Highly active modulators of indole signaling alter pathogenic behaviors in gram-negative and gram-positive bacteria

Article abstract of DOI:10.1002/chem.201303510

Indole is a universal signal that regulates various bacterial behaviors, such as biofilm formation and antibiotic resistance. To generate mechanistic probes of indole signaling and control indole-mediated pathogenic phenotypes in both Gram-positive and Gram-negative bacteria, we have investigated the use of desformylflustrabromine (dFBr) derivatives to generate highly active indole mimetics. We have developed non-microbicidal dFBr derivatives that are 27-2000 times more active than indole in modulating biofilm formation, motility, acid resistance, and antibiotic resistance. The activity of these analogues parallels indole, because they are dependent on temperature, the enzyme tryptophanase TnaA, and the transcriptional regulator SdiA. This investigation demonstrates that molecules based on the dFBr scaffold can alter pathogenic behaviors by mimicking indole-signaling pathways. Phenotype control: Non-microbicidal desformylflustrabromine (dFBr) derivatives that are 27-2000 times more active than indole in modulating biofilm formation, motility, acid resistance, and antibiotic resistance were developed. This investigation demonstrates that molecules based on the dFBr scaffold can alter pathogenic behaviors by mimicking indole-signaling pathways (see scheme).

Full text of DOI:10.1002/chem.201303510

FULL PAPER
DOI: 10.1002/chem.201303510
Highly Active Modulators of Indole Signaling Alter Pathogenic Behaviors in
Gram-Negative and Gram-Positive Bacteria
[
a]
Marine J. Minvielle, Kristen Eguren, and Christian Melander*
Abstract: Indole is a universal signal
that regulates various bacterial behav-
iors, such as biofilm formation and an-
tibiotic resistance. To generate mecha-
nistic probes of indole signaling and
control indole-mediated pathogenic
phenotypes in both Gram-positive and
Gram-negative bacteria, we have inves-
tigated the use of desformylflustrabro-
mine (dFBr) derivatives to generate
highly active indole mimetics. We have
developed non-microbicidal dFBr de-
rivatives that are 27–2000 times more
active than indole in modulating bio-
film formation, motility, acid resistance,
and antibiotic resistance. The activity
of these analogues parallels indole, be-
cause they are dependent on tempera-
ture, the enzyme tryptophanase TnaA,
and the transcriptional regulator SdiA.
This investigation demonstrates that
molecules based on the dFBr scaffold
can alter pathogenic behaviors by mim-
icking indole-signaling pathways.
Keywords: desformylflustrabro-
mine · mechanistic probes · natural
products
· phenotype control ·
structure–activity relationships
[
11]
Introduction
sistance,
induces the expression of multidrug exporter
[12,13]
genes and increases antibiotic resistance.
In addition,
Bacteria coordinate collective behaviors by using different
cell–cell communication systems to protect the community
when subjected to adverse conditions. Quorum sensing (QS)
describes one such behavior, in which secreted chemical sig-
nals, called autoinducers (AI), are used to coordinate gene
indole has been shown to decrease E. coli biofilm formation
in a non-toxic manner by repressing motility, chemotaxis,
[14,15]
and cell adherence,
whereas it promotes biofilm forma-
[1,2]
expression based on population density.
clude several classes of molecules encompassing acylhomo-
QS signals in-
[3]
serine lactones (AHLs) in Gram-negative bacteria, autoin-
[4]
ducing peptides (AIPs) in Gram-positive bacteria and au-
toinducer-2 (AI-2) shared by both Gram-negative and
[5]
Gram-positive species (Figure 1A).
Outside of AI-2 communication pathways, indole signal-
ing has recently received attention as a putative universal
[6]
signaling network. First to be identified in the indole-posi-
tive bacterial species Escherichia coli, indole is synthesized
in high concentration by the action of tryptophanase (Tna),
which converts l-tryptophan into indole, pyruvate, and am-
[7]
monia during the stationary phase of growth. To date, over
5 bacterial species, both Gram negative and Gram positive,
8
have been reported to produce indole, whereas both indole-
producing and non-indole-producing strains of bacteria will
[8,9,10]
adapt their behavior in response to extracellular indole.
It has been shown that indole controls a variety of key
pathogenic phenotypes. In E. coli, indole decreases acid re-
[
a] M. J. Minvielle, K. Eguren, Prof. Dr. C. Melander
Department of Chemistry, North Carolina State University
Raleigh, NC, 27695-8204 (USA)
Fax : (+1)919-515-5079
E-mail: ccmeland@ncsu.edu
Figure 1. A) General structures of major classes of autoinducers.
B) Flustramine family inspiration for the design of a synthetic dFBr ana-
logue. C) General scaffold of the desformylflustrabromine library.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201303510.
Chem. Eur. J. 2013, 19, 17595 – 17602
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
17595

tion in the non-indole-producing bacterial strain Pseudomo-
strains similarly, we hypothesized that compound 1 and our
new generation of analogues may mimic other indole-depen-
dent bacterial behaviors. Our ultimate goal is to develop
highly active modulators of indole signaling that control
pathogenic behavior through indole-signaling pathways in
both Gram-negative and Gram-positive bacteria, to employ
these molecules to address fundamental questions about
indole signaling in disparate bacterial species, and to explore
the impact of disrupting indole signaling both in vitro and in
vivo.
[16]
nas aeruginosa.
In E. coli, phenotypical changes, such as
biofilm formation and antibiotic resistance, have been found
[13]
to be more significant at 258C. At 378C, indole is believed
to control biofilm formation through the transcriptional reg-
ulator SdiA although the exact mechanism has not yet been
[14]
determined.
Many natural and synthetic indole derivatives have been
shown to control bacterial phenotypes in a variety of Gram-
positive and Gram-negative bacteria and have been investi-
gated as potential candidates for antivirulence thera-
[17,18,19,20]
pies.
Because indole derivatives are believed to
modulate a large panel of bacterial behaviors by competing
with indole or promoting indole biding to its signal receptor,
indole-based natural products have been employed in our
group as structural inspiration for the design of small mole-
cules to potentially mimic indole signaling and modulate
pathogenic phenotypes. In previous work, we investigated
the potential of flustramine metabolites, isolated from the
marine invertebrate Flustra foliacea, containing a pyrroloin-
doline or indolic core as a template to construct small mole-
Results and Discussion
The introduction of alternative aromatic substituents off the
aliphatic nitrogen to access analogues of compound 1 was
achieved by alkylation of the tryptamine derivative 3
(Scheme 1). All second-generation dFBr analogues of
1 were accessed from tryptamine 2 in five steps. The alkyla-
tion of nosyl-protected tryptamine 3 was followed by instal-
lation of the reverse prenyl group at the C2 position by ap-
plying Danishefskyꢁs tert-prenylation methodology to give
[21]
cules that inhibit bacterial biofilm formation (Figure 1B).
[22]
We observed the inhibition of E. coli and Staphylococcu-
s aureus biofilm formation by the natural products flustrami-
ne C and desformylflustrabromine, analogous to the effect
of indole itself. Tuning the flustramine scaffold by develop-
ing a desformylflustrabromine (dFBr) library generated the
most potent analogue 1, which inhibited biofilm formation
by S. aureus and E. coli 10–1000 times more potently than
indole itself through a non-microbicidal mechanism, with
IC50 values of 5.9 mm and 53 mm, respectively. Mechanistic
studies in wild-type and knockout E. coli strains have shown
that, parallel to indole itself,
intermediates 5a–f in 34–54% yield.
Treatment of 5a–f
[23]
with N-bromosuccinimide (NBS) in AcOH/HCO H (3:1)
2
gave selective bromination at the C6 position and gave in-
termediates 6a–f in 11–61% yield. Deprotection of the ali-
phatic nitrogen was carried out under Fukayama conditions
by using thiophenol with potassium carbonate to give final
[24]
compounds 7a–f in 80–90% yield.
dFBr analogues inhibit biofilm formation of E. coli, Aci-
netobacter baumannii, S. aureus and methicillin-resistant
Staphyloccocus aureus (MRSA): by synthesizing this second
the activity of compound 1 is
dependent on temperature,
TnaA, and the transcriptional
regulator SdiA, demonstrating
the capacity of 1 to modulate
indole-based signaling path-
ways. This indicated that we
may be mimicking indole sig-
naling with our dFBr deriva-
tives.
Encouraged by the biological
activity of compound 1, we
elected to further explore the
structure–activity relationship
(
SAR) of 1 and synthesize
a second generation of ana-
logues by introducing different
aromatic substituents on the ali-
phatic nitrogen and increasing
the tryptamine chain length
(
Figure 1C). Considering the
fact that indole and compound
control biofilm formation of
1
wild-type and knockout E. coli Scheme 1. Synthetic route introducing structural diversity on the aliphatic nitrogen.
17596
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Chem. Eur. J. 2013, 19, 17595 – 17602

Highly Active Modulators of Indole Signaling
FULL PAPER
[
6,14]
generation of analogues, our goal was to delineate addition-
al SAR parameters on the dFBr scaffold and to develop
highly active modulators of indole signaling with antibiofilm
activity that surpass compound 1, while still acting through
a non-toxic mechanism. Compounds were screened to inves-
tigate their effects on biofilm development by using A. bau-
mannii (ATCC 19606), E. coli (K12 ER2718), S. aureus
1000 mm,
our most potent compounds 7a and b are 27–
130 times more active than indole. In the same manner, ana-
logues 7a and b inhibited biofilm formation of A. baumannii
with 2 to 2.5-fold greater efficacy compared to compound 1,
with IC50 values of 26.3 and 34.9 mm, respectively. In addition
to these two hits, compound 7d presented an increased ac-
tivity against A. baumannii biofilm formation in comparison
to 1. Unfortunately, this new generation of analogues did
not exhibit increased antibiofilm activity towards Gram-pos-
itive bacteria except for 7e, which presented an IC50 value
of 2.3 mm against S. aureus.
The impact of chain length on biological activity was also
investigated while preserving the structural features proven
to be fundamental for biological activity (the bromine atom
at the C6 position and the reverse prenyl group at the C2
position). For these new analogues, the aromatic substitu-
ent appended to the aliphatic nitrogen was selected by con-
sidering the three most potent compounds of the dFBr li-
brary in terms of antibiofilm activity against E. coli. Com-
mercially available carboxylic acid 8 was treated with ethyl
chloroformate and triethylamine in dry tetrahydrofuran
leading to the corresponding amide 9. This intermediate was
reduced by using lithium aluminum hydride to give the cor-
(
ATCC 29213), and MRSA (ATCC BAA44) as our clinical-
ly relevant model bacteria, and effects were quantified by
determining IC50 values. Herein, we define the compound
concentration required to inhibit 50% of biofilm formation
relative to an untreated control as the IC50 value. Growth-
curve analysis was performed for each active compound to
determine their toxicity towards planktonic bacteria. This
analysis revealed if a compound inhibited biofilm formation
through a non-toxic mechanism similar to indole, or acted
through a bactericidal mechanism. Because bacteria tolerate
and adapt to microbicidal therapies, we aimed to develop
non-toxic compounds to avoid evolutionary pressure and
thereby impart a reduced risk of resistance development.
The results of these studies are summarized in Table 1.
[21]
Table 1. Inhibition activity of dFBr analogues. All IC50 values are report-
ed at mm concentrations.
[
25]
responding 3-indoylalkylamine 10 in 92% yield,
which
was then nosyl-protected, alkylated, prenylated, and bromi-
nated by using the same conditions, as was previously de-
scribed. This synthetic route gave final products 11a–
c (Scheme 2).
These new compounds were screened for antibiofilm ac-
tivity against E. coli, A. baumannii, S. aureus and MRSA.
We found that extending the tryptamine chain with one ad-
ditional carbon led to either a conservation of the antibio-
film activity or a decrease up to fourfold for all four bacteri-
al strains. Because compounds 1, 7a and b presented the
best antibiofilm activity, these analogues were selected to
further mechanistic studies.
Compound
E. coli
A. baumannii
S. aureus
MRSA
[
[
[
a]
b]
b]
[b]
1
53.0
15.6
18.5
19.2
19.7
31.4
90.8
16.3
32.1
52.5
70.3
26.3
34.9
5.9
7.7
11.7
11.9
14.7
2.3
4.3
14.6
10.3
14.3
[b]
[a]
7
7
7
7
7
7
1
1
1
a
b
c
d
e
f
1a
1b
1c
[b]
[a]
[
b]
[a]
200
49.0
103.5
[
[
b]
b]
[b]
[a]
14.1
6.3
[
a]
>200
18.4
13.4
9.8
37.0
12.0
9.0
[
[
[
b]
b]
b]
[b]
[b]
[a]
56.7
39.4
73.2
[b]
10.8
15.7
[
a] Indicates inhibition of biofilm formation through a toxic mechanism
as was determined by growth-curve analysis. [b] Indicates not complete
toxicity, bacterial-growth delay was noted in the first 8 h, bacterial densi-
ty was identical at 24 h.
Indole and dFBr analogues reduce the biofilm formation of
E. coli to a greater extent at 258C: It has been established
All the synthesized com-
pounds (except 7 f) equaled or
surpassed the antibiofilm activi-
ty of compound
1
against
E. coli. Although many ana-
logues affected the planktonic
cell growth of A. baumannii,
S. aureus, and MRSA, none of
these analogues presented a bac-
tericidal effect on E. coli. Com-
pounds 7a and b inhibited bio-
film formation of E. coli with 3
to 3.5-fold greater efficacy than
compound 1 with IC50 values of
15.6 and 18.5 mm, respectively.
Because indole is an active
signal that inhibits E. coli and
S. aureus biofilms at 250– Scheme 2. Synthesis of dFBr series 11a–c.
Chem. Eur. J. 2013, 19, 17595 – 17602
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C. Melander et al.
that temperature can have an
important effect on bacterial
[26,27]
signaling.
Indole signaling
has been primarily observed at
temperatures lower than 378C.
It has been shown that indole-
mediated reduction of E. coli
biofilm formation is greater at
25 than 378C, and indole con-
trols biofilm formation through
the transcriptional regulator
[13]
SdiA at 378C.
In our previ-
ous work, mechanistic studies
based on biofilm inhibition of
E. coli at different temperatures
demonstrated that the activity
of analogue 1 paralleled indole.
because its activity was depen-
dent on temperature, SdiA, and
[21]
TnaA. Based on this activity,
we first established temperature
and gene-dependent biofilm-in-
hibition studies with lead com-
pounds 7a and b and compared
their activity to indole and
dFBr analogue 1 by using the
wild-type
E. coli
strain
BW25113 and the isogenic
knockout mutants DtnaA and
DsdiA (Figure 2). Similar to
indole, we found that these
second generation of dFBr ana-
logues inhibited biofilm forma-
tion in
a
dose-dependent
manner that was more pro-
nounced at 25 than at 378C for
all three strains of bacteria
without affecting planktonic
bacterial growth. At 258C,
wild-type E. coli biofilm was re-
duced by 82% in the presence
of 100 mm analogue 7a com-
Figure 2. Biofilm inhibition activity of indole and dFBr analogues against wild-type and knockout E. coli
strains at 25 and 378C. Concentrations are reported in mm.
pared to 33% at 378C. Because the DtnaA mutant does not
produce indole, this strain exhibited biofilm inhibition to
a greater extent than the wild-type upon addition of exoge-
nous indole, which was amplified at 258C. The same result
was observed upon addition of compounds 1 and 7b, which
inhibited biofilm formation of the DtnaA mutant by 79 and
Indole and dFBr analogues promote P. aeruginosa PAO1
biofilm formation: Although P. aeruginosa does not produce
indole, it has been shown that indole is a signal that stimu-
[6,19]
lates biofilm formation of this strain at 30 and 378C.
To
evaluate the effect of indole and our dFBr analogues on
P. aeruginosa biofilm formation, we tested different concen-
trations of indole and synthetic compounds for their ability
to modulate PAO1 biofilm formation after 24 h at 258C, be-
cause at this temperature, the biofilms formed by this bacte-
rial species have been found to be more robust than biofilms
formed at 378C. Given that P. aeruginosa may encounter
95%, respectively at 258C, compared to 69 and 59% for the
wild-type at 100 mm. At 378C, the inhibition of wild-type
bacteria by our analogues, especially 1, was more pro-
nounced than their effects on the DsdiA knockout mutant
strain. At a concentration of 100 mm, analogue 1 inhibited
biofilm formation of the wild-type strain by 81 versus 52%
for the DsdiA knockout mutant strain. These results indicate
that the activity of these compounds depend on both TnaA
and SdiA at 378C.
[
19]
E. coli in different environments,
indole at concentrations comparable to those produced by
we decided to test
[28]
E. coli in a rich medium. Indole promoted P. aeruginosa
biofilm formation in a dose-dependent manner. At 500 mm it
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Chem. Eur. J. 2013, 19, 17595 – 17602

Highly Active Modulators of Indole Signaling
FULL PAPER
Figure 3. Biofilm formation of PAO1 at 258C; effect of indole and dFBr
analogues on PAO1 biofilms.
increased biofilm formation by 1.3-fold compared to the
negative control (Figure 3), and did not affect planktonic
cell growth at this concentration.
Paralleling this activity, the three dFBr analogues in-
creased PAO1 biofilm formation to a greater extent than
indole at lower concentrations. Compound 1 increased bio-
film formation by 35% at 50 mm compared to the 500 mm
indole required to achieve the same increase. Similarly at
150 mm, analogues 7a and b elicited a 1.8-fold and 2.5-fold
increase in P. aeruginosa biofilm formation compared to the
negative control. In comparison, it requires a minimum of
1.0 mm indole to promote P. aeruginosa biofilm formation to
a similar degree.
Indole and dFBr analogues repress E. coli and P. aeruginosa
motility: Motility has been shown to be dependent on
indole signaling and has an essential role in the colonization
of new environments. In both E. coli and P. aeruginosa,
[19,29]
indole has been shown to decrease bacterial motility.
To
investigate the effect of our dFBr analogues on motility,
compounds 1, 7a and b were added to motility agar and
bacterial movement was quantified. In addition to the wild-
type bacterial strain and the DsdiA knockout mutant, the
isogenic mutant strains DtnaA, DtrpE, and DtnaC were used,
because these genes are also involved in E. coli indole syn-
thesis. The experiments were performed at 378C and motili-
ty was measured at 8 h (see the Supporting Information)
and 24 h (Figure 4A). Bacteria that do not express the tnaA
gene are unable to produce indole, whereas the DtrpE or
DtnaC knockout mutants produce less indole than the wild-
type E. coli. Therefore, the motility of the wild-type strain is
[14]
repressed in comparison with its knockout mutants.
As
expected, we observed that the DtnaA isogenic knockout
mutant is more motile than the wild-type strain of E. coli,
which produces indole. The presence of 500 mm indole in the
motility agar reduced the motility of all strains, whereas an
equivalent or greater effect was observed in the presence of
Figure 4. A) Motility of wild-type E. coli and knockout mutants after
2
4 h. B) Motility of P. aeruginosa PAO1 after 24 h.
5
0 mm of dFBr analogues. For example, with the DtnaA
alogue 1, and analogues 7a and b were 1.3 to 1.74-fold more
effective at suppressing motility in the wild-type E. coli
strain in comparison to the DsdiA mutant (33 and 47% in-
hibition for the wild-type compared to 22 and 27% for the
DsdiA mutant in the presence of 500 mm of indole and 50 mm
of 7b, respectively). Therefore, similar to indole, the activity
of our dFBr analogues is partially dependent on SdiA at
378C. Indole and our dFBr analogues both decreased the
mutant the swimming halo diameters at 24 h for this bacteri-
al strain were 1.63Æ0.31 (negative control), 0.99Æ0.19
(
0
indole), 0.78Æ0.24 (analogue 1), 0.50Æ0.28, and 0.53Æ
.04 cm (analogues 7a and b, respectively).
As expected, the effect of indole and our synthetic ana-
logues on motility was considerably muted for the DsdiA
mutant in comparison to the other E. coli strains. Indole, an-
Chem. Eur. J. 2013, 19, 17595 – 17602
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C. Melander et al.
[
12]
swimming motility of P. aeruginosa (Figure 4B). The motili-
ty halo diameter was decreased by 1.4-fold in the presence
of 500 mm of indole, whereas a decrease of 1.7-fold, 2-fold,
and 1.8-fold was observed for compounds 1 and 7a and b,
respectively at a concentration ten times lower than indole.
through the upregulation of multidrug exporter genes, we
assayed the antibiotic resistance of wild-type BW25113
grown to log phase in presence of indole (1 and 2 mm) or
different concentrations of dFBr analogues. Norfloxacin and
ampicillin belong to two different classes of broad spectrum
antibiotics that kill growing cells with distinct modes of
action; the fluoroquinolone norfloxacin inhibits DNA repli-
cation, whereas the b-lactam ampicillin inhibits cell-wall
synthesis. We performed our experiments in log phase to di-
rectly compare the phenotypical changes controlled by
growth in exogenous indole to our synthetic compounds.
The survival of E. coli was evaluated after three hours at
Indole and dFBr analogues decrease acid resistance of
E. coli: Indole has been shown to repress several E. coli
acid-resistance genes and in the process, significantly reduce
the bacterium acid tolerance. We evaluated the effect of our
dFBr analogues on acid resistance by submitting wild-type
E. coli BW25113 to an acid challenge at pH 2.5 (Figure 5).
3
78C (Figure 6A and B). As was expected, we observed
that indole and our dFBr compounds induced antibiotic re-
sistance of E. coli against the two antibiotics.
In comparison to the negative control, growth in 2 mm
indole increased survival rate by 27% when the bacteria
À1
were subjected to 5 mgmL ampicillin (Figure 6A) and
À1
5
1
0% for 0.25 mgmL norfloxacin (Figure 6B). Analogues
and 7a–b increased antibiotic resistance of BW25113 in
a dose-response manner. Growth in 1 mm indole increased
the survival rate by 13% in the presence of norfloxacin and
9
% in the presence of ampicillin, whereas an equivalent or
greater effect was observed upon growth in 10 mm of com-
pound 7b in presence of both antibiotics.
Conclusions
The desformylflustrabromine scaffold was used to develop
analogues that are able to control multiple bacterial behav-
iors. We have shown that our dFBr analogues are able to
1
) inhibit biofilm formation of E. coli in a manner depen-
dent on temperature, SdiA, and TnaA; 2) promote P. aerugi-
nosa biofilm formation; 3) repress E. coli and P. aeruginosa
motility; 4) decrease acid resistance of E. coli; and 5) in-
crease the antibiotic resistance of E. coli, all in a manner
that parallels indole itself (Figure 7). Because our analogues
are upwards of 2000 times more potent than indole itself at
altering pathogenic behaviors, the dFBr scaffold presents
potential for the design of small molecules to probe antivir-
ulence therapies in vivo. In addition to modulating indole-
controlled bacterial behaviors, compounds 1 and 7a and
b can be used as mechanistic probes to potentially deconvo-
lute indole signaling in Gram-positive and Gram-negative
bacteria to further delineate fundamental questions about
indole signaling itself.
Figure 5. Acid resistance of wild-type E. coli at pH 2.5 at 378C.
Because indole is a signal produced during the stationary
phase of growth, we performed our experiments in log
phase to directly observe the phenotypical changes attribut-
ed to growth in exogenous indole or synthetic compound.
Growth in indole or compounds 1 and 7a and b decreased
acid resistance in a dose-response manner. Growth in the
presence of 2 mm indole produced a decrease of 319-fold in
acid resistance, whereas 10 mm of analogue 7a was required
to achieve a similar effect. Only 1 or 5 mm of synthetic com-
pounds 7b and 1 were necessary to produce a decrease of
Experimental Section
5
29-fold and 650-fold, respectively, making our dFBr ana-
Bacterial strains and culture conditions: A. baumannii (ATCC 19606),
E. coli (K12 ER2718), S. aureus (ATCC 29213), MRSA (ATCC BAA44)
and P. aeruginosa (PAO1) strains were used for biofilm inhibition assay.
Wild-type E. coli (BW25113) and the isogenic deletion mutations DtnaA
logues 200–2000 times more active than indole itself in me-
diating acid-resistance of E. coli.
(
(
JW3686–7), DtrpE (JW1256–1), DtnaC (JW3685–1), and DsdiA
JW1901–5) from the Keio collection were used for mechanistic studies.
Indole and dFBr analogues increase antibiotic resistance of
E. coli: Because indole confers drug resistance to E. coli
Luria–Bertani (LB) broth and agar were used as the growth media for
17600
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Highly Active Modulators of Indole Signaling
FULL PAPER
Figure 7. Phenotypes regulated by indole and dFBr analogues.
0
.5% yeast extract) broth (LBNS) was used as the growth media for
P. aeruginosa. Bacteria were streaked from a À808C glycerol stock on
the appropriate agar plate, and a single colony was inoculated in 3 mL of
media and cultured at 378C overnight with agitation at 200 rpm. Prior to
the antibiotic- and acid-resistance assays, the overnight cultures were di-
luted in LB in the presence of indole or dFBr analogue and were re-
grown to mid-log phase (A600 =1.0).
Biofilm-inhibition procedure: The inhibitory effects of compounds
against biofilm formation was determined under static conditions by
[
30]
using a crystal-violet (CV) reporter assay. Inhibition assays were per-
formed by taking an overnight culture and sub-culturing it to an OD600 of
0
.01 into the appropriate media; LB was used for A. baumannii and
E. coli and TSB supplemented with 5% Glucose (TSBG) was used for
S. aureus and MRSA and LBNS for P. aeruginosa. Stock solutions of pre-
determined concentrations of the test compounds were then made in
DMSO (biology grade) and added to the inoculum to give the desired
concentrations, untreated inoculum served as the control. This was ali-
quoted (100 mL) into the wells of a 96-well PVC microtiter plate. Sample
ꢂ
plates were wrapped in GLAD Press nꢁ Seal followed by incubation
under stationary conditions for 24 h at 258C for P. aeruginosa and 378C
for all the other strains of bacteria. After incubation, the media was dis-
carded and the plates were washed with water. The sample plates were
then stained with 110 mL of 0.1% solution of CV and incubated at RT for
3
0 min. The CV stain was then discarded and the plates were washed
with water. The remaining stain was solubilized with 200 mL of 95% eth-
anol for 10 min and a sample of 125 mL of solubilized CV-stained ethanol
was transferred from each well into the corresponding wells of a polystyr-
ene microtiter dish. Biofilm inhibition was quantified by measuring the
OD540 of each well. A negative control lane with no biofilm was mea-
sured to determine background staining and was subtracted out. The per-
cent inhibition was calculated by the comparison of the OD540 for control
wells versus treated wells under identical conditions.
Motility-test procedure: Motility was determined by using plates contain-
[
31]
ing agar (0.3%) with tryptone (1%) and NaCl (0.25%). Bacteria were
grown from diluted overnight cultures to a turbidity of 1.0 at 600 nm, and
1
.5 mL of the bacterial culture was placed into the center of the agar
plate. The motility halos were measured at 8 h and 24 h. The effect of
indole and synthetic compounds was tested by adding indole (500 mm) or
compounds (50 mm) dissolved in DMSO (biological grade) to the agar.
An equivalent volume of DMSO was added as the negative control.
Each experiment was performed in duplicate by using two independent
cultures.
Acid-resistance assay: LB overnight cultures were diluted 1:100 and
grown with indole (1 mm or 2 mm) or synthetic compound dissolved in
DMSO (biological grade) at different concentrations. When the turbidity
reached 1.0 at 600 nm, the culture was then diluted 40-fold into a phos-
phate-buffered saline solution (pH 7.2) or in acidified LB (pH 2.5). Bac-
teria in acidic media were incubated at 378C during 1 h without shaking.
An equivalent volume of DMSO was added as the negative control. The
number of colony-forming units (CFU) per milliliter was determined by
plating serial dilutions on agar plates. The percentage of cells surviving
the acid challenge was calculated as the number of CFU after acid treat-
ment divided by the CFU before treatment. Each experiment was per-
formed in triplicate by using two independent cultures.
Figure 6. A) Effect of indole and dFBr analogues on norfloxacin resist-
À1
ance (0.250 ngmL ) after 3 h. B) Effect of indole and dFBr analogues
À1
on ampicillin resistance (5 mgmL ) after 3 h.
the wild-type E. coli strains and were supplemented with kanamycin
À1
(
32 mgmL ) for the use of the knockout mutants. Tryptic soy (TSB)
broth supplemented with 5% glucose and tryptic soy agar were used for
growth of the S. aureus and MRSA strains. No salt LB (1% tryptone+
Chem. Eur. J. 2013, 19, 17595 – 17602
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
17601

C. Melander et al.
Antibiotic resistance: LB overnight cultures were diluted 1:100 and
grown to a OD600 of 1.0 in the presence of indole (2 mm), different con-
centrations of synthetic compound or a corresponding volume of DMSO
[12] H. Hirakawa, Y. Inazumi, T. Masaki, T. Hirata, A. Yamaguchi, Mol.
Microbiol. 2005, 55, 1113–1126.
[13] J. Lee, X. Zhang, M. Hedge, W. Bentley, A. Jayaraman, T. Wood,
ISME J. 2008, 2, 1007.
[14] J. Lee, A. Jayaraman, T. Wood, BMC Microbiol. 2007, 7, 42.
[15] T. Bansal, D. Englert, J. Lee, M. Hegde, T. Wood, A. Jayaraman,
Infect. Immun. 2007, 75, 4597.
[16] M. Hentzer, H. Wu, K. Riedel, T. B. Rasmussen, N. Bagge, N.
Kumar, M. A. Schembri, Z. Song, P. Kristoffersen, M. Manefield, J.
Costerton, S. Molin, L. Eberl, P. Steinberg, S. Kjelleberg, N. Hoiby,
M. Givskov, EMBO J. 2003, 22, 3805–3815.
(
0
biological grade) as
.25 mgmL and ampicillin 5 mgmL ) were mixed with cells and incu-
a
negative control. Antibiotics (norfloxacin
À1
À1
bated for three hours at 378C with shaking. The percentage of cells sur-
viving the antibiotic treatment was calculated as the number of CFU
after antibiotic treatment divided by the CFU before treatment. Each ex-
periment was performed in triplicate by using two independent cultures.
[
17] R. Worthington, J. Richards, C. Melander, Org. Biomol. Chem.
012, 35, 310–315.
2
Acknowledgements
[
[
18] J. H. Lee, M. H. Cho, J. Lee, Environ. Microbiol. 2011, 13, 62–73.
19] J. Lee, C. Attila, S. L. G. Cirillo, J. D. Cirillo, T. K. Wood, J. Microb.
Biotechnol. 2009, 2, 75.
20] J.-H. Lee, Y.-G. Kim, M. H. Cho, J.-A. Kim, J. Lee, FEMS Micro-
biol. Lett. 2012, 329, 36.
The authors would like to thank the National Institutes of Health
GM055769) for their support.
(
[
[
[
21] C. Bunders, M. Minvielle, R. Worthington, M. Ortiz, J. Cavanagh, C.
Melander, J. Am. Chem. Soc. 2011, 133, 20160.
22] J. M. Schkeryantz, J. C. Woo, S. Danishefsky, J. Am. Chem. Soc.
[
[
1] A. Camilli, B. Bassler, Science 2006, 311, 1113–1116.
2] S. Rutherford, B. Bassler, Cold Spring Harbor Perspect. Med. 2012,
2
, a012427.
1
995, 117, 7025.
[
[
3] G. D. Geske, R. J. Wezeman, A. P. Siegel, H. E. Blackwell, J. Am.
Chem. Soc. 2005, 127, 12763.
[
[
[
23] T. Lindel, L. Brauchle, G. Golz, P. Bohrer, Org. Lett. 2007, 9, 283.
24] T. Fukuyama, C. Jow, M. Cheung, Tetrahedron Lett. 1995, 36, 6373.
25] S. H. Ang, P. Kratsel, S. Y. Leong, L. J. Tan, W. L. J. Wong, B. Yeug,
B. Zou, PCT Int. Appl., WO 2009132921A1 20091105, 2009.
26] H. Hasegawa, A. Chatterjee, Y. Cui, A. K. Chatterjee, Appl. Envi-
ron. Microbiol. 2005, 71, 4655–4663.
4] P. Mayville, G. Ji, R. Beavis, H. Yang, M. Goger, R. Novick, T. W.
Muir, Proc. Natl. Acad. Sci. USA 1999, 96, 1218–1223.
5] M. Miller, B. Bassler, Annu. Rev. Microbiol. 2001, 55, 165.
6] J. Lee, T. Bansal, A. Jayaraman, W. Bentley, T. Wood, Appl. Envi-
ron. Microbiol. 2007, 73, 4100.
7] N. Vega, K. Allison, A. Samuels, M. Klempner, J. Collins, Proc.
Natl. Acad. Sci. USA 2013, 110, 1218–1223.
8] J. H. Lee, J. Lee, FEMS Microbiol. Rev. 2010, 34, 426.
9] E. Nikaido, E. Giraud, S. Baucheron, S. Yamasaki, A. Wiedemann,
K. Okamoto, T. Takagi, A. Yamaguchi, A. Cloeckaert, K. Nishino,
Gut Pathog. 2012, 4, 5.
[
[
[
[
27] T. H. Han, J. H. Lee, M. H. Han, T. K. Wood, J. Lee, Res. Microbiol.
[
2
011, 162, 108–116.
[
[
28] G. Li, K. Young, Microbiology 2013, 159, 402–410.
29] Z. Ma, S. Gong, H. Richard, D. L. Tucker, T. Conway, J. W. Foster,
Mol. Microbiol. 2003, 49, 1309–1320.
[
[
[
[
30] G. A. OꢁToole, R. Kolter, Mol. Microbiol. 1998, 30, 295.
31] V. Sperandio, A. G. Torres, J. B. Kaper, Mol. Microbiol. 2002, 43,
[
10] D. Kim, I. Sitepu, Y. Hashidoko, Appl. Environ. Microbiol. 2013, 79,
845–4852.
8
09–821.
4
[
11] J. Lee, R. Page, R. Garcia-Contreras, J-M. Palermino, X.-S. Zhang,
Received: September 5, 2013
O. Doshi, T. K. Wood, W. Peti, J. Mol. Biol. 2007, 373, 11–26.
Published online: November 14, 2013
17602
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Chem. Eur. J. 2013, 19, 17595 – 17602