]18. Here, we used a C. elegans
strain expressing PGP-1::GFP, a labelled ATP binding-cassette
transporter, whose expression is restricted to the apical plasma
membrane of the intestinal epithelium^[180]35, to test if
MYb115-derived SLs are involved in mitigating Bt-induced damage.
Indeed, in Bt247 infected worms on arabinose-induced MYb115 P[BAD]sga,
we found a clear reduction of the relocalisation of the PGP-1::GFP
marker to intracellular vesicles (Fig. [181]5 and Supplementary
Data [182]9), which is regarded as a response to membrane damage caused
by PFTs^[183]36. In contrast, there was no difference in the numbers of
intracellular vesicles between infected worms on E. coli OP50 and the
MYb115 ΔsgaA mutant (Fig. [184]5), suggesting that the SLs produced by
MYb115 P[BAD]sga contribute to protection of the intestinal barrier
following Bt infection.
Fig. 5. MYb115-derived SL contribute to intestinal barrier protection.
[185]Fig. 5
[186]Open in a new tab
Visualisation and quantification of vesicular structures following
Bt247 infection. Worms were raised on either E. coli OP50, P.
fluorescens MYb115 ΔsgaA or P. fluorescens MYb115 P[BAD]sga + arabinose
for 72 h and then infected with Bt247. Confocal images of PGP-1::GFP
were captured 4 h after exposure to Bt247 mixed with either OP50,
MYb115 ΔsgaA or MYb115 P[BAD]sga + arabinose. For each worm all
PGP-1::GFP positive vesicles were scored and categorised into either of
the three groups “0 vesicles”, “
[MATH: ≤ :MATH]
10 vesicles” or “
[MATH: > :MATH]
10 vesicles”. Representative images of worms are shown, highlighting
magnified regions of PGP-1::GFP positive vesicles (indicated by white
arrows) following Bt247 infection. Scale bar: 100 µm. The proportions
of worms in each category are displayed as stacked bar plots for each
replicate. Population size varied between 14 and 25 individuals
(n = 3). Source data are provided in Supplementary Data [187]9.
MYb115-derived SLs alter host fatty acid and SL metabolism
Mouse lipid metabolism was previously shown to be affected by gut
microbiota-derived SLs^[188]37. Moreover, in a C. elegans Parkinson
disease model, the probiotic B. subtilis strain PXN21 protects the host
against protein aggregation by modulating SL metabolism^[189]38. Thus,
we hypothesised that MYb115-derived SLs impact host metabolism. To test
this hypothesis, we integrated the transcriptomic data into the
iCEL1314 genome-scale metabolic model of C. elegans^[190]39 to create
context-specific models, simulating metabolite flow through the C.
elegans reactions network under specific treatment conditions (see
methods). Statistical analysis of the reaction fluxes resulted in 16
(Bt247 +) and 16 (Bt247 −) significant differences when comparing
MYb115 ΔsgaA and MYb115 worms (Supplementary Data [191]10). Through a
pathway enrichment analysis of the significant reactions against a
background of our model pathways, we found that in the absence of
Bt247, animals colonised by MYb115 or MYb115 ΔsgaA varied in the
activity of multiple pathways linked with SL precursor production, such
as monomethyl branched chain fatty acid biosynthesis, as well as SL
metabolism itself (Fig. [192]6A and Supplementary Data [193]10). In the
presence of Bt247, propanoate metabolism was most strongly affected
(Fig. [194]S15A and Supplementary Data [195]10). Under both infection
and non-infection conditions, the valine, leucine, and isoleucine
degradation pathway were significantly enriched. This pathway degrades
branched-chain amino acids and is directly connected with propanoate
metabolism that provides components for the synthesis of the C15iso
fatty acid, which is the precursor for SLs in C. elegans^[196]40. We
also focused on SL metabolism directly: Flux variability
analysis^[197]41 revealed a significant difference in upper bound
values for the SL metabolism reactions in worms infected with Bt247 on
MYb115 versus MYb115 ΔsgaA (t-test p-value < 0.001). Among those
reactions, six reactions that all have ceramide as a substrate or
product had the strongest changes (Fig. [198]S15B and Supplementary
Data [199]10). Overall, these findings suggest that worms colonised by
MYb115 versus MYb115 ΔsgaA have a significantly reduced capacity to
generate SLs.
Fig. 6. MYb115-derived SLs modulate host SL metabolism.
[200]Fig. 6
[201]Open in a new tab
A Enriched metabolic pathways if the C. elegans (iCEL1314) metabolic
model were identified following a comparison of worm models integrated
with transcriptome data from worms treated with MYb115 with worms
treated with MYb115 ΔsgaA. Significant reactions obtained by
calculating two-sided p-values from linear regression models (data ~
treatment) of FVA centres and OFD data layers were used for Flux
Enrichment Analaysis (FEA) against the background of all reactions
within the iCEL1314 C. elegans metabolic model. Benjamini-Hochberg was
applied only for FEA output due to high pathway/reaction collinearity.
Source data are provided in Supplementary Data [202]10. B Reduced SL
contents in worms exposed to MYb115 compared to worms exposed to MYb115
ΔsgaA. The heatmap shows the differences in ratio of detected SLs
between the mean of MYb115 ΔsgaA and the mean of MYb115. The boxplot
shows the difference in ratio of Sphingomyelin (t43:1) in worms exposed
to MYb115 ΔsgaA and MYb115, all remaining boxplots can be found in
Fig. [203]S16. Boxplots display the median (line), the first and third
quartiles (box edges), and whiskers extending to the smallest and
largest values within 1.5× the interquartile range. Points beyond this
range are shown as outliers. Statistical analysis was done with a
two-sided Welch’s t- test (n = 5), * p-value < 0.05, ** p-value < 0.01.
Dihydroceramides (DhCer), Ceramides (Cer), Sphingomyelins (SM),
Hexosylceramides (HexCer), with hydroxylated fatty acyls (t) or
non-hydroxylated fatty acyls (d), Hexosylceramides with
phytosphingosine base and hydroxylated fatty acyls (HexCer(q)),
monomethyl phosphoethanolamine glucosylceramide (mmPEGC(q)). Source
data are provided in Supplementary Data [204]11.
MYb115-derived SLs interfere with C. elegans complex SLs
The metabolic network analysis revealed that SL metabolism reactions
show differential activity between MYb115 and MYb115 ΔsgaA. To confirm
that MYb115-derived SLs affect C. elegans SL metabolism, we performed
lipidomic profiling of C. elegans exposed to MYb115 or MYb115 ΔsgaA. We
identified C. elegans SLs by manual interpretation of MS^1 and MS^2
data and used SLs that have previously been described in C. elegans
containing a C17iso-branched chain sphingoid base and different length
of N-Acyl chains as input^[205]42 (Supplementary Data [206]11). Since
the employed analytical method cannot separate between different
hexoses attached to the SL, they were annotated as hexosylceramides
(HexCers), which showed the neutral loss of 162.052275 Da.
Monomethylated phosphoethanolamine glucosylceramides (mmPEGCs), a class
of C. elegans phosphorylated glycosphingolipids, were identified based
on fragments as previously described^[207]43,[208]44.
We were not able to detect MYb115-derived sphinganine in worms on
MYb115. Likewise, we did not detect any SLs based on sphinganines
produced by MYb115. A possible explanation is that bacterial
sphinganine concentrations in worms are below the detection limit.
However, we found different complex host SLs based on the
C17iso-branchend chain sphingoid base typical for C. elegans with
N-acyl sides of length 16–26 without or with hydroxylation. In addition
to previously established SLs, we identified HexCer with an additional
hydroxyl group instead of the double bond in the sphingoid base. In
total, we identified 40 C. elegans SLs from different SL classes. We
did not observe a difference in C. elegans C17iso sphinganine or C17iso
sphingosine, but in certain dihydroceramide (DhCer) and ceramide (Cer)
species between worms on MYb115 or MYb115 ΔsgaA. Also, complex SLs
downstream of ceramides, i.e., sphingomyelins (SMs) and HexCers were
increased in worms on MYb115 ΔsgaA, and some even significantly
increased (Fig. [209]6B). Individual SL profiles are shown in
Fig. [210]S16. Most of the significant changes occurred at the lower or
upper end of the detected N-acyl chain length. No changes occurred in
SLs containing an N-acyl of 22 or 24 carbon length. However, the series
of SM(d33:1, d35:1, d37:1), showed a consistent and significant
increase. Additionally, SM(t37:1) and SM(t43:1) as well as the
corresponding HexCer(t37:1) and SM(t37:1) increased significantly.
Notably, we found the highest fold-changes between MYb115 and MYb115
ΔsgaA-exposed worms for mmPEGC. However, changes were not significant
and so far, the biosynthesis pathway of mmPEGCs is unknown.
Together, our data suggest that MYb115-derived SLs interfere with C.
elegans SL metabolism mainly at the conversion of dihydroceramide and
ceramide to sphingomyelins and hexosylceramides.
Modifications in C. elegans SL metabolism affect defence against Bt247
infection
Since MYb115 affects host SL metabolism and protects the worm against
Bt infection, we next asked whether alterations in nematode SL
metabolism affect C. elegans survival following Bt infection. We
performed survival experiments using several C. elegans mutants of SL
metabolism enzymes (Figs. [211]7A–C and [212]S17 and Supplementary
Data [213]12). We assessed the general involvement of SL metabolism in
the response to Bt infection in the presence of the non-protective lab
food E. coli OP50. We found that mutants of the C. elegans serine
palmitoyl transferases sptl-1(ok1693) and sptl-3(ok1927), which
catalyse the de novo synthesis of the C17iso sphingoid base, and the
ceramide synthase mutants hyl-1(ok976) and hyl-2(ok1766)) showed
increased survival on Bt in comparison to wildtype N2 worms
(Fig. [214]7C), whereas the survival phenotype of two ceramide
metabolic gene mutants, namely cgt-1(ok1045) and cerk-1(ok1252), was
variable (Fig. [215]7C). cgt-1 encodes one of three C. elegans ceramide
glucosyltransferases that generate glucosylceramides (GlcCers). cerk-1
is a predicted ceramide kinase that catalyses the phosphorylation of
ceramide to form ceramide-1-phosphate (C1P). In contrast, the
sms-1(ok2399) mutant was clearly more susceptible to Bt247 infection
than wildtype worms. sms-1 encodes a C. elegans sphingomyelin synthase
that catalyses the synthesis of sphingomyelin from ceramide.
Accordingly, the asm-3(ok1744) mutant, which lacks the enzyme that
breaks down sphingomyelin to ceramide, showed increased resistance to
Bt247 (Fig. [216]7C). Notably, the ceramidase mutants asah-1(tm495) and
asah-2(tm609) were also significantly more susceptible to Bt247
infection than the C. elegans control (Fig. [217]7C). asah-1 encodes a
C. elegans acid ceramidase that converts ceramide to
C17iso-sphingosine, which is subsequently phosphorylated by the
sphingosine kinase SPHK-1 to C17iso-sphingosine-1-phosphate^[218]45.
Together, these results suggest that inhibition of de novo synthesis of
ceramide and inhibition of the conversion of ceramide to GlcCer or C1P
increases survival of C. elegans infected with Bt247, while inhibition
of the conversion of ceramide to sphingomyelin or sphingosine decreases
survival of Bt247-infected animals.
Fig. 7. Modulations in C. elegans SL metabolism affect survival after Bt247
infection.
[219]Fig. 7
[220]Open in a new tab
A Overview of SL metabolism in C. elegans. C. elegans produces
sphingoid bases which are derived from a C17 iso-branched fatty acid
and are thus structurally distinct from those of other animals with
mainly straight-chain C18 bases^[221]40. C. elegans SLs consist of a
sphingoid base backbone derived from C15iso-CoA and serine, which is
N-acylated with fatty acids of different lengths as well as different
functional groups at the terminal hydroxyl group. Dihydroceramides
(DhCers) are formed from C17iso sphinganine and fatty acids or
2-hydroxy fatty acids. Desaturation at the 4th carbon yields ceramides
(Cers), which are the precursors of complex SLs such as sphingomyelin
(SM) and glucosylceramide (HexCer). Mutants of SL metabolism genes in
bold were tested in survival assays shown in (C). B Schematic survival
comparing N2 wildtype (solid line) versus mutant strains (dashed
lines), the difference of the area under the survival curve (AUC) is
shaded in brown when the mutants are more susceptible to the infection
than the control and in green when the mutants are more resistant to
the infection. C Heatmap represents the ΔAUC of the survival of the C.
elegans SL metabolism mutants versus average of the wildtype N2 strain.
Each box represents an independent experiment, consisting of three to
four technical replicates (individual bars). The intensity of the bar
colour reflects the overall summary across all experiments, while the
statistical analysis was performed separately for each experiment.
Statistical analyses were carried out using the GLM framework with a
binomial distribution. All tests were two-sided, and p-values were
adjusted for multiple comparisons using the Bonferroni correction.
Significance is indicated as *p < 0.05, **p < 0.01, ***p < 0.001. Each
individual survival curve can be found in Fig. [222]S17A, B. Source
data and exact p-values are provided in Supplementary Data [223]12.
To elucidate the role of specific SLs in defence against Bt infection
we supplemented Bt infected worms with the commercially available SLs
ceramide, sphingomyelin, and sphingosine-1-phosphate. We found that
supplementation with C18 and C20 ceramide significantly improved
survival rates, while C22 ceramide, C16 sphingomyelin, C18
sphingomyelin, d-sphingosine, and S1P did not affect survival
(Fig. [224]S18 and Supplementary Data [225]13). These findings and the
phenotypic differences between the ceramide metabolic gene mutants
imply that the inhibition of ceramide metabolism (and the associated
increase in ceramide content) is not the only factor determining
susceptibility to infection.
We additionally assessed C. elegans SL metabolism mutant survival on
the protective microbiota isolate MYb115. MYb115 and the inhibition of
de novo synthesis of ceramide or the breakdown of sphingomyelin to
ceramide protect worms against infection with Bt247. Therefore, we did
not expect to see an effect of MYb115 on the increased survival
phenotype of the sptl-1, -3, hyl-1, -2, and asm-3 mutants. Our results
are fully consistent with these expectations, the mutants were more
resistant to Bt infection also on MYb115 (Fig. [226]7C). However, both
ceramidase mutants asah-1(tm495) and asah-2(tm609), which were more
susceptible to Bt247 infection on E. coli OP50, were as susceptible as
and even more resistant than wildtype worms on MYb115, respectively
(Fig. [227]7C). Notably, MYb115 also ameliorated the susceptibility
phenotype of the sms-1(ok2399) mutant (Figs. [228]7C and [229]S17).
These data indicate that MYb115 interacts with host SL metabolism at
least at the conversion of ceramide to sphingomyelin and
C17iso-sphingosine.
MYb115-mediated protection is independent of the C. elegans mitochondrial
surveillance response and a Bt toxin glycosphingolipid receptor
Our data suggest a mechanistic link between microbiota-mediated
alterations in host SL metabolism and protection against Bt infection.
As a step towards exploring the underlying mechanisms, we tested two
potential links between SLs and C. elegans defence against Bt
infection. First, we explored the possible involvement of the
mitochondrial surveillance response, which requires ceramide
biosynthesis^[230]46 and second we explored the role of complex
glycosphingolipids that are receptors of the Bt Cry toxin
Cry5B^[231]47. Bt247 infection did not induce the expression of the
mitochondrial stress-induced hsp-6p::gfp reporter, neither did MYb115
(Fig. [232]S19A), indicating that mitochondrial surveillance is not
involved in MYb115-mediated protection against Bt247 infection. Also,
the Bt-toxin resistant (bre) mutants bre-2(ye31) and bre-3(ye26), which
are defective in the biosynthesis of the Cry5B glycosphingolipid
receptor^[233]47, are susceptible to Bt247 infection and still
protected by MYb115 (Fig. [234]S19B and Supplementary Data [235]14). We
thus confirm previous results^[236]48 and can exclude an involvement of
the bre genes in MYb115-mediated protection against Bt247.
Discussion
Understanding microbiota-host interactions at the level of the
molecular mechanism requires the identification of individual
microbiota-derived molecules and their associated biological activities
that mediate the interaction. In this study we demonstrate that P.
fluorescens MYb115-mediated host protection^[237]18, depends on
bacterial-derived SLs. We show that MYb115 produces protective SLs by a
BGC encoding an iterative PKS and an AOS with SPT activity. This
finding is important since eukaryotes and all currently known
SL-producing bacteria depend on the serine palmitoyl transferase (SPT)
enzyme, which catalyses the initial step in the de novo synthesis of
ceramides, for SL production as primary metabolites. Indeed, the SPT
gene is conserved between eukaryotes and prokaryotes and its presence
in bacterial genomes has been used as an indication of SL production.
While SL production is ubiquitous in eukaryotes, it is thought to be
restricted to few bacterial phyla. Known SL-producing bacteria include
the Bacteroidetes and Chlorobi phylum, and a subset of Alpha- and
Delta-Proteobacteria^[238]49. More recently, two additional key enzymes
required for bacterial ceramide synthesis have been identified,
bacterial ceramide synthase and ceramide reductase^[239]31.
Phylogenetic analysis of the three bacterial ceramide synthetic genes
has identified a wider range of Gram-negative bacteria, as well as
several Gram-positive Actinobacteria with the potential to produce
SLs^[240]31. However, our finding that P. fluorescens MYb115 produces
SLs by the BGC-encoded PKS/AOS PfSgaAB, was previously unknown and
therefore indicates that there are non-canonical ways of producing SLs
as secondary metabolites in bacteria. Moreover, our analysis of the
distribution of PfSgaAB in bacteria revealed that homologous putative
PKS clusters are present in bacteria that are so far unknown SL
producers. This finding strongly suggests that PKS cluster-dependent
biosynthesis of SLs is prevalent across bacteria.
By comparing the C. elegans transcriptome response to MYb115 with the
response to the MYb115 PKS mutant in a metabolic network analysis, we
observed an effect of MYb115-derived SLs on host fatty acid and SL
metabolism. Our C. elegans lipidomic profiling corroborated the
transcriptomic data, providing evidence that MYb115-derived SLs alter
C. elegans SL metabolism, resulting in the reduction of certain complex
SL species. A similar effect of gut microbiota-derived SLs on host
lipid metabolism was previously observed in mice: Bacteroides
thetaiotaomicron-derived SLs reduce de novo SL production and increase
ceramide levels in the liver^[241]37. Also, B. thetaiotaomicron-derived
SLs alter host fatty acid and SL metabolism and ameliorate hepatic
lipid accumulation in a mouse model of hepatic steatosis^[242]50. In
humans, bacterial SL production correlates with decreased host-produced
SL abundance in the intestine and is critical for maintaining
intestinal homeostasis^[243]51. Thus, interference with host SL
metabolism may be a general effect of bacterial-derived SLs.
What role do MYb115-derived SLs play in host protection against Bt? The
current study reveals that MYb115-derived SLs protect the C. elegans
intestinal barrier, affect the activation of pathogen defence genes,
and affect host fatty acid and SL metabolism. Consistent with an
important role of host SLs in C. elegans defence, earlier studies have
provided evidence for the involvement of SL metabolism in the host
response to infection with Pseudomonas aeruginosa and Enterococcus
faecalis^[244]52,[245]53. We previously described an association
between modulations in fatty acid and SL metabolism and increased
tolerance to Bt infection^[246]48. In line with this, we here
demonstrate that modulations in SL metabolism strongly affect survival
of infected animals. We do, however, not yet understand how exactly
susceptibility to Bt infection is affected by SL modifications. Our
functional genetic analysis of C. elegans SL metabolism enzymes shows
that inhibition of de novo synthesis of ceramide and inhibition of the
conversion of ceramide to glucosylceramides or ceramide-1-phosphate
increases survival of C. elegans infected with Bt247, while inhibition
of the conversion of ceramide to sphingomyelin or sphingosine decreases
survival of Bt247-infected animals. Supplementation with C18 and C20
ceramide increased survival after Bt infection. These results and the
phenotypic differences between the ceramide metabolic gene mutants
imply that the enhanced susceptibility to infection is influenced yet
not exclusively caused by the inhibition of ceramide metabolism (and
the associated increase in ceramide content) in these mutant
backgrounds. The enzymes responsible for SL production and turnover
comprise a complex metabolic network that gives rise to numerous
bioactive molecules, which participate in highly complex and
interconnected pathways influencing a multitude of physiological
processes^[247]54,[248]55. Also, SL metabolism shares common substrates
with other metabolic routes and is, for example, highly connected to
other lipid metabolic networks. Consequently, imbalances in SL
metabolism in a mutant may have far-reaching consequences for host
physiology.
MYb115 interacts with host SL metabolism at least at the conversion of
ceramide to sphingomyelin and sphingosine, since the susceptibility
phenotypes of the respective ceramide metabolic gene mutants are
ameliorated or even abrogated in MYb115-treated animals, respectively.
However, the effect of MYb115 on the phenotype of a SL metabolism
mutant (increased survival after Bt infection) may not be directly
linked to its effect on wildtype worms (decrease in sphingomyelin and
other SLs). The conclusions we can draw from our data are that
SL-producing MYb115 decreases certain host SL species, including
sphingomyelin species, in comparison to non-SL-producing MYb115 and
that MYb115 ameliorates the survival phenotype of C. elegans ceramide
metabolic gene mutants following Bt infection. Indeed, we cannot
exclude that this effect is indirect or due to other effects of MYb115
on the host.
SLs are not only required for the integrity of cellular membranes, but
can also act as bioactive signalling molecules involved in regulation
of a myriad of cell activities, including pathogen and stress defence
pathways^[249]54. For example, many bacterial pathogens, including Bt,
produce virulence factors that target and damage
mitochondria^[250]56,[251]57. A C. elegans surveillance pathway, which
detects mitochondrial defects and activates xenobiotic-detoxification
and pathogen defence genes, requires ceramide biosynthesis^[252]46.
Since we, however, did not find any evidence of mitochondrial
surveillance activation by Bt247 infection or MYb115 (Fig. [253]S19A),
it is unlikely that this defence pathway is involved in MYb115-mediated
protection. Also, in C. elegans, glucosylceramide deficiency was linked
to an increase in autophagy^[254]58,[255]59, which plays an important
role in cellular defence after attack by certain Bt PFTs^[256]60.
Notably, glucosylceramides serve as a source for the synthesis of
complex glycosphingolipids. In C. elegans, the BRE proteins BRE-2,
BRE-3, BRE-4, and BRE-5 are required for further glucosylation of
glucosylceramide, leading to complex glycosphingolipids that are
receptors of the B. thuringiensis Cry toxin Cry5B^[257]47. However,
Bt247 only expresses the unique toxin App6Ba^[258]61, which belongs to
the PFT class of alpha helical pesticidal proteins^[259]62. These
proteins are unrelated to Cry5B at the level of their primary sequences
and structure^[260]63. Indeed, we could previously exclude an
involvement of the bre genes in C. elegans defence against Bt247, given
that bre mutants are susceptible to Bt247 infection (Fig.
[261]S19B^[262]48) and did not find evidence of an effect on
MYb115-mediated protection (Fig. [263]S19B). Still, MYb115-mediated
interference with SL metabolism might affect membrane organisation and
dynamics, as well as vesicular transport, which in turn might affect
other membrane-associated Bt toxin receptors through modifying their
localisation in the plasma membrane. C. elegans is thus an ideal
experimental system to study the downstream impact of
microbiota-derived SLs in the context of pathogen protection, an area
that is still largely unexplored^[264]64.
Methods
C. elegans strains and growth conditions
The wildtype C. elegans strain N2 (Bristol)^[265]65 and all SL mutant
strains were purchased as indicated in Table [266]1. Worms were grown
and maintained on nematode growth medium seeded with the E. coli strain
OP50 at 20 °C, according to the routine maintenance protocol^[267]66.
Worm populations were synchronised and incubated at 20 °C. Since we did
not cross out the mutant strains, we confirmed the mutations in all C.
elegans sphingolipid mutant strains via PCR (Fig. [268]S20). The
following primer were used for genotyping: asah-1: forward
AGTGGGTGTTCGGATTGGAGG, reverse GGTTGGTGCGGGATGACAAG; sptl-3: forward
AGCCGTGGCAAATGGAAAGTG, reverse ATGGAGTTTCTGCGCGATTGATG; cgt-1: forward
ACTTCAGCTACCACTCCTTCATCAC, reverse AACTTTCCTTTCGATTCCTGGACC; asah-2:
forward CGCCGAAGTGCTTGACGTAC, reverse CCAACATTGGCGCGAGTAAGC; sms-1:
forward TGGTTGCGTTTCTGATGCTCG, reverse TGAGACCGAGCCCGAACATG; for
sptl-1, asm-3, hyl-1, hyl-2 and cerk-1 already published primers were
used^[269]46 (Supplementary Data [270]15).
Table 1.
Worm strains used in this study
Worm strain Genotype Origin
N2 CGC
RB1036 hyl-1(ok976) CGC
RB1498 hyl-2(ok1766) CGC
RB1487 asm-3(ok1744) CGC
RB1465 sptl-1(ok1693) CGC
RB1579 sptl-3(ok1927) CGC
RB1854 sms-1(ok2399) CGC
FX00495 asah-1(tm495) NBRP Tokyo Japan
FX00609 asah-2(tm609) NBRP Tokyo Japan
RB1203 cerk-1(ok1252) CGC
VC693 cgt-1(ok1045) CGC
GK70 dkls37[act-5p::GFP:pgp-1] ^[271]35
FX03036 tir-1(tm3036) NBRP Tokyo Japan
AU3 nsy-1(ag3) CGC
KU4 sek-1(km4) Ewbank Lab
KU25 pmk-1(km25) CGC
KB3 kgb-1(ums3) CGC
SJ4100 zcIs13 [hsp-6p::GFP + lin-15(+)] CGC
HY494 bre-2(ye31) Aroian lab
HY483 bre-3(ye26) Aroian lab
[272]Open in a new tab
Bacterial strain and growth conditions
The standard laboratory food source E. coli OP50 was previously
obtained from the CGC. The natural microbiota isolate P. fluorescens
MYb115 (NCBI Reference Sequence: [273]NZ_CP078138.1) isolated from the
natural C. elegans strain MY379 was used^[274]16.
The promoter-exchange strain MYb115 P[BAD]sga for targeted
in-/activation of the sgaAB BGC was generated via insertion of the
inducible P[BAD] promoter upstream of the BGC following an established
protocol^[275]20. The resulting plasmid (pCEP_kan_sgaA) was transformed
into the conjugation host E. coli ST18 via electroporation and
introduced into MYb115 via conjugation^[276]67. The promoter was
induced by adding 0.02% (w/v) arabinose (ara) to the culture medium and
repressed by adding 0.05% glucose (glc) to the growth medium. Deletions
of the single genes sgaA and sgaB were carried out following a
previously established protocol based on conjugation and homologous
recombination^[277]67,[278]68. Briefly, fragments upstream and
downstream of the target gene were amplified by PCR and assembled into
a plasmid using the pEB17 vector^[279]69. The resulting plasmids
(pEB17_kan_ΔsgaA and pEB17_kan_ΔsgaB) were subsequently transformed
into the conjugation host E. coli via electroporation and the plasmid
was introduced into MYb115 via conjugation^[280]67, sequences are shown
in Supplementary Data [281]16. For the complementation of MYb115 ΔsgaA
and MYb115 ΔsgaB we conducted a series of experiments, inserting the
vanillic acid-inducible P[vanCC] promoter upstream of sgaA or the
complete sgaAB BGC on the plasmid pSEVA631, which was then introduced
into the MYb115 mutants. In detail: genomic DNA (gDNA) of MYb115 was
isolated via Monarch® Genomic DNA Purification Kit (NEB) and used as
the template for PCR amplification. The corresponding gene fragments of
sgaA and sgaAB, together with the pSEVA631
([282]https://seva-plasmids.com/find-your-plasmid/) plasmid backbone
with overhangs suitable for Gibson cloning, were amplified using Q5®
High-Fidelity DNA Polymerase (NEB) and then purified by gel extraction
using the Monarch® DNA Gel Extraction Kit (NEB). The following primers
are used for PCR amplification: sgaA forward
CTAGAGAAAGAGGGGAAATACTAGTTGACAAAGCGTAGACAGGTAG, sgaA_reverse
CAGGGTTTTCCCAGTCACGACTCACTCAATCAAACGGTTAGGTG; sgaAB_forward
TTGACAAAGCGTAGACAGGTAG, sgaAB_reverse
CAGGGTTTTCCCAGTCACGACTCACCCAATCTTCGCCAATTC; pSEVA631_backbone_forward
GTCGTGACTGGGAAAACCCT, pSEVA631_backbone_reverse
CTAGTATTTCCCCTCTTTCTCTAGT. Gibson cloning employing NEBuilder® HiFi DNA
Assembly Cloning Kit (NEB) assembled the constructed plasmids, which
were subsequently transformed by electroporation into electro competent
E. coli DH10B. Finally, plasmids were isolated by the PureYield™
Plasmid Miniprep System (Promega).
Plasmids were transformed via electroporation into electro competent
MYb115 mutants. At least three different colonies were selected for
further small-scale production analysis. Cells were cultivated
overnight in LB media with 75 µg/mL gentamicin. Afterwards, 100 µL
overnight grown culture were inoculated in 5 ml XPP medium^[283]69
containing 75 µg/mL gentamicin and 100 µM vanillic acid. The cells were
cultivated for 3 days at 28 °C and 200 rpm.
One hundred microlitres of the cultures were taken and extracted with
methanol at a 1:1 ratio by shaking 10 min at room temperature. Followed
by further diluting the mixtures 1:10 with methanol and centrifuged at
13,000 rpm for 30 min. Cleared supernatants were used for further
HPLC/MS analysis. HPLC/MS analysis was conducted on an UltiMate 3000
system (Thermo Fisher) coupled to an AmaZonX mass spectrometer (Bruker)
with an ACQUITY UPLC BEH C18 column (130 Å, 2.1 mm × 100 mm, 1.7-μm
particle size, Waters) at a flow rate of 0.4 mL/mL (5–95%
acetonitrile/water with 0.1% formic acid, vol/vol,16 min, UV detection
wavelength 190–800 nm) and an electrospray ionization (ESI) source set
to positive ionization mode.
Only the complementation that included the complete sgaAB BGC restored
SL production (detection of compounds 1 (m/z 414.4 [M + H]^+), 2 (m/z
386.4 [M + H]^+), and 3 (m/z 442.4 [M + H]^+)) by HPLC/MS analysis and
only in the MYb115 ΔsgaA mutant (Fig. [284]S21). The resulting MYb115
ΔsgaA /P[vanCC]sgaAB strain was tested in C. elegans survival assays,
for which bacteria were first grown overnight at 28 °C with shaking
(180 rpm) in 5 mL of Tryptic Soy Broth (TSB) supplemented with
gentamicin (75 µg/mL). This was followed by a three-day cultivation in
XPP medium^[285]69 containing gentamicin and 100 µM vanillic acid to
induce the P[vanCC] promoter. All other bacteria were grown on Tryptic
Soy Agar (TSA) plates at 25 °C and liquid bacterial cultures were grown
in TSB in a shaking-incubator overnight at 28 °C.
Of note: We could confirm targeted activation of SL production in
MYb115 P[BAD]sga by arabinose supplementation (Fig. [286]1C and
[287]https://metaspace2020.org/dataset/2025-02-27_13h37m58s),
suggesting that the P[BAD] promoter is not leaky in this system. In
contrast, SL production was observed in cultures of two MYb115
ΔsgaA/P[vanCC]sgaAB strains even without induction by vanillic acid
supplementation, indicating leakiness of the P[vanCC] promoter under
certain conditions. However, the addition of vanillic acid usually led
to a further significant increase in SL production
([288]https://metaspace2020.org/dataset/2025-02-27_13h37m58s). All
primer sequences can be found in Supplementary Data [289]1. For
survival assays with B. thuringiensis, we used the strain MYBt18247,
MYBt18679 (Bt247 and Bt679, respectively, our lab strains) and
Bt407^[290]70 as non-pathogenic control^[291]48,[292]71. Spore aliquots
of both strains were obtained following a previously established
protocol^[293]72 with minor modifications^[294]18.
Transcriptome analysis using RNA-seq
Roughly 500 synchronised N2 worms were raised on PFM plates inoculated
with MYb115 or MYb115 ΔsgaA (OD[600nm] of 10) from L1 to L4 stage. At
L4 stage worms were transferred to control plates or infection plates
(microbiota mixed with Bt247 spores 1:100). Transcriptomic response was
assessed 24 h post-transfer, with three independent replicates. Worms
were washed off the plates with M9-T (M9 buffer + 0.02% Triton X-100),
followed by three gravity washing steps. The worm pellets were
resuspended in 800 µL TRIzol (Thermo Fisher Scientific, Waltham, MA,
United States). Worms were broken up prior to RNA extraction by
treating the samples with four rounds of freeze-thaw cycles using
liquid nitrogen and a thermo block at 46 °C. The RNA was extracted
using Direct-zol™ RNA MicrolPrep (Zymo Research, R2062) and stored at
−80 °C.
The RNA was processed by Lexogen (Vienna, Austria) using the 3’ mRNAseq
library prep kit and sequenced on an Illumina NextSeq2000 on a P3 flow
cell in SR100 read mode. FASTQ files were checked for their quality
with MultiQC^[295]73, filtered and trimmed with cutadapt^[296]74 and
aligned to the C. elegans reference genome WBcel235 with the STAR
aligner (Spliced Transcripts Alignment to a Reference^[297]75) followed
by an assessment using RseQC^[298]76. Ultimately, HTseq-count
v0.6.0^[299]77 generated the raw gene counts. The count normalization
with the median of ratios method for sequencing depth and RNA
composition as well as the analysis for differential expression by a
generalised linear model (GLM) was performed using DESeq2^[300]78. Raw
data and processed data have been deposited in NCBI’s Gene Expression
Omnibus^[301]79 and are accessible through GEO Series accession number
[302]GSE245296.
Liquid chromatography-mass spectrometry (LC-MS) analysis of MYb115
For LC-MS analysis, 1 mL liquid culture was harvested via
centrifugation (1 min, 20 °C, 17,000 × g). The cell pellet was
resuspended in 1 mL MeOH and incubated at 30 °C for 30 min. The
resulting extract was separated from the cell debris via centrifugation
(30 min, 20 °C, 17,000 × g), diluted and submitted to LC-MS
measurements. LC-MS measurements were performed on a Dionex Ultimate
3000 (Thermo Fisher Scientific) coupled to an Impact II qToF mass
spectrometer (Bruker Daltonics). Five microlitres sample were injected
and a multistep gradient from 5 to 95% acetonitrile (ACN) with 0.1%
formic acid in water with 0.1% formic acid over 16 min with a flow rate
of 0.4 mL/min was run (0–2 min 5% ACN; 2–14 min 5–95% ACN; 14–15 min
95% ACN; 15–16 min 5% ACN) on a Acquity UPLC BEH C18 1.7 µm column
(Waters). MS data acquisition took place between minutes 1.5 and 15 of
the multistep LC gradient. The mass spectrometer was set to positive
polarity mode with a capillary voltage of 2.5 kV and a nitrogen flow
rate of 8 L/min. We compared the MS^2 data of compounds 1–3 to the MS^2
data obtained from commercially available sphinganines (sphinganine
(d18:0) and sphinganine (d20:0), Avanti Polar Lipids).
Expression and purification of PfSgaB
pET28a-PfsgaB (synthesised and cloned by Genscript) was used to
transform chemically-competent E. coli BL21 (DE3) cells via the
heat-shock method. Colonies were developed overnight on VLB-kanamycin
agar plates (50 µg mL^−1). A single colony was propagated in
LB-kanamycin media (50 mL) and incubated overnight (37 °C) with
agitation. The cells were subcultured (OD[600nm] = 0.1, 37 °C) in fresh
VLB-kanamycin media (500 mL) until mid-log phase. The cultures were
cooled to room temperature and protein expression was induced by the
addition of IPTG (0.1 mM). Protein expression proceeded overnight at
20 °C with rigorous agitation. The biomass was harvested by
centrifugation using a Fiberlite F14-6 × 250 y fixed-angle rotor
(7000 rpm, 5 min), combined into 2–5 g yellow-tinged pellets using a
Fiberlite F15-8 × 50cy fixed angle rotor (5000 rpm, 10 min) and stored
at −20 °C. When needed, cell pellets were defrosted and resuspended
(10% w/v) in ice-cold Binding/Storage buffer containing HEPES (50 mM,
pH 7.5), NaCl (250 mM), glycerol (10% v/v) and PLP (25 µM). Benzamidine
hydrochloride (1 mM) was added to the resuspension and the cells were
lysed by sonication (10 s pulse/second cooldown, 15 cycles) on ice.
Cell debris was pelleted by high-speed centrifugation using a Fiberlite
F15-8 × 50cy fixed angle rotor (13,000 rpm, 40 min, 4 °C). The
cell-free extract was collected and clarified by filtration (Millex-HP
0.45 µm polyethersulfone, Merck). PfSgaB was purified from the
cell-free extract using a HiTrap TALON Crude 1 mL column. The bound
PfSgaB protein was washed with copious Binding buffer (20 mL) and
eluted with imidazole (150 mM). Yellow fractions containing PfSgaB were
pooled and further purified using a HiLoad Superdex S200 16/600 pg
(120 mL) SEC column, using Binding/Storage buffer as mobile phase.
Purified fractions were concentrated by centrifugal concentration
(50 kDa MWCO, <4 mL). For long-term storage, aliquots of PfSgaB were
flash frozen in liquid nitrogen and stored at −80 °C.
Detection of external aldimine formation by UV–vis
A reaction mixture (1000 µL) containing purified PfSgaB (10 µM) and
L-serine (0.3125–10 mM) was prepared in a reaction buffer containing
HEPES (50 mM, pH 7.5), NaCl (250 mM) and glycerol (10% v/v). The
mixture was incubated at room temperature for 5 min and analysed using
a pre-blanked spectrophotometer (300–700 nm). External aldimine
λ[max] = 413 nm.
DTNB activity assay
A reaction mixture (200 µL) containing purified PfSgaB (5 µM), L-serine
(10 mM) and DTNB (250 µM) was initiated by the addition of acyl-CoAs
7–9 (100 µM) in a reaction buffer containing HEPES (50 mM, pH 7.5),
NaCl (250 mM) and glycerol (10% v/v). Negative controls were prepared
by the replacement of the reaction component(s) with buffer. Amino acid
specificity was determined by the replacement of L-serine with
L-alanine (10 mM) or glycine (10 mM). Absorbances were measured over
the course of 20–60 min using a BioTek Synergy HXT (28 °C, 412 nm),
configured for pathlength correction. A molar attenuation coefficient
of 14150 M^−1 cm^−1 was used to convert absorbance into concentration
using Beer’s law.
PfSgaB-catalysed 3-KDS formation using LC/ESI-MS
A reaction mixture (200 µL) containing purified PfSgaB (10 µM),
L-serine (10 mM) and acyl-CoA (100 µM) was prepared in a reaction
buffer containing HEPES (50 mM, pH 7.5), NaCl (250 mM) and glycerol
(10% v/v). The reactions were incubated at 28 °C for 18 h with rigorous
shaking. The reactions were quenched by the addition of ice-cold LC-MS
grade MeOH (200 µL) containing formic acid (2% v/v). Precipitate was
removed by microcentrifugation (13,300 rpm, 5 min). The supernatant was
sampled for LC/ESI-MS analysis in positive ion mode using a Waters
SYNAPT G2 HDMS, equipped with a Waters ACUITY Premier CSH C18 column
(1.7 µm particle size, 2.1 mm ID, 100 mm length). Analytes were
resolved using a water/ACN gradient (5–95% ACN) over 12 min. 0.1%
formic acid was used as the mobile phase modifier.
Bioinformatics
The P. fluorescens MYb115 (NCBI accession: [303]NZ_CP078138) SL BGC was
identified and annotated using antiSMASH^[304]80. Sequence homologues
were retrieved using BLASTp and UniProt. Multiple sequence alignments
were generated using ClustalOmega^[305]81 and visualised using ESPript
3.0^[306]82. AlphaFold3^[307]83 was used for predictive structural
modelling. Structural models were visualised and analysed using
ChimeraX (v1.8)^[308]84.
Labelling experiments
Bacterial cultures producing the sphinganine compounds were grown in
ISOGRO®-^13C and ISOGRO®-^15N (Sigma Aldrich) medium and subsequently
analysed by LC-MS to determine the number of carbon and nitrogen atoms,
respectively. To confirm the incorporation of serine into the
sphinganines, MYb115 P[BAD]sga cultures were grown in XPP
medium^[309]69 with addition of all proteinogenic amino acids (Carl
Roth GmbH + Co. KG, Karlsruhe) except serine. To test the
incorporation, either ^13C[3]^15N-labelled (Sigma Aldrich) serine or
regular serine (Carl Roth GmbH + Co. KG, Karlsruhe) displaying the
usual isotopic abundances were used. This should result in the
production of two isotopologues of each sphinganine. With addition of
^13C[3]^15N-labelled serine, the isotopologue that is
m[monoisotopic] + 3 should be labelled with two ^13C isotopes and one
^15N isotope, since one carbon atom is lost through the elimination of
CO[2] during the condensation. In the cultures with regular serine, the
isotopologue that is m[monoisotopic] + 3 should be labelled with three
^13C isotopes because of the higher natural abundance of ^13C compared
(1.1%) to ^15N (0.4%). The two isotopologues, ^13C[3] and ^13C[2]^15N,
were distinguished by their respective masses.
Metabolic modelling
For the metabolic model analysis, transcriptomic data was integrated
into the iCEL1314 C. elegans metabolic model using the MERGE
pipeline^[310]39 in MATLAB (version: 9.11.0.1769968 (R2021b)) using the
COBRA toolbox^[311]85) to create context-specific (CS) models of each
sample using iMAT++^[312]27. This method not only integrates
transcriptomic data into the model, but also simulates the optimal flux
distribution (OFD) for the fitted transcriptomic data, as well as
provides a flux variability analysis (FVA)^[313]41 output that
describes the minimum (lb) and maximum (ub) flux values that each
reaction can take within each CS model under the same in silico dietary
conditions. Gene categorization was performed in Python^[314]86
(version 3.10.6) using 0.7816 (mu1), 4.856 (mu2) and 8.15 (mu3), as
rare, low, and high expression category cutoffs, respectively. We would
like to point out that the iMAT++ algorithm used to integrate the
transcriptomic data into the iCEL1314 metabolic model is done on a
sample basis, therefore any statistical comparisons of gene expression
are not taken into account during this process. This means that
comparing the differences in simulation results of reactions encoded by
a certain gene and the logFC values of this gene might not directly
match in direction. This is a desirable attribute of metabolic
modelling since we can predict metabolic requirements of an organism
that are in conflict with the gene expression differences—these might
be caused by post-translational modifications or other effects.
Differences between generated metabolic models were assessed by fitting
a linear regression model (data ~ treatment) using FVA^[315]41 centres
(([ub-lb]/2)) and OFD values (equivalent to parsimonious FBA solution)
from each model. We subsequently contrasted MYb115 and MYb115 ΔsgaA,
combining unique significant reaction names (alpha = 0.01) across the
different simulation data layers (OFD and centres), and performed a
Flux Enrichment Analysis (FEA)^[316]61 using these names to obtain
significantly affected metabolic model pathways. For SL metabolism
pathway analysis, FVA was performed on all reactions, with biomass
objective minimum set to 50%. Upper bound values were grouped by
pathway, then normalized against the mean on the MYb115 flux values for
each reaction. Lower bound values were not analysed due to the
unidirectional nature of most reactions (lb = 0).
P. fluorescens MYb115 lipidomics
For the bacterial lipidomics experiment, we adapted the extraction
method from Brown et al. ^[317]51. 5 mL liquid cultures were incubated
for 24 h at 30 °C. The equivalent of 1 mL OD[600nm] of 5 was harvested
by centrifugation (1 min, 20 °C, 17,000 × g). The cell pellet was
resuspended in 0.4 mL H[2]O. 1.5 mL CHCL[3]/MeOH (1:2) were added and
the extracts were mixed by vortexing. The cell mixture was incubated at
30 °C with gentle shaking, after 18 h 1 mL CHCl[3]/H[2]O (1:1) was
added. After phase separation, the organic phase was dried using a
nitrogen evaporator and stored at −20 °C.
The relative quantification and annotation of lipids was performed by
using HRES-LC-MS/MS. The chromatographic separation was performed using
a Acquity Premier CSH C18 column (2.1 × 100 mm, 1.7 μm particle size,
VanGuard) a constant flow rate of 0.3 mL/min with mobile phase A being
10 mM ammonium formate in 6:4 ACN:water and phase B being 9:1 IPA:ACN
(Honeywell, Morristown, New Jersey, USA) at 40 °C. For the measurement,
a Thermo Scientific ID-X Orbitrap mass spectrometer was used.
Ionisation was performed using a high temperature electrospray ion
source at a static spray voltage of 3500 V (positive) and a static
spray voltage of 2800 V (negative), sheath gas at 50 (Arb), auxiliary
gas at 10 (Arb), and ion transfer tube and vaporiser at 325 and 300 °C,
respectively.
Data dependent MS^2 measurements were conducted applying an orbitrap
mass resolution of 120,000 using quadrupole isolation in a mass range
of 200–2000 and combining it with a high energy collision dissociation
(HCD). HCD was performed on the ten most abundant ions per scan with a
relative collision energy of 25%. Fragments were detected using the
orbitrap mass analyser at a predefined mass resolution of 15,000.
Dynamic exclusion with an exclusion duration of 5 s after 1 scan with a
mass tolerance of 10 ppm was used to increase coverage. For lipid
annotation, a semi-quantitative comparison of lipid abundance and
annotated peaks were integrated using Compound Discoverer 3.3 (Thermo
Scientific). The data were normalised to the maximum peak area sum of
all samples, the p-value per group ratio calculated by a one-way ANOVA
with Tukey as post-hoc test, and the p-value adjusted using
Benjamini-Hochberg correction for the false-discovery rate^[318]87. The
p-values were estimated by using the log-10 areas. The normalized peaks
were extracted and plotted using R (4.1.2) within RStudio using the
following packages: ggplot2 (3.4.0), readxl (1.4.1), grid (4.1.2),
gridExtra (2.3) and RColorBrewer (1.1-3). Metabolomics data have been
deposited to the EMBL-EBI MetaboLights database^[319]88 with the
identifier [320]MTBLS8694.
PKS distribution analysis
The monomodular PKS (KW062_RS19805) and the AOS aminotransferase
(KW062_RS19800) in P. fluorescens MYb115 ([321]NZ_CP078138) were
searched against the non-redundant (nr) National Center for
Biotechnology Information (NCBI) database using cblaster
(1.3.18)^[322]89. PKS clusters encoded by various bacterial genera were
aligned and visualised using clinker^[323]90.
C. elegans lipidomics
For lipidomic profiling, N2 worms exposed to MYb115 or MYb115 ΔsgaA
were used. Approximately 10,000 worms were raised on either of the
bacteria for 70 h until they were young adults. Excess bacteria were
removed by three gravity washing steps using M9 buffer. The buffer was
thoroughly removed, and the samples were snap-frozen in liquid
nitrogen.
Extraction and analysis of lipids were performed as described
previously^[324]91. Worm pellets were suspended in MeOH and homogenised
in a Precellys Bead Beating system (Bertin Technologies,
Montigny-le-Bretonneux, France), followed by addition of MTBE. After
incubation water was added and through centrifugation the organic phase
was collected. The aqueous phase was re-extracted using MTBE/MeOH/H[2]O
(10/3/2.5 v/v/v). Organic phases were combined and evaporated to
dryness using a SpeedVac Savant centrifugal evaporator (Thermo
Scientific, Dreieich, Germany). Proteins were extracted from the
residue debris pellets and quantified using a BCA kit (Sigma-Aldrich,
Taufkirchen, Germany). Lipid profiling was performed using a Sciex
ExionLC AD coupled to a Sciex ZenoTOF 7600 under control of Sciex OS
3.0 (Sciex, Darmstadt, Germany). Separation was achieved on Waters
Cortecs C18 column (2.1 mm × 150 mm, 1.6 µm particle size) (Waters,
Eschborn, Germany). 40% H[2]O/60% ACN + 10 mM ammonium formate/0.1%
formic acid and 10% ACN / 90% iPrOH + 10 mM ammonium formate/0.1%
formic acid were used as eluents A and B. Separation was carried out at
40 °C at a flow rate of 0.25 mL/min using a linear gradient as
followed: 32/68 at 0.0 min, 32/68 at 1.5 min, 3/97 at 21 min, 3/97 at
25 min, 32/68 at 25.1 min, 32/68 at 30 min. Analysis was performed in
positive ionisation mode.
Dried samples were re-dissolved in H[2]O/ACN/iPrOH (5/35/60, v/v/v)
according to their protein content to normalise for differences in
biomass. Ten microlitres of each sample were pooled into a QC sample.
The remaining sample was transferred to an autosampler vial. The
autosampler temperature was set to 5 °C and 5 µL were injected for
analysis. MS^1 ions in the m/z range 70–1500 were accumulated for 0.1 s
and information dependent acquisition of MS^2 was used with a maximum
number of 6 candidate ions and a collision energy of 35 eV with a
spread of 15 eV. Accumulation time for MS^2 was set to 0.025 s yielding
a total cycle time of 0.299 s. ZenoTrapping was enabled with a value of
80,000. QC samples were used for conditioning of the column and were
also injected every 5 samples. Automatic calibration of the MS in MS^1
and MS^2 mode was performed every 5 injections using the ESI positive
Calibration Solution for the Sciex X500 system or the ESI negative
Calibration Solution for the Sciex X500 system (Sciex, Darmstadt,
Germany).
Data analysis was performed in a targeted fashion for SLs
(Supplementary Data [325]11). SLs were identified by manual
interpretation of fragmentation spectra following established
fragmentation for different SL classes: m/z 268.263491, 250.252926 and
238.252926 for C17iso sphingosine and m/z 270.279141, 252.268577 and
288.289706 for C17iso sphinganine based derived SLs. Data analysis was
performed in Sciex OS 3.0.0.3339 (Sciex, Darmstadt, Germany). Peaks for
all lipids indicated below were integrated with a XIC width of 0.02 Da
and a Gaussian smooth width of 3 points using the MQ4 peak picking
algorithm. All further processing was performed in R 4.2.1 within
RStudio using the following packages: tidyverse (v1.3.2), readxl
(1.4.1), ggsignif (0.6.4), ggplot2 (3.3.6), scales (1.2.1).
Significance was tested using a two-sided Welch-Test within ggsignif.
Metabolomics data have been deposited to the EMBL-EBI
MetaboLights^[326]88 database with the identifier [327]MTBLS8440.
Bacterial SL peak intensity
Bacterial biomass was screened for the presence of SLs via MALDI mass
spectrometry spot assays. Briefly, 1 µl of each cell pellet was
transferred on a microscopy slide and air dried. The microscopy slide
with spots of cells was covered with a matrix (sDHB) using a pneumatic
sprayer (HTX Science). The spots were analysed using the AP-SMALDI5 AF
source (Transmit, Giessen) connected to a QExactive HF (Thermofisher)
as described previously^[328]22.
A step size of 100 µm in X and Y direction was used to image all spots
in positive ionisation mode. The MS settings where as followed:
positive ionisation mode, mass range m/z 300–1200 Da, S-Lens 100,
capillary voltage 4 kV, mass resolution 240000 at m/z 200 Da. The raw
data was uploaded to figshare 10.6084/m9.figshare.29093192.v1 and
transformed into imzml and deposited to metaspace2020.org for browsing
of images (datasets: MPIMM_514_QE_P
[329]https://metaspace2020.org/dataset/2025-02-27_13h37m58s). For
relative quantification of each compound 1–6, a region of interest
(ROI) was drawn around each bacterial spot and the peak intensity for
M + H+ (see Supplementary Data [330]1) per pixel was averaged within
this ROI.
Bt survival assay
B. thuringiensis survival assays were performed as described previously
with minor adjustments^[331]18,[332]92,[333]93. N2 wildtype worms and
the SL mutants were synchronised and grown on PFM plates seeded with
1 mL MYb115 or OP50 (OD[600nm] of 10) until they reached the L4 stage.
Infection plates were inoculated with each of the bacteria adjusted to
OD[600nm] of 10 mixed with Bt247 spores or Bt407. For the infection L4
worms were washed off the plates with M9 buffer and 30 worms were
pipetted onto infection plates and incubated at 20 °C. To assess
survival, all worms were counted as either alive or dead 24 h after
infection. Worms were considered dead if they did not respond to light
touch with a platinum wire picker. We plotted all survivals as survival
curves (Fig. [334]S17) but provided a summary of the data in a heatmap
(Fig. [335]7C). The area under the survival curve (AUC) was calculated
for the C. elegans mutant strains and the mean AUC of C. elegans
wildtype N2. The AUC for the mutant strain was then subtracted from the
mean AUC of wildtype worms (ΔAUC). Based on the ΔAUC values, the
shading for the heatmap was determined (Fig. [336]7B). To test the
effect of SLs on the survival of C. elegans we supplemented the worms
with a range of different commercially available SLs. C18, C20 and C22
ceramide, as well as C16 and C18 sphingomyelin, were prepared in
ethanol at concentrations of 0.5 mg/ml or 10 mg/ml, respectively. Prior
to inoculating the Bt assay plates with Bt-OP50 mixture, 60 µg of the
SLs were inoculated onto PFM plates and thoroughly dried. A stock
solution of 25 mM of D-Sphingosine in EtOH was prepared, for the assay
either 50 or 100 µM were used for each infection plate.
Sphingosine-1-phosphate was diluted in MeOH (2 mM) and 20 µM was used
for each infection plate. In all survival assay equal amounts of EtOH
or MeOH was used as control treatment. All SLs were obtained from
Biomol GmbH - Life Science Shop, Germany.
Bt survival assays were done each with three to four replicates per
treatment group and around 30 worms per replicate for each independent
experiment. Statistical analyses were performed with RStudio (Version
4.1.2)^[337]94. GLM analysis with Tukey multiple comparison
tests^[338]95 and Bonferroni^[339]96 correction were used for all
survival assays individually. Graphs were plotted using ggplot2^[340]97
and were edited in Inkscape (Version 1.4).
Bacterial colonisation assay
To test for differences in colonisation of C. elegans L4 and young
adults by MYb115 and MYb115 ΔsgaA, colonisation was quantified by
counting colony forming units (CFUs). Worms were exposed to MYb115 and
MYb115 ΔsgaA from L1 to L4 larval stage or additionally 24 h until
worms reached young adulthood. To score the CFUs, worms were washed off
their plates with M9-T (M9 buffer + 0.025% Triton-X100) followed by
five gravity washing steps with M9-T. Prior to soft bleaching, worms in
M9-T were paralysed with equal amounts of M9-T and 10 mM tetramisole to
prevent bleach solution entering the intestine. Worms were bleached for
2 min with a 2% bleach solution (12% NaClO diluted in M9 buffer).
Bleaching was stopped by removing the supernatant and washing the
samples with PBS-T (PBS: phosphate-buffered saline + 0.025%
Triton-X100). A defined number of worms was transferred into a new tube
with PBS-T. A subsample of this was used as a supernatant control,
while the remaining sample was homogenised with sterile zirconia beads
(1 mm) using the BeadRuptor 96 (omni International, Kennesaw Georgia,
USA) for 3 min at 30 Hz. Homogenised worms were diluted (1:10/1:100)
and plated onto TSA plates, as well as the undiluted supernatant as
control. After 48 h at 25 °C, colonies were counted and the CFUs per
worm were calculated. To determine significant differences, we
performed a t-test.
Pumping behaviour
To score the pumping rate, i.e., the back and forth movement of the
grinder, worms were exposed to either MYb115 or MYb115 ΔsgaA. Pumping
was scored at L4 larval stage, young adults and young adults infected
with Bt247 (1:100). Only worms that were on the bacterial lawn were
counted for a period of 20 s. 15–20 worms per condition were counted.
To determine significant differences, we performed pairwise Wilcoxon
test.
C. elegans intestinal integrity
To visualise intestinal morphology and integrity of C. elegans upon
infection with Bt247 the worm strain GK70 (dkls37[Pact-5::GFP:pgp-1])
was synchronized. L1 larvae were exposed for 72 h to E. coli OP50, P.
fluorescens MYb115 ΔsgaA and MYb115 P[BAD]sga supplemented with
arabinose, followed by a 4 h infection with Bt247. Worms were picked
into 10 mM tetramisole on agar-padded object slides. The number of
membrane vesicles was counted and sorted into either of the three
categories: “0 vesicles”, “
[MATH: ≤ :MATH]
10 vesicles” or “
[MATH: > :MATH]
10 vesicles”. For each treatment populations of 14–25 worms were
scored, the experiment was repeated three times.
Reporting summary
Further information on research design is available in the [341]Nature
Portfolio Reporting Summary linked to this article.
Supplementary information
[342]Supplementary Information^ (3.2MB, pdf)
[343]41467_2025_60234_MOESM2_ESM.docx^ (26KB, docx)
Description of Additional Supplementary Files
[344]Supplementary Dataset 1^ (49.3KB, xlsx)
[345]Supplementary Dataset 2^ (21.3KB, xlsx)
[346]Supplementary Dataset 3^ (14.8KB, xlsx)
[347]Supplementary Dataset 4^ (14.5KB, xlsx)
[348]Supplementary Dataset 5^ (14.9KB, xlsx)
[349]Supplementary Dataset 6^ (15.2KB, xlsx)
[350]Supplementary Dataset 7^ (34.3KB, xlsx)
[351]Supplementary Dataset 8^ (33.2KB, xlsx)
[352]Supplementary Dataset 9^ (11.2KB, xlsx)
[353]Supplementary Dataset 10^ (24.6KB, xlsx)
[354]Supplementary Dataset 11^ (24.3KB, xlsx)
[355]Supplementary Dataset 12^ (62.6KB, xlsx)
[356]Supplementary Dataset 13^ (25.9KB, xlsx)
[357]Supplementary Dataset 14^ (16.7KB, xlsx)
[358]Supplementary Dataset 15^ (10.4KB, xlsx)
[359]Supplementary Dataset 16^ (18.4KB, txt)
[360]Reporting Summary^ (111.1KB, pdf)
[361]Transparent Peer Review file^ (339.4KB, pdf)
Acknowledgements