Imaging mass spectrometry and genome mining reveal highly antifungal virulence factor of mushroom soft rot pathogen

Article abstract of DOI:10.1002/anie.201206658

Soft rot diseases caused by a variety of bacteria account for severe losses in agriculture, devastating fruits, vegetables, and cultivated mushrooms. After bacterial infection, often owing to direct contact or transmission by insects, virulence factors and lytic enzymes cause degradation of plant and mushroom tissues, thereby turning crop into mush. In many cases, the chemical mediators of soft rot diseases have remained elusive, as in the long-known mushroom pathogen Janthinobacterium agaricidamnosum. This motile Gram-negative bacterium has been found to be the causative agent of soft rot disease of the cultured button mushroom, Agaricus bisporus. Typical symptoms of the infection are lesions turning into sticky blotches on the cap surface and a complete dissolution of the mushroom within only a few days (Figure 1A, B). We reasoned that knowledge on the causative agent of the soft rot would have a double benefit. Foremost, it could aid in understanding the pathobiology of the mushroom pathogen, which may be a starting point for protective measures. Second, there is an increasing need for novel antifungals, since the incidents of severe and even lethal fungal infections and resistance towards antifungals are on the rise. We hypothesized that mushroom soft rot bacteria could excrete antifungal agents as virulence factors, which might also be active against human pathogens. Herein we report the discovery and full characterization of a highly antifungal virulence factor from the soft rot pathogen Janthinobacterium agaricidamnosum guided by imaging mass spectrometry and genome mining.

Full text of DOI:10.1002/anie.201206658

Angewandte
Chemie
DOI: 10.1002/anie.201206658
Natural Products
Imaging Mass Spectrometry and Genome Mining Reveal Highly
Antifungal Virulence Factor of Mushroom Soft Rot Pathogen**
Katharina Graupner, Kirstin Scherlach, Tom Bretschneider, Gerald Lackner, Martin Roth,
Harald Gross, and Christian Hertweck*
Soft rot diseases caused by a variety of bacteria account for
severe losses in agriculture, devastating fruits, vegetables, and
cultivated mushrooms. After bacterial infection, often owing
to direct contact or transmission by insects, virulence factors
and lytic enzymes cause degradation of plant and mushroom
To gain insight into the metabolic potential of J. agarici-
damnosum, we subjected genomic DNA of the bacterium to
shotgun sequencing. Bioinformatic mining of the J. agarici-
damnosum draft genome sequence revealed several gene loci,
[
1–3]
tissues, thereby turning crop into mush.
In many cases, the
chemical mediators of soft rot diseases have remained elusive,
as in the long-known mushroom pathogen Janthinobacterium
[
4]
agaricidamnosum. This motile Gram-negative bacterium
has been found to be the causative agent of soft rot disease of
the cultured button mushroom, Agaricus bisporus. Typical
symptoms of the infection are lesions turning into sticky
blotches on the cap surface and a complete dissolution of the
[
4]
mushroom within only a few days (Figure 1A,B). We
reasoned that knowledge on the causative agent of the soft
rot would have a double benefit. Foremost, it could aid in
understanding the pathobiology of the mushroom pathogen,
which may be a starting point for protective measures.
Second, there is an increasing need for novel antifungals,
since the incidents of severe and even lethal fungal infections
[
5]
and resistance towards antifungals are on the rise. We
hypothesized that mushroom soft rot bacteria could excrete
antifungal agents as virulence factors, which might also be
active against human pathogens. Herein we report the
discovery and full characterization of a highly antifungal
virulence factor from the soft rot pathogen Janthinobacterium
agaricidamnosum guided by imaging mass spectrometry and
genome mining.
[*] K. Graupner, Dr. K. Scherlach, T. Bretschneider, Dr. G. Lackner,
Dr. M. Roth, Prof. Dr. C. Hertweck
Department of Biomolecular Chemistry
Leibniz Institute for Natural Product Research and Infection
Biology, HKI
Beutenbergstrasse 11a, 07745 Jena (Germany)
E-mail: christian.hertweck@hki-jena.de
Figure 1. A) Pictures of button mushroom before and after (72 h)
infection with Janthinobacterium agaricidamnosum (micrograph,
center). B) Imaging mass spectrometry of a mushroom fruiting body
inoculated with J. agaricidamnosum; dried mushroom slice showing
bacteria-induced lesion (left), enlarged view of the lesion and imaging
Prof. Dr. H. Gross
Eberhard Karls University, Tꢀbingen (Germany)
Prof. Dr. C. Hertweck
Friedrich Schiller University, Jena (Germany)
[
**] We thank A. Perner, F. Rhein, and H. Heinecke for MS and NMR
measurements, N. Ueberschaar for GC-MS, M.-G. Schwinger, M.
Ishida, C. Weigel, and H.-M. Dahse for bioactivity testing, G.
Krauter, J. Schçnemann, K. Willing, and M.-G. Schwinger for
fermentations. We are grateful for financial support by the Pakt fꢀr
Forschung und Innovation and the ILRS as part of the excellence
graduate school Jena School for Microbial Communication (JSMC).
area (right). The dashed area was laser-scanned with a raster width of
+
100 mm. The occurrence of the ion [M+H] 1181 (for jagaricin) is
visualized in green. C) Architecture of the jag biosynthesis gene locus
GenBank accession nr. HE967328); NRPS and accessory genes (blue
and black), regulator genes (yellow), transporter gene (pink), other
gray). D) RTq-PCR expression analysis of NRPS gene jagA; a) non-
(
(
producing conditions, b) producing conditions. E) HPLC profiles of
wild-type J. agaricidamnosum in standard growth medium (a), same in
modified production medium (b), DjagA mutant (c).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201206658.
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13173

.
Angewandte
Communications
such as the jag gene locus (Figure 1C), that code for
thiotemplate systems composed of modular polyketide syn-
thase (PKS) and nonribosomal peptide synthetase (NRPS)
modules. Thus, the mushroom pathogen would in principle
have the capacity to produce complex secondary metabolites.
Nonetheless, by quantitative reverse-transcriptase PCR
Thr-Thr-Tyr-Dhb-Gln-Gly-Thr-His (Dhb, dehydrobutyrine)
and an N-terminal acyl residue (C H O ).
1
4
26
2
To obtain a sufficient amount of jagaricin for a full
structural characterization and biological evaluation, the
fermentation was upscaled to 50 L. Size exclusion chroma-
tography followed by repeated preparative RP-HPLC yielded
29 mg of pure jagaricin. 1D and 2D NMR studies confirmed
the architecture of the jagaricin peptide backbone that was
(
RTq-PCR) we found that the PKS and NRPS gene loci are
silent or downregulated under standard cultivation conditions
Figure 1D). This is in line with the observation that
n
(
predicted by MS and bioinformatics analyses (Figure 2A).
J. agaricidamnosum grown under these conditions does not
produce any secondary metabolites (Figure 1E, lane a).
Apparently, the production of these cryptic natural products
requires a particular trigger, and thus we aimed at catching
the pathogen in action while infecting a mushroom fruiting
body.
NMR spectroscopy data also revealed that the N-terminal
acyl moiety is derived from b-hydroxymyristic acid (HMA).
The absolute configuration of HMA obtained by acid
hydrolysis of jagaricin was determined to be R in the 3-
position of HMA by employing GC–MS analysis of the
corresponding Mosher ester and authentic (R)- and (S)-HMA
[
6]
For this purpose imaging mass spectrometry appeared to
[
7,8]
be a promising method.
First, we inoculated slices of
Agaricus bisporus with J. agaricidamnosum and monitored
the formation of bacteria-induced tissue lesions. Next, a liquid
matrix containing a-cyano-4-hydroxy cinnamic acid was
sprayed onto the surface of the infected mushroom. The
imaging area was automatically scanned with a raster width of
1
00 mm in XY recording 1000 spectra at each spot with a laser
frequency of 1000 Hz. MALDI-MS measurements were
performed using an ultrafleXtreme mass spectrometer in
the positive reflector mode collecting data in the range of m/z
9
00–2000 Da. The resultant sum spectrum was evaluated
manually, and the mass of interest was visualized in the
logarithmic scale by picking the peak with 1 Da mass range
using the brightness optimization as implemented in
flexImaging. In this way we visualized the production of
+
a new compound with m/z 1181 [M+H] within the damaged
mushroom tissue (Figure 1B). We next aimed at stimulating
the formation of the metabolite in liquid media. From
a variety of media screened we found that traces of the new
compound were produced in minimal medium containing
mushroom cubes. Higher yields were, however, obtained
À1
À1
À1
using a complex culture medium (5 gL glycine, 10 gL
À1
À1
yeast extract, 10 gL glucose, 10 gL corn steep, 10 gL
CaCO ), which also proved to be more convenient when
3
increasing the culture volume. From high-resolution mass
spectrometry measurements (Exactive, Thermo Scientific) we
inferred the elementary composition of C H N O for the
5
6
85 12 16
new metabolite, named jagaricin. Considering the size and
elementary composition of the molecule, we assumed that the
corresponding gene cluster would harbor NRPS genes. The
best candidate for mediating jagaricin biosynthesis would be
the jag gene locus (Figure 1C), since the size of the putative
product of the encoded synthase is similar to jagaricin. RTq-
PCR measurements confirmed that the jag genes were
substantially upregulated under these producing conditions
(
Figure 1D). Open reading frames (orfs) jagA–H code for
Figure 2. Full structure elucidation of jagaricin. A) Key results from 2D
NMR spectroscopy, degradation experiments, and application of
Marfey’s method. B) Stereochemical elucidation of HMA (as Mosher
ester) by GC–MS analysis and comparison with equally derivatized (R)-
and (S)-HMA references (lower traces); C) Phylogeny of TE domains
to deduce connectivity and lactone ring size of jagaricin; the ring size
of the cyclopeptide (number of aa) and the molecule where the
formation of the ester bond occurs are shown; b-OH FA=hydroxy
fatty acid; Pip=pipecolin.
a nonamodular NRPS (jagA–D) as well as several accessory
and regulatory components (jagE–H). By employing the
modular structure of the encoded NRPS, the adenylation (A)
domain specificities inferred in silico (see the Supporting
Information), and MS/MS analyses we predicted that jagar-
icin is a cyclic lipopeptide with the amino acid sequence Dhb-
1
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references (Figure 2B). The absolute configurations of the
free amino acids were elucidated by derivatization with
Marfeyꢀs reagent and HPLC analyses using 1-fluoro-2,4-
dinitrophenyl-5-l-alanine-amide (l-FDAA) conjugates of d-
and l-amino acids as references (see the Supporting Infor-
mation).
main is clearly missing in module 2. Considering the Thr
specificity of the upstream A domain, it is well conceivable
that it loads the second Thr residue, too. This scenario would
be reminiscent of the yersiniabactin synthetase, where a single
A domain in the second module loads cysteine units onto two
[
12,13]
individual downstream T domains.
Next, the stereochem-
Whereas the architecture and absolute configuration of
the lipopeptide backbone could be rigorously determined by
means of NMR spectroscopy, MS , and chemical degradation,
ical analysis showed unequivocally that d-Tyr is incorporated
into the peptide chain, yet no epimerization domain can be
found in module 4 (Figure 3). A detailed analysis of the
n
the position of the ester bond remained ambiguous. Several
Thr residues could in principle provide the hydroxy residue,
yet no diagnostic couplings were observed by 2D NMR
spectroscopy. For this reason we performed a phylogenetic
analysis of thioesterase (TE) domains from various NRPSs
producing cyclopeptides of different ring sizes. In the clado-
gram the jag TE falls into a clade of TEs producing
cyclooctapeptides (Figure 2C). We thus concluded that the
ester bond is located between the His carboxy group and the
hydroxy moiety of the first Thr residue. To corroborate this,
we peracetylated jagaricin, and indeed NMR data of O-
acetylated jagaricin were in full agreement with the predicted
position of the ring closure (see the Supporting Information).
We have thus fully elucidated the structure of jagaricin,
a novel lipopeptide containing two Dhb residues that are
rather uncommon for NRPS products. Actually, these en-
amino acids are a hallmark of ribosomally produced lanti-
biotics, where they mainly occur as precursors of the
C domain in the downstream module revealed the signature
d
sequences of a C domain, which is known to link an l-amino
L
[
14]
acid to a d-amino acid. Hence, the upstream A domain
seems to activate d-Tyr, which is likely generated by a race-
mase. Notably, there is only a small number of known
pathways where d-amino acids are incorporated into NRPs
[
15–18]
by this mechanism.
Another surprising finding was that
module 8 possesses an additional C domain. It has been
suggested that the second C domain catalyzes the epimeriza-
tion of the Thr side chain, thereby leading to the insertion of
[19]
l-allo-Thr into the growing peptide chain. The fact that l-
allo-Thr is incorporated by the jag NRPS at this particular
position supports this prediction. Remarkably, only two
NRPSs are known that harbor the same unusual domain
[
19,20]
structure.
The expression studies and the clear correlation of
structure and NRPS architecture already indicate a link
between the jag gene locus and the production of jagaricin.
However, for a rigorous assignment a gene knockout was
essential. To achieve this goal proved to be most challenging,
since neither genetic tools nor protocols were available for
this extraordinary bacterium. Eventually, we succeeded in
transforming J. agaricidamnosum and disrupting the NRPS
gene jagA by insertion of a kanamycin resistance cassette. The
[9]
lanthionine building blocks. In contrast, the mechanism
and timing of Dhb formation in NRPS-derived peptides is still
unknown. Overall, the structure of jagaricin correlates well
with the encoded NRPS assembly line (Figure 3). However,
the jagaricin synthetase has some interesting features that
[
10,11]
breach with canonical NRPS functions.
First, an A do-
Figure 3. Deduced NRPS thiotemplate and model for the biosynthesis of jagaricin. The timing of Dhb formation from Thr is approximate.
C=condensation domain; A=adenylation domain; T=thiolation domain; E=epimerization domain; TE=thioesterase domain.
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successful mutant construction was confirmed by PCR, and
indeed the jagaricin formation was completely abrogated in
the DjagA mutant (Figure 1E, lane c). Thus, the identity of
the jag gene cluster was unequivocally confirmed. Moreover,
when growing the DjagA mutant on mushroom tissue, no
jagaricin could be detected by imaging MS. To further
corroborate the role of jagaricin in the infection process we
applied pure jagaricin onto mushroom tissue. We noted that
the lipopeptide alone caused a superficial lesion that is
symptomatic to the wild type of the soft rot pathogen
concept that the elucidation of virulence factors involved in
mushroom diseases may lead to the discovery of natural
products that are also active against human pathogenic fungi.
Further biological evaluations will reveal possible applica-
tions of jagaricin as a therapeutic.
Herein we have reported the discovery of a new anti-
fungal virulence factor, jagaricin, the discovery of which
proved to be challenging, because the jag biosynthesis genes
are downregulated or silenced under regular cultivation
conditions. In the pathobiological context jagaricin produc-
tion is triggered upon contact of the bacterium (J. agarici-
damnosum) with the mushroom (A. bisporus). To elucidate
the cryptic natural product we employed a combination of
imaging mass spectrometry and genome mining. Imaging
mass spectrometry has proven to be a valuable tool for the
discovery of microbial and plant natural products, in partic-
(
Figure 4A). These results indicate that jagaricin is clearly
[
24–26]
ular in symbioses.
Coupling this technique to genome
[27–29]
mining is a new approach,
and to our knowledge we
describe the first case where this combination enabled the
systematic discovery and full characterization of a cryptic
natural product. Furthermore, the revelation of a virulence
factor by imaging mass spectrometry and genomics is
unprecedented. For the rigorous elucidation of the antifungal
principle the combination of physicochemical methods,
chemical derivatization, and bioinformatics analysis of the
encoded NRPS assembly line was most effective. Herein,
predicting the lactone ring size by a TE phylogeny proved to
be a particularly valuable, new approach that will certainly aid
in solving related structural riddles. Furthermore, comparison
of the jagaricin structure and the thiotemplate system
revealed several unusual features of the jag NRPS. In this
regard it should be highlighted that the identity of the jag
biosynthesis gene locus was unequivocally proven by a gene
knockout, which proved to be challenging considering that
this unusual bacterium has been genetically fully unexplored.
On ecological and agricultural grounds the discovery of the
virulence factor and the corresponding genes is significant,
because it helps understanding the pathobiology of bacteria-
induced soft rot. From a chemical point of view, our study
highlights the impact of blending modern analytics with
genetics to unveil cryptic natural products from neglected
Figure 4. Biological evaluation of jagaricin. A) Induction of mushroom
tissue lesions with pure jagaricin solution. The arrow indicates the
position of the lesion. B) Antifungal activity of jagaricin against
a) Candida albicans, b) Aspergillus fumigatus, c) Aspergillus terreus (agar
À1
diffusion assay with c=500 mgmL ); minimal inhibitory concentration
(
MIC100) values in mm.
involved in the infection process. However, jagaricin is
apparently not the only pathogenicity factor, as enzymes
such as chitinases may contribute to the further degradation
of the fruiting bodies. This finding is interesting in light of the
other known mechanisms of mushroom pathogens. Inves-
tigation of brown blotch disease caused by Pseudomonas spp.
[21]
revealed tolaasin as the sole virulence factor,
whereas
[30]
degradative enzymes have been shown to be the only
microorganisms. The general approach of combining imag-
ing mass spectrometry with genome mining holds promise to
be generally applicable to the discovery of cryptic natural
products including chemical mediators such as virulence
factors that are only produced in a particular (patho)biolog-
ical context.
virulence factor in the cavity disease caused by Burkholderia
[22]
gladioli pv. agaricicola. An analogous case where secondary
metabolites are not the only determinants for pathogenicity
but support infection has been described for the plant
[23]
pathogen Pseudomonas syringae.
Since jagaricin exerts a clear antifungal effect on the
mushroom, we next tested our hypothesis that the virulence
factor may also act as an agent against pathogenic fungi. For
this purpose jagaricin was subjected to an antimicrobial
profiling. By using a panel of representative test strains we
found that jagaricin has only little or no antibacterial activity.
In contrast, the lipopeptide is highly active against the major
human pathogens Candida albicans, Aspergillus fumigatus,
and Aspergillus terreus even at submicromolar concentrations
Received: August 17, 2012
Revised: October 22, 2012
Published online: November 19, 2012
Keywords: biosynthesis · natural products ·
.
Janthinobacterium agaricidamnosum · lipopeptides ·
structure elucidation
(
Figure 4B). At 3–10x higher concentrations jagaricin exhib-
its antiproliferative and cytotoxic activity (see the Supporting
Information). Taken together, this finding is a proof of
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