A bioluminogenic substrate for in vivo imaging of β-lactamase activity

Article abstract of DOI:10.1002/anie.200701931

Reporting live: A caged β-lactam-D-luciferin (Bluco) conjugate can image β-lactamase activity in vivo through a two-step enzymatic process. This novel bioluminogenic probe should facilitate the detection of β-lactamase and widen its applications as a reporter. (Chemical Equation Presented).

Full text of DOI:10.1002/anie.200701931

Angewandte
Chemie
DOI: 10.1002/anie.200701931
Imaging Probes
A Bioluminogenic Substrate for In Vivo Imaging of b-Lactamase
Activity**
Hequan Yao, Min-kyung So, and Jianghong Rao*
b-Lactamases are an important family of bacterial enzymes
for research in bacterial infection and biotechnology. Bacte-
rial resistance to antibiotics is becoming a growing global
health problem, and bacteria expressing b-lactamases are
resistant to b-lactam antibiotics because b-lactamases effi-
ciently hydrolyze b-lactam antibiotics such as penicillins and
cephalosporins.[1] Rapid and sensitive detection of b-lacta-
mase activity in vitro and in vivo is thus clinically important
for the identification of bacterial pathogens that are resistant
to b-lactam antibiotics. A number of colorometric and
fluorometric substrates have been developed for the detec-
tion of b-lactamase in vitro and in living cells,[2–7] but a
substrate that can detect b-lactamase in living animals has not
yet been reported. Highly sensitive imaging of bacteria will
find many health and environmental applications.[8–11] In
contrast, b-lactamases have been developed as a powerful
tool with a number of biotechnology applications. For
example, TEM-1 b-lactamase (Bla), a small (29 kDa) and
monomeric enzyme, is a sensitive reporter system for detect-
ing and imaging biological processes and interactions in single
living mammalian tissue culture cells. It has been applied for
examining promoter/regulatory elements activity,[12] imaging
RNA splicing,[13,14] monitoring viral infection,[15] and detecting
protein–protein interactions.[16,17] The b-lactamase enzymatic
activity has recently also been applied to control formation of
supramolecular hydrogels.[18] To extend the reporter utility of
Bla to whole living animals is highly desirable but has been
impeded by lack of a suitable substrate that can image the Bla
activity in vivo. Herein, we report the first bioluminogenic
substrate for Bla and demonstrate its application for in vivo
imaging of the Bla activity.
(Scheme 1). Firefly luciferase catalyzes the oxidation of its
substrate d-luciferin in the presence of O2, ATP, and Mg2+ and
Scheme 1. The structure of Bluco and proposed two-step reaction for
detection of Bla.
Our detection strategy utilizes the bioluminescent enzyme
firefly luciferase (fLuc) to image the Bla activity in vivo,
because luciferase-based bioluminescence imaging offers
great sensitivity for probing biology in small living animals[19]
emits light, which can be imaged with a sensitive CCD
camera. The 6-hydroxy group of d-luciferin is critical to its
oxidation by fLuc and has been modified to design probes for
detection of target enzyme activity, for example, b-galactosi-
dase,[20] monoamine oxidase,[21] and caspases.[22] We thus
designed the bioluminogenic substrate by coupling this
hydroxy group to the 3’ position of the cephalosporin through
an ether bond (Scheme 1). The resulting conjugate should be
a poor substrate for fLuc but remain as a substrate for Bla.
The opening of the b-lactam ring by Bla would trigger
spontaneous fragmentation, leading to the cleavage of the
ether bond at the 3’ position and subsequent release of free d-
luciferin that can now be oxidized by fLuc in a light-producing
reaction. The Bla activity can thus be imaged from the light
emission. To improve the stability of the conjugate, the sulfide
group on the cephalosporin was oxidized to sulfoxide.[6] The
final structure was termed Bluco (beta-lactam and d-luciferin
conjugate) and was efficiently prepared by a multiple-step
[*] Dr. H. Yao,[+] M.-K. So,[+] Prof. J. Rao
Molecular Imaging Program at Stanford
Department of Radiology, Biophysics, Cancer Biology Programs
Stanford University School of Medicine
1201 Welch Road, Stanford, CA 94305-5484 (USA)
Fax: (+1)650-736-7925
E-mail: jrao@stanford.edu
Homepage: http://raolab.stanford.edu
[+] H. Yao and M.-K. So contributed equally to this work.
[**] This work was supported by the National Institute of Health
(EB3803), the Career Award at the Scientific Interface from the
Burroughs Wellcome Fund, and the NCI’s Small Animal Imaging
Resource Program (SAIRP R24A92862).
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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organic synthesis (see the Supporting Information). The
purity of the final substrate was validated by the HPLC
analysis (see the Supporting Information).
Bluco displayed a maximal fluorescence emission at
440 nm, and, after the Bla treatment, the maximal emission
red-shifted to 540 nm (Figure 1). This fluorogenic feature of
of Bla + fLuc and fLuc cells was more than 55-fold. As the
concentration of Bluco increased, the bioluminescent emis-
sion from Bla + fLuc cells proportionally increased, but
reached a maximum after the Bluco concentration was
higher than 500 mm, which might reflect the saturation of
the Bla activity (Figure 2). The bioluminescent emission from
Bla + fLuc cells was also proportionally dependent on the cell
numbers, and as few as 1500 intact cells were detected in the
presence of 500 mm Bluco (Figure 3). These results demon-
strate that Bluco is cell-permeable and able to detect the Bla
activity in living mammalian cells.
Figure 1. Fluorescence emission spectra of Bluco (1 mm) in 10 mm
PBS before (dashed line) and after (solid line) Bla treatment
(lex =350 nm).
Bluco can be applied to measure its hydrolysis kinetics by Bla:
Km = 90 mm and kcat = 1.0 sÀ1 (see the Supporting Informa-
tion). The small value of kcat suggests that the leaving group d-
luciferin is not as efficient as thiolphenol used in CCF2.[5]
Subsequent addition of fLuc to the Bla-treated Bluco solution
produced strong light emissions, validating our design of the
bioluminogenic substrate for Bla.
Since Bluco is a polar molecule containing two carboxyl-
ate groups, we first evaluated whether Bluco was cell-
permeable and able to detect the Bla activity in intact cells.
We added Bluco (62.5 mm in phosphate buffered saline (PBS))
to 1 ” 105 intact COS7 (monkey kidney) cells cotransfected
with Bla and fLuc (Bla + fLuc), and observed strong biolu-
minescence emission (Figure 2). In comparison, control
COS7 cells transfected only with fLuc displayed similar
fLuc activity but produced little light emission upon addition
of Bluco (Figure 2). The contrast between the luminescence
Figure 3. Dependence of luminescence emission on cell number.
Serially diluted solutions of Bla+fLuc (solid line) and fLuc cells
(dashed line) were exposed to Bluco (500 mm) in 10 mm PBS.
To test whether Bluco can work in vivo, we injected 1 ” 106
COS7 cells transiently cotransfected with Bla and fLuc into
the left rear thigh of a nude mouse. The same number of only
fLuc transfected cells was injected into the right rear thigh as
a control. Two hours after the implantation, we injected 78 mg
of Bluco (5 mmolkgÀ1) via the tail vein into each mouse and
measured the bioluminescent signal. The left injection site
exhibited a strong emission, which was about 15–25-fold
higher than the emission from the right injection site
(Figure 4). The emission peaked at about 30 min and lasted
for approximately 30 min, then gradually decreased by 40%
over 1 h. At the end of the Bluco measurement, d-luciferin
was injected into the same mice for measuring the fLuc
activity at both injection sites. Both sites showed similar
bioluminescent emission (Figure 4), indicating that the fLuc
activity was similar and that the observed contrast from the
Bluco imaging was not due to different fLuc activity at each
site. All these data demonstrate that Bluco can image the Bla
activity in vivo in the presence of fLuc.
Finally, we applied Bluco to image the RNA-splicing
reaction in vivo. We have previously reported a Bla-based
RNA-splicing reporter for imaging Tetrahymena group I
intron ribozyme-splicing activity in living cells (Figure 5a).[13]
This ribozyme reporter construct RzB was transiently trans-
fected with COS7 cells that stably expressed fLuc (RzB +
fLuc). An inactive mutant of RzB with a single mutation at
the catalytic site (RzBm) was used as the control.[13] The
Figure 2. Bioluminescence emission of 1”105 Bla+fluc (solid line)
and fLuc (dashed line) COS7 cells after incubation for 10 min with
various concentrations of Bluco in 10 mm PBS.
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Figure 4. Imaging of Bla activity in living mice (n=4; n is number of
mice). 1”106 COS7 cells were cotransfected with Bla and fLuc and
injected into left rear thigh of a nude mouse, and the same number of
cells transfected with fLuc only were injected at the right rear thigh.
After implantation for 2 h, Bluco (78 mg) was injected intravenously
into each mouse for bioluminescence imaging of the Bla activity (left
image), and then d-luciferin (0.14 mg) was injected to measure the
fLuc activity at both sites (right image). Intensity unit: photo-
nsÀ1 cmÀ2 steradianÀ1
.
cis splicing of RzB was confirmed by in vitro RT-PCR
(reverse-transcription polymerase chain reaction) analysis
(see the Supporting Information). The in vitro Bluco assay
revealed that the Bla activity from transfected cells was 4
times higher than that of the control (Figure 5b). As shown
previously,[13] the Bla activity resulting from the RzB splicing
was able to be imaged with CCF2/AM in single live cells
under a fluorescence microscope (Figure 5c). The blue
fluorescence indicated Bla activity from the splicing reaction,
and the mutant construct (RzBm) did not emit any blue
fluorescence but only green fluorescence from uncleaved
CCF2.
Figure 5. Detection and imaging of the Tetrahymena ribozyme-splicing
activity in vitro, in single living cells, and in whole living mice with Bla
as reporter. a) Schematic of the Tetrahymena ribozyme-splicing reporter
RzB. b) Bluco assay of 1 ”105 intact COS7 cells transfected with
indicated constructs (500 mm Bluco; 30 min incubation). c) Imaging
the ribozyme splicing in single living cells. COS7 cells were transfected
with indicated constructs, and red fluorescent protein (dsRed) was
used as the transfection marker. Cells were incubated with CCF2/AM
for 1 h. Top panel images are an overlay of frames captured at 530 nm
(green emission) and 460 nm (blue emission), and images in lower
panel are dsRed emission at 605 nm with excitation at 560 nm.
d) Representative image of mice (n=4) implanted with COS7 cells
expressing RzB+fLuc (left rear thigh) and RzBm+fLuc (right rear
thigh) and imaged with the similar procedure described in Figure 4.
We then injected these transfected COS7 cells (1 ” 106)
into nude mice at the left (transfected with RzB + fLuc) and
right rear thighs (transfected RzBm + fLuc as the control).
Two hours after the cell implantation, Bluco (0.3 mg) was
injected intravenously into each mouse for bioluminescence
imaging of the Bla activity (Figure 5d). The left injection site
exhibited about twofold emission of that from the right
injection site (see the Supporting Information). We verified
the fLuc activity at both sites to be identical through separate
d-luciferin injection and imaging. These data indicate that
Bluco can image the Bla activity produced from the splicing
of Tetrahymena ribozyme in vivo. This example also demon-
strates that with the availability of Bluco and fluorogenic
substrates for individual experimental settings, the Bla
reporter assay can now be performed in vitro, in single
living cells, and in whole living animals.
In conclusion, the work presented herein describes the
design and evaluation of a new bioluminogenic substrate
Bluco for imaging the Bla activity in vivo. Together with
previously reported fluorogenic substrates,[5–7] the biolumino-
genic substrate reported herein completes the reporter
enzyme Bla as a unified reporter for imaging in vitro, in
single cultured living cells, and in whole living animals, and
should enable many in vivo applications of the Bla reporter.
Although this demonstration focused on mammalian cells,
Bluco may also be applied for in vivo imaging of b-lactam-
resistant bacteria pathogens that express b-lactamase.
Received: May 2, 2007
Published online: August 3, 2007
Keywords: bioluminescence imaging · firefly luciferase ·
.
imaging agents · lactams
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