Real time detection of ESKAPE pathogens by a nitroreductase-triggered fluorescence turn-on probe

Article abstract of DOI:10.1039/c7cc07050k

The identification of bacterial pathogens is the critical first step in conquering infection diseases. A novel turn-on fluorescent probe for the selective sensing of nitroreductase (NTR) activity and its initial applications in rapid, real-time detection and identification of ESKAPE pathogens have been reported.

Full text of DOI:10.1039/c7cc07050k

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Real Time Detection of ESKAPE Pathogens by a Nitroreductase-
triggered Fluorescence Turn-on Probe
Shengnan Xu,a Qinghua Wang,ab Qingyang Zhang,ab Leilei Zhang,b Limin Zuo,c Jian-Dong Jiangac
and Hai-Yu Hu*ab
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Identification of bacterial pathogens is the critical first step in real time imaging of enzyme activities with a high signal-to-
conquering infection diseases. A novel turn-on fluorescent probe noise ratio in live bacterial cells. Furthermore, these probes
for selective sensing nitroreductase (NTR) activity, and its initial also would be used for bacterial species identification via the
applications in rapid, real time detection and identification of differences of fluorescence turn-on response of each pathogen
ESKAPE Pathogens have been reported.
to the probe.
Nitroreductases (NTRs),
a family of flavin-containing
Bacterial infections represent the leading cause of morbidity
and mortality worldwide; especially, the ESKAPE pathogens
enzymes, are able to metabolize nitrosubstituted compounds
using the reducing power of nicotinamide adenine
dinucleotide (NAD(P)H).7 These enzymes are present in a wide
range of bacterial genera and, to a lesser extent, in eukaryotes
expect hypoxic tumour cells and tissues, which make it a
potential bacterial imaging target. Multiple sequence
alignments of NTR proteins from E. coli and ESKAPE pathogens
showed low similarity between each sequence pair (Fig. S1a,
ESI). Moreover, the phylogenetic tree constructed from amino
acid sequences of NTR proteins also indicates the NTRs of
these seven species are not closely related (Fig. S1b, ESI).
Based on statistical analysis of sequences, we envisioned that
it would be possible to diagnose ESKAPE pathogens by
evaluating NTR activity detecting.
(
E
nterococcus faecium,
S
taphylococcus aureus,
cinetobacter baumannii, seudomonas
Enterobacter species) cause the majority of
Klebsiella
pneumoniae,
A
P
aeruginosa, and
hospital infections and effectively “escape” the effects of
antibacterial drugs.1 In addition to novel treatment options,
there is a strong need to better diagnose bacterial infections
via non-invasive molecular imaging methods.2 To this end,
various methods have been developed for detection and
identification of pathogens by nuclear imaging and optical
imaging.3 Among these methods, fluorescence imaging has
attracted much attention due to its high selectivity, good
spatial resolution, and non-invasive mode of action.4 A number
of bacteria targeting fluorescent probes have been developed
in the past and several are on the way to the clinic.5 A common
problem with these probes is the background fluorescent
signal, which requires extensive and time-consuming washing
steps to remove free fluorophore. Therefore, we hypothesized
that bacteria specific enzyme-triggered fluorescence turn-on
probes would provide a superior diagnostic capability for
bacterial infections.6 These probes exhibit only low
background fluorescence in a free state and, as a result, enable
Detection of NTR activities within hypoxic tumour cells and
tissues using fluorogenic methods has been well described
recently.8 In comparison, only a few optical probes have been
developed for detecting NTR activities in E. coli and/or
bacterial lysates, and there appears to have been no real time
NTR enzymatic activity detected and evaluated in bacterial
pathogens using a fluorogenic method. In an effort to detect
NTRs which could be used for real time rapid detection and
identification of ESKAPE pathogens; herein, we designed a
bacterial cell permeable fluorogenic probe which could
selectively and rapidly be triggered by NTRs. Furthermore, the
probe was successfully applied for imaging of NTRs in living
bacterial cells and visualized the difference of NTRs level in
ESKAPE pathogens by fluorescence confocal microscopy for
the first time.
a.State Key Laboratory of Bioactive Substances and Function of Natural Medicine,
Institute of Materia Medica, Peking Union Medical College and Chinese Academy
of Medical Sciences, Beijing, 100050, China. haiyu.hu@imm.ac.cn.
b.Beijing Key Laboratory of Active Substances Discovery and Drugability Evaluation,
Institute of Materia Medica, Peking Union Medical College and Chinese Academy
of Medical Sciences, Beijing, 100050, China.
c. Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences &
Peking Union Medical College, Beijing, China.
Electronic Supplementary Information (ESI) available: Synthetic procedures and
spectra data for all new compounds, and statistical analysis of NTRs sequences,
UV-VIS, FI spectral studies and confocal imaging studies. See
DOI: 10.1039/x0xx00000x
In our preliminary studies, we found the lipophilic, near-
infrared fluorescent cyanine dye Cy 5.5 with one positive
charge could penetrate both Gram positive and Gram negative
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Journal Name
fluorescence
ON
OFF
N
N
N
N
Nitroreductase, NADH
Tris buffer pH 7.4
O
O
NH2
NO2
NH
NH
N
N
N
N
HO
OH
1
probe
product 1
Scheme 1 The structure of probe 1 and its reaction with NTR.
bacterial cell membranes and label ESKAPE pathogens (Fig. S2,
ESI); therefore, Cy 5.5 was chosen as a fluorochrome. The
y = 3.21× 105 x + 3.12 × 104
R2 = 0.9888
fluorogenic probe
1 was synthesized by the conjugation of a
nitro-imidazole group (quencher) to Cy 5.5 and its structure
1
was characterized by H NMR, 13C NMR and HRMS (see ESI for
details). We expected the reduction of the nitro group into the
amino group by NTR would result in an off-on fluorescence
response (Scheme 1).
We first investigated the function response of probe
purified E. coli NTR in vitro by fluorescence emission
spectroscopy. Probe was incubated in 0.05 M Tris buffer
solution at pH 7.4, containing 500 µM NADH. As depicted in Fig.
S3b (ESI), the latent fluorescent probe itself shows a very
weak emission at 699 nm and the adding of NTR leads to an
obvious fluorescence enhancement. The reduction product
was determined by high resolution mass spectrometry and
confirmed by comparison to chemical synthesized product
1 to
1
1
Fig. 1 (a) Fluorescence spectra of probe
1 (15 µM) reacting
1
with NTR at varied concentrations (0.05, 0.10, 0.15, 0.20, 0.25,
0.30, 0.35, 0.40, 0.45 and 0.50 µg/mL) in the presence of 500
µM NADH at 37 °C for 30 min. (b) Linear correlation between
the concentration of NTR and the fluorescence intensity of the
reaction mixture (λex/λem = 658/699 nm), data shown as mean
± s.d (n = 3).
1
with HPLC (Fig. S4, S15) and the deduced process is proposed
in Scheme S4 (ESI)8a. Furthermore, effects of pH and
temperature on the reaction between probe
1 and NTR were
studied (Fig. S5 and Fig. S6, ESI), which reveals that the probe
functions best under physiological conditions (about pH 7.4
and 37 °C). Under the optimized conditions, the fluorescence
also implying that the probe is stable in the detection system
(Fig. S3b, ESI).
response of probe
1 to NTR at varied concentrations is shown
Next, we investigated the kinetic parameters for the
in Fig. 1. A gradual increase in fluorescence intensity (FL.
Intensity) was observed with increase in the NTR
concentrations, and a good linearity was obtained in the
concentration range of 0.05 to 0.5 µg/mL, with a linear
equation of F = 3.21×105 C (µg/mL) + 3.12×104 (R = 0.9944).
The detection limit (3σ/k, in which σ is the standard deviation
of blank measurements, n = 11, and k is the slope of the linear
equation) is determined to be 8.4 ng/mL NTR.
enzyme-activated reaction of probe 1. The Lineweaver-Burk
plot of 1/V (V represents the initial reaction rate) versus the
reciprocal of the probe concentration was established (Fig. S8,
ESI). By fitting the data with the Michaelis-Menten equation,
the corresponding Michaelis constant (Km) and maximum of
initial reaction rate (Vmax) for the enzyme-activated reaction
were determined to be 7.21 µM and 0.020 µM s-1,
respectively, which was comparable to the values reported
previously.10
The apparent kinetic parameters of probe
were additionally determined. The rate of product
from probe triggered by NTR in the presence of NADH (500
µM) as an electron donor was measured by adding a known
concentration of probe (7.5–25 µM) to the reaction mixture.
As shown in Fig. S7 (ESI), fluorescence intensities increase with
the level-up of the concentrations of probe and nearly reach
a plateau in about 30 min, which indicates that higher
concentrations of probe result in stronger fluorescence
intensity. In contrast, the fluorescence of probe without NTR
1
catalysis by NTR
1
released
The specificity of probe
utilized for imaging NTR activity in bacteria. In order to explore
the specificity of probe to NTR, two sets of experiments
1 is extremely important when it is
1
1
1
based on interference factors and NTR inhibitor were carried
out. First, considering the complexity of the bacterial
intracellular environment, the interferences of various
bioanalytes were studied, including reductive biothiols (Cys,
DTT, GSH and homocysteine), arginine (Arg), ascorbic acid (Vc),
glucose, reactive oxygen species (ClO-, H2O2) and inorganic
salts (CaCl2, MgCl2, KCl and NaCl). Fluorescence responses of
1
1
1
(control) hardly alters during the same period of reaction time,
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Following the demonstration of the specificity of probe
NTR, we examined whether probe could be used to detect
NTR activity in bacterial lysates. Grown E. coli cultures were
treated with 5 µM of probe at three different initial
1 to
1
250000
200000
150000
100000
50000
0
1
concentration and subjected to subsequent lysis. As shown in
Fig. 3, a gradual increase in fluorescence intensity is observed
with increase in the bacterial concentration. Moreover,
addition of dicoumarin (0.1 mM) to the bacterial culture
before treatment with the probe significantly reduced the
fluorescence intensity of the live E. coli, further demonstrating
the specificity of the probe towards NTR.
Encouraged by the in vitro imaging data, the ability of the
NTR probe
1 for real time detection and identification of
medically important bacterial pathogens of the ESKAPE panel,
comprising the Gram negative species K. pneumoniae, A.
baumannii, P. aeruginosa, E. cloacae and E. coli, and as well as
the Gram positive species E. faecium and S. aureus, was
Fig. 2 Fluorescence responses of probe
1 (15 µM) to various
species: NTR (1 µg/mL), Cys (1 mM), DTT (1 mM), GSH (1 mM),
Hcy (1 mM), Arg (1 mM), Vc (1 mM), glucose (10 mM), ClO- (10
µM), H2O2 (10 µM), CaCl2 (2.5 mM), MgCl2 (2.5 mM), KCl (10
mM) and NaCl (10 mM). All spectra were acquired in 0.05 M
Tris buffer (pH 7.4) with 1.5% DMSO as co-solvent at 37 °C
(λex/λem = 658/699 nm).
probed. 5 µM probe
1
was incubated with seven bacterial
C for 1 h,
strains in 0.05 M Tris buffer (pH 7.4) at 37
°
probe 1
probe 1
Hoechst
(a)
Hoechst
(b)
+ inhibitror
probe
1 to tested species showed fluorogenic probe 1 only
could be triggered by NTR but other interference factors,
including reductive biothiols at a high concentration (Fig. 2).
These experiments provide evidence that probe
1 is highly
specific to NTR and hence can be used for NTR detection.
Next, we investigated the effect of a common inhibitor of
NTR, dicoumarin,11 on the activity of the enzyme. When probe
(d)
(c)
1
was pre-treated with 2.5 µM dicoumarin and then mixed
with NTR, the fluorescence intensity was much lower than that
without dicoumarin (Fig. S9, ESI). Moreover, increasing the
concentration of dicoumarin led to a progressive decrease in
10
m
(e)
100
the fluorescence intensity of a mixture of probe
1 and NTR,
probe 1
probe 1 + inhibitor
confirming that the NTR activity was effectively inhibited by
dicoumarin. These results confirmed that the enhanced
80
60
40
20
0
fluorescence response of probe
1 to NTR arises from the
enzyme-catalyzed reduction reaction.
150000
E.coli lysate
E.coli lysate + probe 1
E.coli lysate + probe 1 + Inhibitor
100000
50000
0
Fig. 4 Confocal microscopic images of E. coli (a, b) and E.
faecium (c, d) incubated with probe in the absence (a, c) or
Buffer
OD600 = 0.5 OD600 = 1.0 OD600 = 2.0
1
presence (b, d) of NTR inhibitor, respectively. DNA was stained
with Hoechst 33258 (white), red: probe Cy 5.5 signals. (e)
Quantification of fluorescence intensity of probe 1 incubated
with E. coli and ESKAPE pathogens in the absence (red bars) or
presence (green bars) of NTR inhibitor, respectively. Bars
represent mean values ± s.d (n = 3).
Fig. 3 Fluorescence intensity of the clarified cell lysate of E. coli
before (blue bars) and after incubating with probe (+/- NTR
inhibitor, red/green bars) at various initial bacterial
concentration (λex/λem = 658/719 nm), bars represent mean
values ± s.d (n = 3).
1
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1 was analysed by
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bacterial cells. The intensity of the near-infrared fluorescent
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1
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strongest fluorescent signals from S. aureus incubated with
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probe was largely activated by NTR intracellularly after
internalization. We believe the different fluorescence turn-on
response of probe to ESKAPE pathogens most likely be due
1 reduction, suggesting that the
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ESKAPE pathogens with NTR triggered probe
1 might be
possible in the future. However, the exact molecular
mechanism behind this finding remains unknown at this stage.
To further evaluate the bacterial imaging efficiency of probe
we determined the time-dependent fluorescence responses of
probe in live methicillin-resistant S. aureus (MRSA). As
1,
6
1
depicted in Fig. S11a (ESI), visible fluorescent signal was
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Probe displays a highly selective and rapid response of
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within the ESKAPE collection.
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We thank Prof. Ping Zhu, Dr. Shu Xu and their group
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This work was partially supported by the National Natural
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