Mining Plants for Bacterial Quorum Sensing Modulators

Article abstract of DOI:10.1021/acschembio.7b00859

The bacterial plant pathogen Agrobacterium tumefaciens uses quorum sensing (QS) in order to regulate the transfer of DNA into the host plant genome, and this results in the induction of crown gall tumors. The deleterious results of these infections are wi

Full text of DOI:10.1021/acschembio.7b00859

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Article
Mining Plants for Bacterial Quorum Sensing Modulators
Shimrit David, Aviad Mandabi, Shaked Uzi, Asaph Aharoni, and Michael M. Meijler
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00859 • Publication Date (Web): 07 Dec 2017
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Mining Plants for Bacterial Quorum Sensing Modulators
a
a
a
b
a
Shimrit David, Aviad Mandabi, Shaked Uzi, Asaph Aharoni, Michael M. Meijler
a
Department of Chemistry and the National Institute for Biotechnology in the Negev, Ben-Gurion
b
University of the Negev, Be’er-Sheva, Israel. E-mail: meijler@bgu.ac.il Department of Plant and
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Environmental Sciences, Weizmann Institute of Science, Rehovot, Israel
ABSTRACT: The bacterial plant pathogen Agrobacterium tumefaciens uses quorum sensing (QS) in order
to regulate the transfer of DNA into the host plant genome and this results in the induction of crown gall
tumors. The deleterious results of these infections are widespread and affect many species of fruit and
crops. In order to further our understanding of this process and to provide potential solutions we
evaluated a library of 3800 natural products from plant sources and identified potent compounds that
are able to strongly modulate plant-bacterial interactions.
Bacteria use chemical signaling to coordinate
gene expression. This is a phenomenon called
quorum sensing (QS) and it is based on the
secretion of small signaling molecules and their
to exploit plants vulnerabilities by attacking
wounded sites and spread infection has been
thoroughly studied, but to date none of the
current treatments against the disease have
resulted in efficient methods to prevent
bacterial infection the onset of crown gall
disease. Since the QS mechanism of A.
1-3
recognition based on population density, The
plant pathogen Agrobacterium tumefaciens is
one of the most extensively studied bacteria
that use QS to regulate virulence and it can
effectively induce crown gall disease and infect
tumefaciens has
a
crucial effect on
implementing and controlling infection in
plants, a rational approach to deal with this
question is to search for natural plant products
that have evolved to attenuate bacterial QS
4-6
various plant species.
A. tumefaciens
produces and secretes the QS molecule N-3-
oxo-octanoyl-L-homoserine lactone (OC HSL),
8
which at a threshold population density (and
concentration) will bind the primary QS
receptor, TraR, which results in the expression
activity
through
exogenous
chemical
interruption.
7
Due to their enormous structural and chemical
versatility, natural products (secondary
metabolites) in general and plant metabolites in
particular can be used as a potential tool kit
both to study and mimic plant-bacterial
interactions.
of virulence genes. One of these genes is
responsible for the conjugal transfer of the
tumor inducing (Ti) plasmid between the
bacteria and their plant host. Each bacterium
holds a Ti plasmid that carries both virulence
gens and a DNA fragment called transfer DNA
(
T-DNA). When the T-DNA is transferred and
Here we report the screening of a library of
integrated into the host genome it will initiate
over expression of phytohormones (i.e. auxin
and cytokinin) and uncontrolled cell division
that eventually will result in a formation of
3
800 purified and characterized natural
products from plant sources on A. tumefaciens
in order to find strong modulators that may
affect the QS system. Based on the initial results
of the screening, we prepared a synthetic
8-9
crown gall tumors. The ability of the bacteria
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library of selected natural products and analogs
that significantly agonize or antagonize the QS
system of A. tumefaciens. We also validated the
efficacy of the most potent modulators in vivo
in a tumor formation model system in carrots.
3
4
Momordica
Ruta
Agonism
Agonism
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RESULTS AND DISCUSSION
Graveoline
5
6
Millettia
Agonism
Agonism
Library screening of natural products from
plants source. Our initial library screening of
the 3800 natural products (acquired from
AnalytiCon Discovery library) was performed
Verbascum
Millettia
systematically using
a
highly sensitive
luminescent A. tumefaciens reporter strain -
A136 (pCF218)(pMV26) (see methods) - that
responds to picomolar concentration of 3-oxo-
C8-AHL (the A. tumefaciens autoinducer). The
compounds that revealed more than 50%
inhibition or stimulation of the QS system with
7
Agonism
8
9
Chrysanthe-mum Antagonism
and without 400 pM 3-oxo-C -AHL (the A.
8
tumefaciens autoinducer) were selected as
potential hits (listed in Table 1). We observed
interesting trends regarding structural features
of active compounds. For instance, many of the
agonists contain the flavonoid scaffold. This is in
line with previous reports on flavonoid activity
in bacteria; they have been shown to act as
Bisdesmethoxycurc
umin
Fissistigma
Antagonism
2
-N-
Phenylethylcinnam
amide
10-11
antimicrobial agents in plants
, while they
1
0
Amorpha
Polygala
Antagonism
Antagonism
also were found to mediate symbiotic
interactions between plants and Rhizobium
12-13
bacteria . Among the stronger antagonists,
most of the hits contain long alkyl chains
substituted with aromatic moieties at the
terminal positions, or polyphenol containing
compounds.
Amorfrutin B
1
1
1
2
Chrysanthe-mum Antagonism
Table 1. Selected active compounds. Compounds
that showed more than 50% inhibition or activation
of QS at 30 µM
1
3
Glycyrrhiza
Glycyrrhiza
Antagonism
Antagonism
No. Structure
(name if available)
Source (genus) QS activity
Licochalcone B
1
2
Panax
Acacia
Agonist
14
Agonism
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7a
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Grevillea
Antagonism inhibitory effects for compounds 8 and 8a,
which is not surprising given that bis-
desmethoxycurcumin (8) and curcumin (8a),
which are found in the flowering plants of the
Antagonism Chrysanthemum genus and in turmeric
Calophyllum
(
Curcuma longa), respectively, have anti-
Altholactone
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microbial effects, among other therapeutic
15,
17-18
properties.
Compound 9,
which
Hits to leads - focused antagonist libraries with
structural refinements. To further investigate
structure-activity relationships among the
antagonists we selected compounds 8, 9, and
originates from plants belonging to the
Fissistigma genus showed moderate inhibitory
effects , but we saw no significant inhibitory
effect for the other analogs 9a-9w. As far as we
know, the effect of these compounds has never
been tested on bacteria before, but in previous
studies it has been shown that this family of
1
7a
(bisdesmethoxycurcumin,
and
N-
phenethylcinnamamide
5,5'-(1,10-
decanediyl)bis[1,3-benzenediol], respectively).
19
The synthesis of compound 8 and its analog 8a
compounds induces apoptosis in cancer cells .
(
curcumin, Scheme 1A) were performed
Compound 17a, which can be found in the
Grevillea genus flowering plants, was examined
along with its analogs 17b and 17c, and while
we observed strong QS inhibition for compound
17a in the initial library screening, we could not
separate the mixture of the three isomers 17a,
14
following the Pabon method with minor
modifications described by Venkateswarlu et
15
al . For analog 8b (Scheme 1A) we used an
alternative method, due to the likelihood of
side reactions on the unprotected catechol
moiety. Instead, we reacted compound 8a
17b and 17c (IC50
= 12.4 µM, preliminary data –
(
curcumin) with boron tribromide to obtain the
full characterization of the purified isomers will
be reported elsewhere). Compound 8b and 9a,
which contain catechol moieties, show
relatively strong inducing effects on the QS
response cascade. It was previously reported
that certain soil microbes are chemoattracted
to phenolic compounds. In A. tumefaciens,
phenolic compounds participate in chemotaxis
based interactions, which could lead to
activation of virulence genes, which may in turn
prompt the Ti plasmid transfer and accelerate
demethylated product. Then, compound 9 and
several analogues were obtained by simple
amide coupling reactions (Scheme 1B). The
synthetic route to compound 17a was designed
based on three main steps (Scheme 1C); a Heck
16
cross coupling reaction described by Li et al.
with slight modifications, hydrogenation of
conjugated double bonds and demethylation.
Interestingly, the Heck reaction resulted in two
additional isomers, which proved hard to
separate (their synthesis and biological
evaluation is part of ongoing investigations).
20-21
the infection process by A. tumefaciens . We
believe that this phenomenon affects the QS
system in an indirect way. We came across
another interesting phenomenon when we
compared the activities of compounds 9a and
Biological evaluation of synthetic library. To
study the activities of the natural and synthetic
derivatives on the QS system of A. tumefaciens,
we used the luminescent reporter strain A136
9
b. It appears that conjugated aromatic
compounds such as compound 9a have a better
inducing effect than the non-conjugated
compound 9b.
(
pCF218)(pMV26) in the presence (antagonist
assay) or absence (agonist assay) of 400 pM 3-
oxo-C -AHL (table 2). We observed mild
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Scheme 1. Synthetic routes for selected natural products and their analogs. A) Synthesis of
bisdesmethoxycurcumin (8) and analogs (8-8b). B) Synthesis of 2-N-phenethylcinnamamide (9) and analogs
(
9a-9w). C) Synthesis of 5,5'-(1,10-decanediyl)-bis-[1,3-benzenediol] (17a) and analogs (17b-17c).
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Table 2. Activities of the natural products and
their synthetic derivatives on the QS system
of A. tumefaciens
9
m
>100 >100
>100 >100
9n
9o
No.
8
Compound
EC50
µM
IC50
µM
>100 >100
0
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>100 33.3
>100 15.0
19.2 >100
9
9
p
q
>100 >100
>100 >100
>100 >100
>100 >100
8
8
a
b
9
r
9
>100
4.8
9
9
a
b
13.6 >100
65.1 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
9s
9
t
>100 >100
>100 77.4
9c
9
u
9
d
9
v
>100 >100
>100 >100
9
e
9
w
9
f
9
9
g
h
In vitro carrot disc infection assay. To assess
the virulence level of A. tumefaciens in the
presence of the most potent anti-QS
compounds, we performed tumor induction
>100 >100
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9i
>100 >100
>100 >100
>100 >100
>100 >100
assays on carrot discs , which provide both
visual and quantitative information on the
selected compounds effect (see methods).
Compounds 8a and 9 were selected as potent
inhibitors based on their QS activity in the
previous tests. The assay was performed on
carrot discs, eight replications for each
treatment under sterile conditions (Figure 1).
The tumor induction on carrots was tested at a
concentration range between 0 µM - 50 µM. A.
9j
9
k
9
l
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tumefaciens induced tumor formation which
could be inhibited strongly by both compounds
at 50 µM (>90%, Figure 2).
SUMMARY AND CONCLUSION
Over the past half a billion years plants have co-
evolved with microbes in general and plant
pathogens in particular. In order to deal with
threats and adjust to changing and often hostile
environments, plants have developed multiple
mechanisms to cope with adverse effects. We
Humans have therefore used plants as a
powerful and rich source of agents to promote
health and combat microbial pathogens. Here
we focus on the pathogen A. tumefaciens which
induces crown gall tumors in plants. Since this
disease is regulated and controlled by the cell
dependent mechanism-QS, using natural
products which are originated from plants could
help finding potential strong anti-QS that will
enable to attenuate infection. To achieve this
goal, we performed a wide library screening of
natural products on the QS system of A.
tumefaciens, we selected potential hits with
strong activity and we generated a synthetic
library of analogs. by evaluating the QS activity
of the synthetic library, we manage to acquire
knowledge on activity based structural
elements of analogs. the strongest QS inhibitors
were selected and tested in vitro by monitoring
over the tumor formation on surfaces of carrot
discs. Based on these results and the
mechanistic QS activity assays we can conclude
that compounds 8a, 9 and 17a are indeed
effective QS system inhibitors. To further
understand their inhibitory role in the QS
complex system in detail, additional studies are
needed. Notably, both the QS reporter and
wild-type virulence assays are somewhat
limited in terms of quantitative assessment of
virulence inhibition, and they do not take in
account the ability of bacteria to swarm or
engage in chemotaxis, which under natural
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conditions contribute greatly to their
pathogenicity.
Figure 1. Carrot disc infection assay after 4 weeks: (A)
carrot disc with LB medium, (B) carrot discs infected by
A. tumefaciens C58. (C) A. tumefaciens C58 in the
present of selected QS inhibitors at 10 µM (upper row)
and 50 µM (bottom row)
Figure 2. The effect of selected QS inhibitors on tumor
formation at different concentrations. The tumors on
each carrot disc were scalped and their weights were
measured. Data are represented as the mean weight
of eight experiments. P values of infected carrots in
the presence of QS inhibitors vs. infected carrots
without the tested compounds: (*) P < 0.02, (**)
P<0.01.
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METHODS
selected (compound that showed more than
0% activation or inhibition effect).
5
Reagents and general procedures. All solvents
and reagents were of reagent grade quality
A. tumefaciens A136 (pCF218) (pMV26) QS
activation assays. The effect of the synthetic
compounds on A. tumefaciens A136 (pCF218)
(pMV26) was measured as described by Brenier
(
Acros, Sigma-Aldrich or Fisher Scientific,
TM
Difco ) and used without further purification.
Luria−Bertani (LB) medium was prepared as
instructed. Compound Handling; Stock solutions
of synthetic compounds (10 mM) were
prepared in DMSO and stored at -20 °C in
sealed vials. The amount of DMSO used in small
molecule screens did not exceed 1 % (v/v).
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et al . an overnight culture of A. tumefaciens
A136 (pCF218) (pMV26) was grown in Luria-
Bertani
broth
(LB)
(Miller's
broth)
-
1
supplemented with 25 μg ml of kanamycin and
-
1
4.5 μg ml of tetracycline, at 28–30 °C for 24-
8 h. This overnight culture was diluted to
absorbance density (OD ) of 0.05 by fresh LB
4
Compound synthesis. Synthetic procedures and
characterization data can be found in the
Supporting Information.
600
medium.
A
white/clear bottom 96-well
microliter plate was prepared with wells
containing test compounds serially diluted into
LB medium (triplicates). 100 µL of the diluted
cells were added to each well. In case of the
A136 (pCF218) (pMV26) strain we performed
two different types of experiments: one in
which the test compounds were incubated with
Library screening -Luminescence Reporter
Assay Protocol. The effect of the library
compounds (AnalytiCon Discovery library) on A.
tumefaciens A136 (pCF218)(pMV26) (lacking Ti
−
plasmid, TraR response regulator, traI and TraI
promoter fused to luxCDABE) was measured as
23
described by Brenier et al . an overnight
the addition of 400 pM of 3-oxo-C -HSL to
8
monitor antagonist properties, and the other
was performed in the absence of exogenous 3-
culture of A. tumefaciens A136 (pCF218)
(
pMV26) was grown in Luria-Bertani broth (LB)
-
1
(Miller's broth) supplemented with 25 μg ml of
oxo-C -HSL to measure agonistic activity of the
8
-
1
kanamycin and 4.5 μg ml of tetracycline, at 28
30 °C for 24-48 h. The overnight culture was
diluted to absorbance density (OD ) of 0.05 by
test compounds. The control contained all the
substances besides the tested compound.
Luminescence was measured every 20 min for
–
600
o
fresh LB medium. The initial library screening of
3800 natural products was performed in
Grenier white 384-well microliter plate. The
activity of each compound was tested at
approximate concentration of 30 µM. Two
types of experiments were performed;
competition assay in the presence of 400 pM 3-
18 h with continuous shaking at 28.5 C, using a
Microtiter Plate Reader (Varioskan Flash,
Thermo). Average luminescence values divided
by OD600 values were plotted against the added
compound concentration.
A. tumefaciens carrot disc infection assay. The
effect of the potent synthetic compounds on A.
oxo-C -HSL and the other was performed in the
8
tumefaciens tumor formation was tested as
absence of 3-oxo-C -HSL to measure agonistic
8
22
described by Trigui et al
with slight
activity of the test compounds. The control
contained all the substances besides the tested
compounds. Luminescence was measured every
modifications. A. tumefaciens C58 strain was
cultured on Luria Bertani (LB) agar medium. A
single colony was transferred into LB broth
2
o
0 min for 18 h with continuous shaking at 28.5
medium
5.7 °C for 24 h. Carrots (Daucas carota) were
disinfected by scrubbing under running water
and
incubated
at
C, using a Microtiter Plate Reader (Varioskan
Flash, Thermo). Based on the luminescence
values of the tested compounds hits were
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and soap with a brush, then immersed in 95%
ethanol for 20 min. and 0.3 % NaClO for another
0 min. The carrots were then rinsed twice with
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