Azetidine-Containing Alkaloids Produced by a Quorum-Sensing Regulated Nonribosomal Peptide Synthetase Pathway in Pseudomonas aeruginosa

Article abstract of DOI:10.1002/anie.201809981

Pseudomonas aeruginosa displays an impressive metabolic versatility, which ensures its survival in diverse environments. Reported herein is the identification of rare azetidine-containing alkaloids from P. aeruginosa PAO1, termed azetidomonamides, which are derived from a conserved, quorum-sensing regulated nonribosomal peptide synthetase (NRPS) pathway. Biosynthesis of the azetidine motif has been elucidated by gene inactivation, feeding experiments, and biochemical characterization in vitro, which involves a new S-adenosylmethionine-dependent enzyme to produce azetidine 2-carboxylic acid as an unusual building block of NRPS. The mutants of P. aeruginosa unable to produce azetidomonamides had an advantage in growth at high cell density in vitro and displayed rapid virulence in Galleria mellonella model, inferring functional roles of azetidomonamides in the host adaptation. This work opens the avenue to study the biological functions of azetidomonamides and related compounds in pathogenic and environmental bacteria.

Full text of DOI:10.1002/anie.201809981

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Accepted Article
Title: Azetidine-Containing Alkaloids Produced by a Quorum-Sensing-
Regulated Nonribosomal Peptide Synthetase Pathway in
Pseudomonas aeruginosa
Authors: Zhilai Hong, Arnaud Bolard, Caroline Giraud, Sébastien
Prévost, Grégory Genta-Jouve, Christiane Deregnaucourt,
Susanne Häussler, Katy Jeannot, and Yanyan Li
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201809981
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Azetidine-Containing Alkaloids Produced by a Quorum-Sensing-
Regulated Nonribosomal Peptide Synthetase Pathway in
Pseudomonas aeruginosa
Zhilai Hong,[a] Arnaud Bolard,[b] Caroline Giraud,[c] Sébastien Prévost,[d] Grégory Genta-Jouve,[e]
Christiane Deregnaucourt,[a] Susanne Häussler,[f] Katy Jeannot,*[b] and Yanyan Li*[a]
Abstract: Pseudomonas aeruginosa displays an impressive
metabolic versatility, which ensures its survival in diverse
environments. Herein we report the identification of rare azetidine-
containing alkaloids from P. aeruginosa PAO1, termed
azetidomonamides, that are derived from a conserved, quorum-
sensing regulated non-ribosomal peptide synthetase (NRPS)
pathway. Biosynthesis of the azetidine motif has been elucidated by
gene inactivation, feeding experiments and biochemical
characterization in vitro, which involves a new S-adenosylmethionine-
dependent enzyme to produce azetidine 2-carboxylic acid as an
unusual building block of NRPS. The mutants of P. aeruginosa unable
to produce azetidomonamides had an advantage in growth at high cell
density in vitro and displayed rapid virulence in Galleria mellonella
model, inferring functional roles of azetidomonamides in the host
adaptation. This work opens the avenue to study the biological
functions of azetidomonamides and related compounds in pathogenic
and environmental bacteria.
fibrosis patients for the deleterious impact on lung function. The
ecological success of P. aeruginosa is largely attributed to its
metabolic plasticity and versatility, frequently linked to quorum-
sensing (QS) regulation. QS is
a cell-cell communication
mechanism allowing bacteria to sense cell density and to
coordinate gene expression at the community level, in response
to environmental conditions. P. aeruginosa has evolved complex
QS systems to control critical processes including biofilm
formation, virulence factor production and antimicrobial
resistance. The knowledge of the chemical nature and biological
function of QS-regulated specialized metabolites is thus of prime
importance and would provide new biomarkers or therapeutic
strategies to progress in combating P. aeruginosa infection[1].
The core genome of P. aeruginosa encodes six non-
ribosomal peptide synthetase (NRPS) biosynthetic gene clusters
(BGCs) as major components of the specialized metabolism[2].
Among them, three are involved in the production of siderophores
such as pyoverdine and pyochelin as well as an antibacterial
antimetabolite, L-2-amino-4-methoxy-trans-3-butenoic acid[3].
Although extensively studied, products of other NRPS pathways
remain elusive. During the course of investigating new
physiological functions associated with polymyxin resistance, we
generated a spontaneous mutant from P. aeruginosa reference
strain PAO1 on colistin (16 mg/L), named AB16.2, and performed
the whole genome expression profile. Among genes that are most
up-regulated in the mutant AB16.2 in comparison with the
parental strain PAO1, we identified the operons pmrAB encoding
a two-component system PmrA-PmrB involved in polymyxin
resistance[4], pqsABCDE coding the Pseudomonas Quinolone
Signal (PQS) belonging to the QS system and one
uncharacterized BGC for non-ribosomal peptide synthesis (Table
S1). This BGC comprises 10 genes (PA3326-PA3335) encoding
notably a bimodular NRPS (the product of PA3327) and
modification enzymes (Figure 1a; Table S2). Worth to note, this
BGC is strictly conserved in all sequenced P. aeruginosa
genomes, with only few exceptions in other Pseudomonas
species, in the Pseudomonas genome database[5]. Moreover,
some genes of this BGC including PA3326, 3327, 3329 and 3332,
have been shown to be part of the core regulon of the las/rhl QS
systems using acyl-homoserine lactone as signals[6]. Given that
pqs genes are overexpressed in the mutant AB16.2, we assessed
if the cluster is also regulated by pqs QS. Quantitative real-time
PCR with reverse transcription analysis showed that in-frame
deletion of pqsA in the mutant AB16.2 resulted in a significant
decrease of PA3327 transcription (Figure S1). Therefore, this
BGC is under the control of three QS systems operating in P.
aeruginosa. Taken together, this suggests that the metabolites
derived from this pathway play an important role in the pathogen’s
physiology.
Pseudomonas aeruginosa is a highly adaptable Gram-negative
bacterium, which thrives in diverse environments including soil,
water, plant and animal hosts. This microorganism is a prominent
nosocomial human pathogen, which is associated with acute
pulmonary infections in immunocompromised patients,
particularly in intensive care units. It is also well-known in cystic
[a]
Z. Hong, Dr. C. Deregnaucourt, Dr. Y. Li
Unité Molécule de Communication et Adaptation des
Microorganismes (MCAM)
Muséum National d’Histoire Naturelle (MNHN), Centre National de
la Recherche Scientifique (CNRS)
CP 54, 57 rue Cuvier, 75005 Paris, France.
E-mail: yanyanli@mnhn.fr
[b]
A. Bolard, Dr. K. Jeannot
Laboratoire de Bactériologie, Centre National de Référence (CNR)
de la Résistance aux Antibiotiques, Centre Hospitalier Régional
Universitaire (CHRU) de Besançon, UMR4269 "Chrono-
Environnement"
Boulevard Fleming, 25030 Besançon, France.
E-mail : katy.jeannot@univ-fcomte.fr
[c]
[d]
Dr. C. Giraud
U2RM Stress/Virulence, Normandy University, UNICAEN
14000 Caen, France.
Dr. S. Prévost
Laboratoire de Synthèse Organique, UMR 7652, CNRS, Ecole
Polytechnique, ENSTA ParisTech, Université Paris-Saclay
828 Bd des Maréchaux, 91128 Palaiseau, France.
Dr. G. Genta-Jouve
C-TAC, UMR 8638, CNRS, Faculté de Pharmacie de Paris,
Université Paris Descartes, Sorbonne Paris Cité
4 Avenue de l'Observatoire, 75006 Paris, France.
Prof. Dr. S. Häussler
[e]
[f]
Institute for Molecular Bacteriology, TWINCORE, Centre for
Experimental and Clinical Infection Research, Hannover, Germany.
Department of Molecular Bacteriology, Helmholtz Centre for
Infection Research, Braunschweig, Germany.
To facilitate compound identification, a PA3327 deficient
mutant of AB16.2 was constructed. After media screening,
Supporting information for this article is given via a link at the end of
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AB16.2 and AB16.2∆27 were grown in selected E2 medium and
their metabolic profiles were compared by liquid chromatography
coupled to high resolution mass spectrometry (LC-HRMS). Two
major peaks with [M+H]+ ion at m/z 237.0868 (1) and 193.0969
(2), respectively, were present only in the extract of AB16.2
(Figure S2). The exact masses indicated molecular formulas of
C11H12N2O4 for 1 and C10H12N2O2 for 2, indicating a CO2 loss in
the latter. Fermentation and subsequent purification allowed the
full elucidation of their structures by HR-MS/MS, UV and NMR
spectroscopy (Supplementary Information; Table S3; Figure S3-
19). Compound 1, named azetidomonamide A, features a
[5.2.0]bicyclocarbamate ring system, whereas 2, termed
azetidomonamide B, has an azetidopyrroline scaffold (Figure 1b).
Based on specific rotation and the comparison between the
measured and theoretical electronic circular dichroism (ECD)
spectra, the absolute configuration was determined to be 5R for
azetidomonamide A (1) and 4R for azetidomonamide B (2),
respectively (Figure S20-21).
Figure 1. a) Gene organization of the aze cluster. b) Structure and proposed biosynthetic pathway of azetidomonamides.
The four-membered azetidine heterocycle presented in
azetidomonamides is rare in natural products (Figure S22), which
raised the question about its biosynthetic origin. Structurally,
carboxylic acid (AZC) or a derivative thereof instead of Pro. To
prove this, in vivo isotope labelling experiments with -Ser,
methionine (Met) and -AZC were performed using an inverse
feeding approach[10]. The rational to feed Met was that Met-
derived S-adenosylmethionine (AdoMet) could be a key precursor
of AZC, similarly to the synthesis of plant nicotianamine[11]. LC-
HRMS and MS/MS analysis showed the incorporation of all three
amino acids into azetidomonamide B (2) (Figure S24-25). As for
L
L-
L
azetidomonamide
A
and
B
are related to bacterial
lipocyclocarbamates[7] and pyrrolizidine alkaloids[8] (Figure S23).
Biosynthesis of these compounds has been characterized to
involve a bimodular NRPS that ligates a serine (Ser) or threonine
(Thr) to
a proline (Pro) residue, and a Baeyer-Villiger
monooxygenase whose action leads to two bicyclic skeletons[8-9]
.
azetidomonamide A (1), its labelling by
L-Ser and L-Met was
Predicted functions of now designated aze genes (Table S2),
suggested that azetidomonamide biogenesis would operate in a
similar way, except the activation by NRPS of an azetidine 2-
observed, but addition of -AZC appeared to reduce significantly
its production level which prevented the detection (Figure S26-27).
Furthermore, in silico analysis of the adenylation (A) domains of
L
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AzeB was carried out. This predicted that AzeB_A1 activates Ser,
whereas the specificity of AzeB_A2 could be Pro but without
certainty (Table S4). To unambiguously establish the building
blocks of AzeB, both A domains together with their cognate
condensation (C) and peptidyl-carrier-protein (PCP) were
produced and purified from Escherichia coli, respectively, for
characterization in vitro (Figure S28). Twenty proteinogenic
amino acids together with additional ones (i. e. L-AZC, D-Pro,
AdoMet and homoserine) were tested as substrates. Using a
hydroxylamine-trapping assay,[12] it was shown that AzeB_A2
azetidomonamides, while complementation of azeJ back to the
mutant restored the production, albeit at a much lower level
(Figure 2b). Interestingly, supply of L-AZC to the medium yielded
only azetidomonamide B (2), which is consistent with the feeding
experiments. It thus appeared that AZC availability regulates
directly the product outcome. To provide direct evidence of the
AzeJ function, recombinant AzeJ was purified from E. coli and
characterized in vitro (Figure S28). Incubation of AdoMet with
AzeJ led to the production of 5'-methylthioadenosine (MTA), as
revealed by HPLC coupled to UV/Vis spectrophotometry (Figure
S31). Detection of AZC was achieved by derivatization with
benzyl chloroformate followed by LC-HRMS analysis. AZC was
observed in the AzeJ reaction and absent in the control with the
heat-inactivated enzyme (Figure 2d). Moreover, AZC was found
only in the extract of E. coli expressing the azeJ gene, compared
to that of control cells with an empty vector (Figure S32). These
experiments showed unambiguously that AzeJ is an AZC
synthase. Under our conditions, AzeJ displayed Kmapp(AdoMet) of
1.3 ± 0.1 mM and kcatapp of 16.7 ± 0.7 min-1, indicating a rather low
catalytic efficiency (Figure S33). Worth to note, a similar enzyme
(VioH) proposed to catalyze the same reaction as AzeJ, has been
identified very recently by gene inactivation in the myxobacterial
vioprolide pathway[16]. Sequence analysis using profile hidden
Markov models pointed out that AzeJ homologs can be found in
disparate bacteria, and occasionally in eukaryotes (e.g. algae) or
archaea (Table S5). The discovery of AZC synthases from
bacteria adds a new reaction to AdoMet-dependent enzymes and
may inform the biosynthetic origin of AZC in plants.
displayed the highest activity towards
substrate promiscuity notably to the structural mimics of AZC such
as -Pro and -Ala (Figure 2a). By a more specific ATP-release
assay[13], we could further confirm the substrate preference of
AzeB_A1 and AzeB_A2 for -Ser and -AZC, respectively (Figure
L-AZC and showed
L
L
L
L
S29-30). Together, these data established that the azetidine motif
comes from L-AZC, which is derived from the Met cycle and is
subsequently incorporated into the NRPS assembly line.
AZC has been known as a non-proteinogenic amino acids
in some plants since 60 years[14]. However, its biosynthetic route
still remains unknown, although feeding studies similarly showed
a link of AZC and the Met metabolism[15]. We envisioned that
L-
AZC can result from the intramolecular cyclisation of AdoMet, as
in the plant nicotianamine biosynthesis[11]. Inspection of the aze
cluster identified azeJ encoding an AdoMet-dependent enzyme
as a candidate for this reaction. Indeed, inactivation of azeJ in the
mutant AB16.2 disrupted completely the production of both
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Figure 2. Biosynthetic origin of the azetidine motif. a) The substrate specificity of AzeB_A2 domain determined in vitro. b) Reaction scheme of AzeJ. c) LC-MS
analysis of azetidomonamide production in P. aeruginosa AB16.2 (grey), the azeJ-deficient strain AB16.2∆35 (dotted line), AB16.2∆35 supplemented with AZC
(black) and the complementation strain AB16.2∆35 attB::PA3335 (dashed line). Extracted ion chromatograms (EICs) of ions m/z 237.0867 ([M+H]+ of 1) and m/z
193.0968 ([M+H]+ of 2) were shown. For clarity, the trace of the complementation strain was enlarged 2 folds. Addition of AZC disrupted the production of 1. d) LC-
MS analysis of AZC production in AzeJ assays in vitro. EICs of m/z 258.0729 ([M+Na]+ of the derivatized AZC product): AZC standard (dashed line), AzeJ reaction
(black) and a control with boiled AzeJ (grey).
Overall, the ensemble of our data consolidates the
proposed biosynthetic route to azetidomonamides (Figure 1),
which is similar to that of related bacterial alkaloids (Figure S34).[8-
9] Our data also extends the current model. The crotonyl side chain
in azetidomonamides is delivered to the NRPS on a dedicated
ACP, contrary to other pathways that use CoA esters. On-line
cyclisation would operate on a dipeptide intermediate containing
a serine-derived dehydroalanine (Dha), a process likely assisted
by the TE domain.[8-9] A plausible cyclisation mechanism would
involve nucleophilic attack of the Dha β-carbon to the carbonyl
group, which is enabled by delocalization of the lone pair of
electrons of the enamine nitrogen. Upon cyclisation, the
azetidopyridin-2-one intermediate would undergo keto-enol
tautomerization to generate a R-configured substrate for AzeC,
which catalyzes Baeyer-Villiger oxidation in a stereospecific
manner. This resembles the biosynthesis of brabantamide A.[9b]
insects[18]. The direct implication for P. aeruginosa would be to
establish a long-term stable relationship with the host, as under
the chronic infection conditions[19]. The underlining molecular
mechanisms are being investigated.
Genome mining revealed that identical aze BGC is present
in two strains of human pathogens, namely Acinetobacter
baumannii AB32_M and Enterobacter cloacae e403 (Figure S37),
indicating a horizontal gene transfer event from P. aeruginosa.
Using the combined presence of azeB, azeC and azeJ as
indication, related aze BGCs can be identified in a handful of
bacteria, some of them are plant-associated (Figure S37).
Consistently, phylogenetic analysis showed that, A2 domains of
the AzeB-like NRPSs, which would activate AZC, form a distinct
group from the Pro-utilizing ones involved in the biosynthesis of
bacterial pyrrolizidine alkaloids and cyclocarbamates[8] (Figure
S38). Together, these analyses open the avenue to study the
diversity and biological functions of azetidomonamide-like
compounds in various ecological settings, particularly in human
pathogens.
More minor products derived from the aze BGC could be
identified by LC-HRMS. In particular, compound 3 ([M+H]+ at m/z
207.1124) and 4 ([M+H]+ at m/z 251.1024) are analogues of
azetidomonamide B and A, respectively (Figure S35-36). The
pyrrolizidine structure of 3 was confirmed by a synthetic reference
(Supplementary Information). These molecules are generated by
incorporating Pro instead of AZC during the assembly-line
synthesis. Such diversity-oriented biosynthesis would imply
distinct or/and synergetic functional roles of these metabolites in
the physiology of P. aeruginosa.
Azetidomonamide A (1), B (2) and the synthetic pyrrolizidine
3 showed no antibacterial activities to tested Gram-negative and
Gram-positive bacteria (E. coli, Micrococcus luteus,
Staphylococcus aureus), no inhibition of human phospholipase
A2, an activity reported for the related 5,5- or 5,7-
lipocyclocarbarmate[7, 17], as well as no cytotoxicity to murine
macrophages cell line J774A.1. This indicates that, at least, these
metabolites are less likely to be involved in microbial competition
or acute virulence. To gain insight into their biological function in
P. aeruginosa, the mutants AB16.2 and AB16.2∆27 were subject
to phenotypic characterization. No significant difference between
them could be observed with regard to biofilm formation,
microcolony morphology and antibiotic resistance. However, the
mutant AB16.2 displayed a reduced growth at high cell density as
compared to AB16.2∆27 in vitro (Figure 3a). The deviation
typically started at the mid- or late logarithmic phase,
corroborating with the growth phase-dependent QS-regulation of
these aze genes[6b]. To note, both strains showed a slower growth
rate than the parental PAO1. When microinjected into a caterpillar
host, Galleria mellonella, the mutant AB16.2 had a delayed killing
phenotype, whereas AB16.2∆27 killed as rapidly and efficiently
as the wildtype PAO1 (Figure 3b). This attenuated virulence effect
manifested by the presence of the aze BGC, likely via the growth
modulation, is reminiscent of a QS-controlled virulence reduction
in an insect endosymbiotic bacterium, following its infection of
Figure 3. Characterization of the functional roles of azetidomonamides. a)
Growth curve in LB medium at 37 °C. b) Kaplan-Meier survival curves of Galleria
mellonella infected with P. aeruginosa strains with an inoculum of 25 (± 5) CFU
per larvae. p<0.001 (log-rank test with Bonferroni correction). PAO1 (black
circles), AB16.2 (black squares) and AB16.2∆27 (grey crosses).
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In conclusion, this work identifies
a
novel class of
specialized metabolites in the major opportunist human pathogen
P. aeruginosa, elucidates their biosynthesis which involves a new
family of AdoMet-dependent enzymes, and provides first insight
into their physiological functions. Further studies of exact roles of
azetidomonamides in host-pathogen interaction with regard to QS
regulation would warrant important mechanistic discoveries.
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Acknowledgements
The bioorganic analytical platform of MNHN and the bacteriology
service in the MCAM unit are acknowledged for giving access to
necessary instruments. We thank Yoann Negre for technical
assistance in caterpillar killing assays, Dr. Gérard Lambeau
(Université Côte d’Azur, France) for providing radioactive E. coli
membranes for phospholipase tests, and Dr. Stéphane Mann
(MNHN, France) for helpful discussions. Z. H. was supported by
a China Scholarship Council PhD fellowship. This work was
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Santé publique France agency.
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Keywords: biosynthesis • natural products • nonribosomal
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Entry for the Table of Contents (Please choose one layout)
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Be more strained: The major
opportunist human pathogen
Pseudomonas aeruginosa produces
azetidine 2-carboxylic acid, a proline
mimic, to be incorporated into a
quorum-sensing controlled
Zhilai Hong, Arnaud Bolard, Caroline
Giraud, Sébastien Prévost, Grégory
Genta-Jouve, Christiane Deregnaucourt,
Susanne Häussler, Katy Jeannot* and
Yanyan Li*
Page No. – Page No.
nonribosomal peptide synthetase
assembly line, yielding constrained
bicyclic alkaloids. These compounds
play important functions in the
physiology of P. aeruginosa.
Azetidine-Containing Alkaloids
Produced by a Quorum-Sensing-
Regulated Nonribosomal Peptide
Synthetase Pathway in Pseudomonas
aeruginosa
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