New peptide-based antimicrobials for tackling drug resistance in bacteria: Single-cell fluorescence imaging

Article abstract of DOI:10.1021/ml400073g

New peptide molecules with metal binding abilities proved to be active against multidrug resistant clinical isolates. One of them labeled with a dansylated lysine has been imaged inside single-multidrug resistant bacteria cells by deep ultraviolet fluorescence, showing a heterogeneous subcellular localization. The fluorescence intensity is clearly related to the accumulation of the drug inside the bacteria, being dependent both on its concentration and on the incubation time with cells.

Full text of DOI:10.1021/ml400073g

Letter
pubs.acs.org/acsmedchemlett
New Peptide-Based Antimicrobials for Tackling Drug Resistance in
Bacteria: Single-Cell Fluorescence Imaging
Jean-Marie Pages,† Slavka Kascakova,‡ Laure Maigre,† Anas Allam,§ Mickael Alimi,§ Jacqueline Chevalier,†
̀
̀
̀
Erwan Galardon,§ Matthieu Refregiers,‡ and Isabelle Artaud*,§
́
́
†UMR-MD1, Aix-Marseille Universite
́
IRBA, Faculte de Medecine, 27 Bd Jean Moulin, 13385 Marseille Cedex 05, France
‡DISCO Beamline, Synchrotron Soleil, L’Orme des Merisiers, 91192 Gif-sur-Yvette, France
§UMR 8601, LCBPT, Universite
́ ́ ̀
Paris Descartes, PRES Paris cite, 45 rue des Sts Peres, 75270 Paris Cedex 06, France
S
* Supporting Information
ABSTRACT: New peptide molecules with metal binding abilities proved to be
active against multidrug resistant clinical isolates. One of them labeled with a
dansylated lysine has been imaged inside single-multidrug resistant bacteria cells by
deep ultraviolet fluorescence, showing a heterogeneous subcellular localization. The
fluorescence intensity is clearly related to the accumulation of the drug inside the
bacteria, being dependent both on its concentration and on the incubation time
with cells.
KEYWORDS: Multidrug resistant, amphipatic peptide, metal binding group, microspectrofluorimetry, fluorescence imaging
he dissemination of multidrug resistant (MDR) Gram-
RWRWOBn, with a benzyl ester in C-terminal position.6
Tnegative bacteria drastically impairs the efficacy of AMPs are cationic, amphipathic peptides that have a broad
antibiotic families and limits their clinical uses.1,2 Actually, it
spectrum of activity associated with membrane permeabiliza-
tion.7,8 Thus, they can facilitate entry of additional molecules
is urgently needed to develop new classes of antimicrobial
from the exterior. To test the efficiency of the sequence, we
prepared derivative AA37 functionalized by the tetrapeptide
with the alternated sequence WRWROBn, and the hexapep-
tides AA36 and AA38 with the sequences RWRWRWOBn and
WRWRWROBn, respectively. Then, to evaluate the efficacy of
the metal binding group, we also synthesized the free N-
acylRWRWOBn tetrapeptide, AA19, and a derivative closely
related to AA08, AA24, in which the TPA moiety has been
replaced by a tris(benzyl)amine (TBA) without any chelating
ability. Finally, for the fluorescence imaging studies, we
prepared AA23 and AA33, fluorescent analogues of AA08
and AA24, respectively, including in the peptide sequence
(K*RWRWOBn), an additional lysine residue labeled on the ε-
amino group with a dansyl moiety.
Major pathways for the synthesis of all the compounds are
depicted in Scheme 1. N((6-(Amino-2-pyridyl)methyl)
N,N’bis((2-pyridyl)methyl)amine (TPA-NH2) was prepared
as described by Berreau et al.9 Condensation of dibenzylamine
with 3-nitrobenzyl bromide, followed by reduction in EtOH
with hydrazine hydrate in the presence of a catalytic amount of
Pd/C afforded 3-((dibenzylamino)methyl)amine (TBA-NH2).
The glutaryl spacer was introduced by reaction of TPA-NH2 or
TBA-NH2 with methyl 5-chloro-5-oxopentanoate in THF,
followed by saponification under mild conditions with LiOH in
THF/H2O, yielding TPA-NHCO(CH2)3COOLi and TBA-
agents to tackle drug resistance in bacteria. Mechanisms
associated with modifications of membrane permeability,
decreasing passive uptake (influx) or increasing active efflux
of antibiotics (efflux), are reported as key contributors of the
bacterial MDR phenotype.3,4 Moreover, today, a major concern
is associated with the lack of connection between the
intracellular location of a molecule and its antibacterial activity.4
This defect tragically impairs our understanding of antibiotic
accumulation inside the resistant bacterial cell and the possible
way to combat them.
Herein, we described the activity of a new class of
antibacterial agents that exhibit a noticeable activity against
resistant bacterial strains. They are dual compounds based on
the covalent binding between a short antimicrobial peptide
analogue, to enhance the permeability, and a metal chelating
group to search for a new activity. They are active against E.
aerogenes, EA289, a MDR clinical isolate, which overexpresses
efflux pumps5 and made this strain insensitive to classical
antibiotics such as ciprofloxacin. One of these molecules
(AA23) presents the original property to be strongly
fluorescent after deep ultraviolet (DUV) excitation. Since
resolution is directly proportional to the wavelength (Rayleigh
criteria), we used this characteristic to determine its intra-
cellular accumulation inside individual MDR EA289 bacteria.
The lead molecule AA08 is a two-component compound,
which contains a tris-(pyridylmethyl)amine metal binding
group (TPA), [N((6-(amino)-2-pyridyl)methyl)N,N’-bis((2-
pyridyl) methyl)amine] linked via a glutaryl spacer to a small
tetrapeptide analogue of antimicrobial peptide (AMP),
Received: February 25, 2013
Accepted: May 7, 2013
© XXXX American Chemical Society
A
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ACS Medicinal Chemistry Letters
Letter
a
by 1H NMR (Figure S1, Supporting Information), mass
spectrometry, and elemental analysis, and their characteristics
are described in the experimental procedures. Complete
syntheses of all intermediates are detailed in the Supporting
Information.
Scheme 1. Synthesis of Peptide Compounds
The activity of the various molecules was evaluated using
recommended bacteriological assays.10 While AA08 displayed
some activity against wild type E. coli AG100, it showed a
noticeable activity against E. aerogenes EA289, a MDR clinical
isolate which has lost its sensitivity toward fleroxacin and
ciprofloxacin. In contrast, the N-acyl tetrapeptide alone, AA19,
as well as AA24, which possesses a tris(benzyl)amine without
any chelating property, were inefficient against both AG100 and
EA289. These results associated with the high binding affinity
of divalent cations such as Zn(II), Cu(II), and Fe(II) for TPA11
support the hypothesis that the efficiency of AA08 is likely
related to its ability either to trap cations essential to bacterial
survival12 or to interact with a presently unknown target in
bacteria. There is no marked difference of activity between TPA
compounds containing a tetrapeptide or a hexapeptide. All
derivatives presented similar activities on the tested strains
(Table 1) being more active than actinonin and usual
Table 1. Antibacterial Activity of AA Series and Antibiotics
on Enterobacterial Strains
MIC (μM)
a
b
E. coli
E. aerogenes
a
(A) (i) piperidine/DMF (20/80%); (ii) DMF/pyridine/Ac2O (7/1/
2, v:v:v); (iii) H2O/TIS/EDT/TFA (1.5:2:2.5:94, v:v:v:v). (B) (i)
piperidine/DMF (20:80, %); (ii) Fmoc-Lys(Dansyl)-OH/PyBOP/
HOBt/DIEA, DMF; (iii) piperidine/DMF (20/80, %); (iv) TPA-
NHCO(CH2)3COOLi or TBA-NHCO(CH2)3COOLi/HBTU/
HOBt/DIEA, DMF; (v) H2O/TIS/EDT/TFA (1.5:2:2.5:94,
v:v:v:v). (C) (i) piperidine/DMF (20/80%); (ii) TPA-NHCO-
(CH2)3COOLi or TBA-NHCO(CH2)3COOLi/HBTU/HOBT/
DIEA, DMF; (iii) H2O/TIS/EDT/TFA (1.5:2:2.5:94, v:v:v:v). (D)
(i) piperidine/DMF, (20/80, %); (ii) Trp-CO2Bn or Arg-CO2Bn/
HBTU/HOBt/DIEA, DMF.
AG100
WT
AG100A
EA289
EA298
molecules
AcrAB-strain
MDR isolate
TolC-strain
c
Act
>128
0.125
0.5
32
2
>128
64
32
d
Cip
0.03
0.125
8
8
e
Fle
>128
16
16
AA08
AA23
AA19
AA24
AA36
AA37
AA38
8
16
8
16
8
64
16
64
8
>128
128−64
16
128−64
64
64
8
8
16
8
8
4
NHCO(CH2)3COOLi (Schemes S1 and S2, Supporting
Information). The peptide was assembled manually through
solid phase peptide synthesis (SPPS) on a low load Wang resin
preloaded with arginine or tryptophane (0.39 mmol·g−1) using
Fmoc synthesis. Each deprotection of Fmoc groups was
performed twice with 20% piperidine/DMF (v:v) and checked
by UV−vis spectroscopy at 300 nm. Coupling was achieved in
DMF with 3 equiv of the amino acids Fmoc-Trp(Boc)-OH,
Fmoc-Arg(Pbf)-OH or Fmoc-Lys(Dansyl)-OH, and TPA-
NHCO(CH2)3COOLi or TBA-NHCO(CH2)3COOLi. HOBt,
and PyBOP or HBTU were used as activators in the presence
of DIEA (aa/HOBt/(PyBOP or HBTU)/DIEA, 1:1:1:2). The
precursor of peptide 6 was acylated with acetic anhydride in a
mixture of pyridine and DMF. After cleavage of the final
peptides from the resin, the side-chain protecting groups were
removed by treatment with TFA/EDT/TIS/water (94/2.5/2/
1.5, v:v:v:v). All the peptides in TFA solution were directly
precipitated into diethyl ether. Then, after dissolution in water,
they were lyophilized to afford the desired peptides 6, 7a−b,
and 8a−e. The last residue Trp-COOBn or Arg-COOBn was
then introduced as previously described,6 in DMF solution
using HOBT and HBTU as activators in the presence of DIEA
to afford AA08, AA19, AA23, AA24, AA33, AA36, AA37, and
AA38. All the final compounds were thoroughly characterized
8
8
16
8
a
b
c
d
Laboratory strains. Resistant clinical isolates. Actinonin. Cipro-
e
floxacin. Fleroxacin.
antibiotics such as ciprofloxacin on clinical MDR isolates.
Moreover, at a difference of one dilution, compound AA37
with the alternate sequence WRWROBn is equipotent as AA08
toward EA289 and EA298. Introduction of a dansylated lysine
in AA08 did not alter the activity, and AA23 was as active as
AA08.
AA23 was further assayed for accumulation and location
determinations inside resistant isolates. DUV microspectro-
fluorimetry spectra recorded with λex 280 nm from single
bacteria treated or untreated with AA23 are shown in Figure S2
(Supporting Information). The emission peak of AA23 around
340 nm overlaps with the tryptophan fluorescence of bacterial
proteins, while the peak centered at 510 nm exclusively
corresponds to AA23 fluorescence. In these conditions, the
resolution is around 180 nm,13 which enables to visualize the
accumulation of the labeled compound in single bacteria cell
with size in the range 0.5 to 5 μm (2 × 10−12 cm3). Thereby, for
single-cell imaging, the fluorescence of AA23 was detected
within a 480−550 nm wavelength range. The concentrations of
B
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ACS Medicinal Chemistry Letters
Letter
AA23 ranged from 0.5 to 3 × MIC, and two incubation times
of 15 and 60 min were selected. For each condition, two
different determinations of localization with a minimum of 30
bacteria per plane were performed. Each bacterium (minimum
size 4 pixels; maximum size 20 pixels) was individually counted
after automatic recognition of bacteria patterns under ImageJ.
Subcellular segregation of the compound inside individual
bacteria is clearly shown in Figures 1 and S3 (Supporting
formation of aggregates inside the bacteria (Figure S4,
Supporting Information).
In summary, we have described synthesis and antibacterial
properties of a new class of peptides with both metal
complexing abilities and permeabilizing properties, which are
active against multidrug resistant bacteria. For the first time, the
biological activity and the location of a new antibacterial
molecule are directly related using antibacterial susceptibility
assays and DUV fluorescence spectroscopy. This combined
approach enables to assess the relationships between the
antibacterial properties of a new molecule with a fluorescent tag
to its internalization. The in situ determination of drug
accumulation gives new clues to dissect the membrane-
associated mechanisms of resistance and to improve the
diffusion profile of new antibacterial molecules.
EXPERIMENTAL PROCEDURES
■
Chemicals. 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyl-uronium
hexafluorophosphate (HBTU), N-hydroxybenzotriazole (HOBt),
benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate
(PyBOP), trifluoroacetic acid (TFA), triisopropylsilane (TIS), ethan-
1,2-dithiol (EDT), anisole, N,N-diisopropylethylamine (DIEA), (N-α-
Fmoc-N-γ-(2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl)-L-ar-
ginine (Fmoc-Arg(Pbf)-OH), N-α-Fmoc-N(in)-Boc-L-tryptophan, N-
alpha-(9-fluorenylmethyloxycarbonyl)-N-in-tert-butyl-oxycarbonyl-L-
tryptophan (Fmoc-Trp(Boc)-OH), N-α-Fmoc-N-ε-[5-
(dimethylamino)naphthalene-1-sulfonyl]-L-lysine (Fmoc-Lys-
(Dansyl)-OH)), and tryptophan benzyl ester (Trp-OBn) were
purchased from Aldrich.
Figure 1. DUV fluorescence of AA23. Fluorescence (on the left) and
transmission (on the right) images of EA289 bacteria after 30 min
incubation with AA23 at 57.5 μM (λex 280 nm; λem 480−550 nm).
The heterogeneous localization of the molecule is clearly seen between
bacterial cells and inside one bacterium.
All the syntheses are detailed in the Supporting Information.
Bacterial Strains, Growth and Susceptibility Determinations.
E. aerogenes strains used: EA289 is a clinical multidrug resistant strain
that overexpresses AcrAB-tolC efflux pumps, and EA298 is its tolC−
derivative. E. coli AG100 is used as a control strain, and its derivative
AG100A is deleted of AcrAB and hypersensitive to chloramphenicol,
tetracycline, ampicillin, and nalidixic acid.14 Strains were routinely
grown at 37 °C on Luria−Bertani (LB) agar or in LB broth,
supplemented with kanamycine (50 μg·mL−1) for EA298 and
AG100A. Minimal inhibitory concentrations (MICs) were determined
with an inoculum of 2 × 105 cfu in 200 μL of Mueller Hinton (MH)
broth containing 2-fold serial dilutions of each antibiotics or
compounds.
Information) with biapical localization in most of the bacteria
retaining the molecule and, for some, a central punctuation.
Even though an absolute quantification of the molecule inside a
bacterium is not yet possible, the fluorescence intensity clearly
depends on the concentration of AA23 used in the incubation
experiments, as well as on the incubation time, as shown in
Figure 2.
For the sake of comparison, we tried the same experiment of
single-cell imaging by fluorescence with AA33, an inactive
compound. Results are clearly different since we observed the
Ciprofloxacin and fleroxacin were used as internal antibiotic
reference. The results were scored after 18 h at 37 °C.
Compound Accumulation in Single Bacterial Cells. Bacteria
growth and incubation have been previously described.10 Briefly, the
bacterial suspension was centrifuged at 6000g for 15 min at 20 °C, and
pellets were resuspended in 1/10 of the volume in a sodium phosphate
buffer (50 mM) at pH 7 supplemented with MgCl2 (NaPi-MgCl2
buffer) to obtain a density of 1010 CFU·mL−1. Bacteria suspension (1.6
mL) was incubated for 15, 30, or 60 min at 37 °C (final volume 2 mL)
with AA23 at different concentrations (8, 40, or 57.5 μM). Bacterial
suspensions incubated without molecules were used as controls.
Suspensions (800 or 400 μL) were then loaded on 1 M sucrose
cushion (1100 or 550 μL, respectively) and centrifuged at 13 000 rpm
for 5 min at 4 °C to eliminate extracellular-adsorbed compound and
collect washed bacteria. To measure the compound uptake by EA289
and EA298 strains, we used the routine fluorimetric method previously
described.10 Briefly, pellets corresponding to 800 μL of suspension
were solubilized with 500 μL of 0.1 M Glycin-HCl pH 3 buffer at least
2 h at room temperature. After a centrifugation for 10 min at 13 000
rpm, 400 μL of lysates was diluted in 600 μL of 0.1 M Glycin-HCl pH
3 buffer and analyzed by spectrofluorimetry. To detect the antibiotic
fluorescence from single bacteria, pellets corresponding to 400 μL of
suspension were resuspended in 200 μL of NaPi-MgCl2 buffer and
analyzed by DUV microspectrofluorimetry or DUV fluorescence
imaging as described in Jamme et al.13
Figure 2. Concentration and incubation time-dependent fluorescence
intensity. This figure shows the fluorescence intensity of EA289
bacteria after incubation times of 15 and 60 min with AA23 at
concentrations corresponding to 0.5 MIC (8 μM) and 2.5 × MIC (40
μM).
C
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ACS Medicinal Chemistry Letters
Letter
(11) Anderegg, G.; Hubmann, E.; Podder, N. G.; Wenk, F.
Pyridinderivate als komplexbildner. XI. Die thermodynamik der
metallkomplexbildung mit bis-, tris- und tetrakis[(2-pyridyl)methyl]-
aminen. Helv. Chim. Acta 1977, 60, 123−140.
ASSOCIATED CONTENT
■
S
* Supporting Information
All the syntheses of peptides combined to a TPA moiety are
1
described. H NMR spectra of AA08, AA19, AA23, AA24,
(12) Ma, Z.; Jacobsen, F. E.; Giedroc, D. P. Coordination chemistry
of bacterial metal transport and sensing. Chem. Rev. 2009, 109, 4644−
4681.
AA33, AA36, AA37, and AA38 are shown in Figure S1. Figure
S2 shows fluorescence emission spectra after excitation at λ =
280 nm recorded on a film of EA289 single bacterial cells
untreated and treated with AA23. Figures S3 and S4 show
DUV fluorescence imaging of single EA289 bacterial cells
treated or not with AA23 and AA33, respectively (λex = 280
nm; 480 ≤ λem ≤ 550 nm). This material is available free of
charge via the Internet at http://pubs.acs.org.
(13) Jamme, F.; Villette, S.; Giuliani, A.; Rouam, V.; Wien, F.;
́ ́
Lagarde, B.; Refregiers, M. Synchrotron UV fluorescence microscopy
uncovers new probes in cells and tissues. Microsc. Microanal. 2010, 16,
507−514.
(14) Viveiros, M.; Jesus, A.; Brito, M.; Leandro, C.; Martins, M.;
Ordway, D.; Molnar, A. M.; Molnar, J.; Amaral, L. Inducement and
reversal of tetracycline resistance in Escherichia coli K-12 and
expression of proton gradient-dependent multidrug efflux pump
genes. Antimicrob. Agents Chemother. 2005, 49, 3578−3582.
AUTHOR INFORMATION
Corresponding Author
*(I.A.) E-mail: isabelle.artaud@parisdescartes.fr. Fax: 33-1-42
86 83 87.
■
Author Contributions
The three groups contributed equally to this work.
Funding
S.K., and L.M. and A.A. received postdoctoral fellowships from
Soleil and ANR METABACT, respectively.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
ANR-2010 BLAN METABACT, CNRS (France), Paris
Descartes and Aix-Marseille Universities, IRBA, and Synchro-
tron Soleil are gratefully acknowledged.
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