Imaging tuberculosis with endogenous β-lactamase
reporter enzyme fluorescence in live mice
Ying Konga, Hequan Yaob,1, Hongjun Renb,1, Selvakumar Subbiana,1,2, Suat L. G. Cirilloa, James C. Sacchettinic,
Jianghong Raob,3, and Jeffrey D. Cirilloa,3
aDepartment of Microbial and Molecular Pathogenesis, Texas A&M Health Sciences Center, College Station, TX 77843;bMolecular Imaging Program at
Stanford, Departments of Radiology and Chemistry, Stanford University, Stanford, CA 94305; andcDepartments of Chemistry and Biochemistry and
Biophysics, Texas A&M University, College Station, TX 77843
Edited by Rino Rappuoli, Novartis Vaccines, Siena, Italy, and approved June 4, 2010 (received for review January 17, 2010)
The slow growth rate and genetic intractability of tubercle bacilli
has hindered progress toward understanding tuberculosis, one of
the most frequent causes of death worldwide. We overcame this
roadblock through development of near-infrared (NIR) fluorogenic
but not by their eukaryotic hosts, to allow real-time imaging of
pulmonary infections and rapid quantification of bacteria in living
animals by a strategy called reporter enzyme fluorescence (REF).
This strategy has a detection limit of 6 ± 2 × 102colony-forming
units (CFU) of bacteria with the NIR substrate CNIR5 in only 24 h of
incubation in vitro, and as few as 104CFU in the lungs of live mice.
REF can also be used to differentiate infected from uninfected mac-
rophages by using confocal microscopy and fluorescence activated
cell sorting. Mycobacterium tuberculosis and the bacillus Calmette–
Guérin can be tracked directly in the lungs of living mice without
sacrificing the animals. Therapeutic efficacy can also be evaluated
through loss of REF signal within 24 h posttreatment by using in
vitro whole-bacteria assays directly in living mice. We expect that
the laboratory is potentially transformative for tuberculosis viru-
lence studies, evaluation of therapeutics, and efficacy of vaccine
candidates. Thisis a uniqueuseof anendogenousbacterialenzyme
probe to detect and image tubercle bacilli that demonstrates REF
is likely to be useful for the study of many bacterial infections.
worldwide by killing nearly 2 million people each year (1).
Emergence of strains resistant to multiple drugs has led to sit-
uations where treatment is no better than before the discovery of
antibiotics (2, 3). Diagnosis of tuberculosis remains a major bar-
rier to control of the disease because the standard method, the
months after transmission occurs. Culture-based techniques are
more sensitive, but still take weeks to obtain results. Similar
problemsplague tuberculosis researchingeneral,andparticularly
in animal models, where data from assays is dependent upon
determination of colony-forming units (CFU). This problem
impacts the pace of virulence studies, evaluation of therapeutics,
and development of vaccines. All tuberculosis research and di-
agnosis would be facilitated by methods to detect tubercle bacilli
of mycobacteria have been developed for detection of mycobac-
teria using fluorescence, luminescence (4–8), and even single
photon emission computed tomography (SPECT) (9, 10), these
methods require specific laboratory strains and do not allow de-
tection of pulmonary tuberculosis by optical imaging directly in
live animals. Recombinant systems have facilitated progress, but
expression of a foreign gene can impact bacterial fitness in un-
expected ways, particularly when expressed from plasmids
(11–13). The ability to detect all strains that cause tuberculosis
directly without expression of foreign genes would have a pro-
uberculosis, caused by Mycobacterium tuberculosis (Mtb),
remains one of the most frequent causes of death in humans
found impact upon the tuberculosis field and would facilitate re-
search with clinical strains that cause tuberculosis. Sensitive
detection of nonrecombinant strains that cause tuberculosis can
also improve clinical diagnosis using sputum and other diagnostic
samples as well as ultimately be applied directly to diagnosis of
infections in patients.
We describe noninvasive detection of natural strains that cause
pulmonarytuberculosis in livinganimals.Detection is based on re-
porter enzyme fluorescence (REF) technology, which uses β-
imaging. Although optical probes for endogenous enzymes have
been used to differentiate cancerous tissue from normal tissue in
the field of oncology (16), they have not been used for infectious
diseases where, due to the presence of enzymatic targets that are
unique to pathogens, this approach has the potential for exquisite
specificity. A great deal is known about β-lactamase enzymes and
substrates because they are the most common mechanism of bac-
hydrolysis of a β-lactam ring (17–20). Comparison of the crystal
structure of theMtb β-lactamase,BlaC, to that of other similaren-
BlaC has an unusually large active site pocket, suggesting that de-
sign of substrates with high specificity should be straightforward
signal at 1,000-fold lower protein levels than fluorescent proteins
(22), β-lactamase REF has the potential for extremely high sensi-
tivity. These characteristics, along with the very high catalytic ac-
tivity (23) of β-lactamase, make this enzyme a nearly ideal choice
for development of specific probes to detect tuberculosis in the
laboratory, live animals, or patients.
Expression of β-Lactamase by Mtb. We evaluated the expression of
β-lactamase in different strains that cause tuberculosis. All strains
ofMtbexaminedexpress BlaC atlevels measurable within2hand
increasing in signal intensity to 20 h (Fig. 1A). As expected,
a specific mutant in Mtb blaC (24) produces greater than 10-fold
lower levels of signal, indicating that BlaC is responsible for the
Author contributions: J.R., and J.D.C. designed research Y.K., H.Y., H.R., S.S., S.L.G.C., J.R.,
and J.D.C. performed research; Y.K., H.Y., H.R., J.C.S., J.R., and J.D.C. contributed new
reagents/analytic tools; Y.K., H.Y., H.R., S.S., S.L.G.C., J.C.S., J.R., and J.D.C. analyzed data;
and Y.K., S.S., S.L.G.C., J.R., and J.D.C. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Freely available online through the PNAS open access option.
1H.Y., H.R., and S.S. contributed equally to this work.
2Present address: W250T, Public Health Research Institute at University of Medicine and
Dentistry of New Jersey, 225 Warren St., Newark, NJ 07103.
3To whom correspondence may be addressed. E-mail: firstname.lastname@example.org or
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| July 6, 2010
| vol. 107
| no. 27
majority of β-lactamase activity in Mtb. The Mtb BlaC is primarily
surface-localized because membrane fractions carry the majority
of the activity (Fig. 1B). The fact that the Mtb BlaC is surface-
localized makes it a nearly ideal candidate for use to detect Mtb
because substrates would not have to traverse the bacterial cell
wall. This is a significant issue for detection of mycobacteria be-
cause their cell wall is thought to be highly selective.
Substrates for Detection of Mtb β-Lactamase. We have developed
substrates that are eukaryotic cell permeable and produce a long
wavelength fluorescent signal when cleavedthatisamenableto use
in live animal imaging due to the ability of long wavelengths to
penetrate mammalian tissue efficiently (25, 26). To detect Mtb us-
ing BlaC, we designed several near-infrared fluorogenic substrates:
switches” or “beacons” are not fluorescent before cleavage due to
Cy5 (CNIR4) or Cy5.5 (CNIR5, CNIR9, and CNIR10) through
a lactam ring, leading to a fluorescence resonance energy transfer
(FRET) quenching effect (Fig. 2). BlaC cleavage triggers sponta-
neous fragmentation, releasing the quencher connected to the 3′
position of the lactam, breaking the FRET process, and restoring
a hydrophilic QSY21 disulfonate that absorbs maximally at 649 nm
and a hydrophobic QSY22 that absorbs maximally at 670 nm to
allow optimal quenching of Cy5 and Cy5.5, respectively. We con-
nected acetylated D-glucosamine to the carboxylate of cysteine
through a γ-amino-butyric acid as the linker in CNIR5 and CNIR9
because we have shown that fully acetylated glucosamine can fa-
cilitate uptake of the highly charged CNIR4 into eukaryotic cells
(25). We designed CNIR10 without the D-glucosamine to directly
display little fluorescence before β-lactamase cleavage and an in-
crease in maximal emission by 8.5- (660 nm, CNIR4), 24- (690 nm,
CNIR5), 9.5- (690 nm, CNIR9), and 10-fold (690 nm, CNIR10)
after cleavage (Fig. S1b). We found that coincubation of each of
these probes with Mtb resulted in direct labeling of the bacteria,
with an increase in fluorescence of 2-fold for CNIR4, 3-fold for
CNIR5, 1.5-fold for CNIR9, and 2-fold for CNIR10 after 18 h
coincubation (Fig. S1C).
Detection and Quantification of Mtb in Vitro. In the case of CNIR5,
which displays the greatest increase in fluorescence during coin-
cubation with Mtb, we found a good correlation (r2= 0.989) be-
tween bacterial numbers and level of fluorescence over a broad
range of bacteria from 102to 106colony forming units (Fig. 3A).
The calculated limit of detection using CNIR5 was 6 ± 2 × 102
CFU. The calculated limit of detection using CNIR5 for other
β-lactamase–producing bacteria was similar: 6 ± 1 × 102CFU for
Pseudomonas aeruginosa and 8 ± 0.7 × 102CFU for Staphylo-
coccus aureus. Incorporation of the fluorescent dye into Mtb was
confirmed by fluorescence confocal microscopy at 24 h coincu-
bation whereas Mtb in the absence of CNIR does not display
fluorescence at 690 nm (Fig. 3B). Mtb within macrophages could
also be detected with CNIR, and there is a good correlation (r2=
0.984) between bacterial numbers and fluorescence intensity
(Fig. 3C), with a threshold of detection of 1–5 × 105bacteria.
Interestingly, the most intense staining with all four CNIR sub-
strates is within compartments in the cytoplasm of macrophages,
suggesting that these substrates efficiently penetrate subcellular
compartments and that the majority of the active Mtb BlaC is
accessible to substrate outside of the bacteria (Fig. 3D), which is
consistent with our fractionation studies. CNIR labeling allows
direct detection of infected versus uninfected macrophages using
microscopy, a useful tool for studies with tissue culture cells.
Cytotoxicity of these substrates was examined as described pre-
viously (27) in J774A.1 cells, and no significant cytotoxicity occurs
with concentrations up to 100-fold higher than those used
for detection of Mtb. We also found that CNIR can be used to
differentiate infected from uninfected cells by fluorescence-
activated cell sorting (FACS; Fig. 3E), making it possible to
quantify infected cells during in vitro studies or in homogenized
tissues from animal studies. The number of cells separated by
FACS using either recombinant green fluorescent protein (GFP)
or CNIR was similar and correlated with the multiplicity of in-
fection (MOI) used.
Real-Time Analysis of Tuberculosis in Living Animals. These obser-
vations indicate that Mtb can be detected and quantified using
REF, but do not address whether CNIR will allow detection di-
rectly in animals. To establish the kinetics of CNIR substrates for
quantification of Mtb in mammalian tissues, we infected BALB/c
mice by s.c. inoculation with Mtb and the closely related vaccine
strain bacillus Calmette–Guérin. We found that CNIR4, CNIR5,
CNIR9, and CNIR10 could detect 107s.c. bacteria and CNIR5
could readily detect 106bacteria (Fig. 4). Maximal fluorescence
producing the highest levels of signal followed by CNIR10,
CNIR4, and CNIR9. Fluorescent signal is not significantly differ-
ent from background for any substrate after 96 h postadministra-
tion, allowing the substrate to be readministered to the same
animals for subsequent imaging. Both bacillus Calmette–Guérin-
and Mtb-infected animals display nearly identical kinetics and
signal strength with all CNIR substrates. The improved signal
observed for CNIR5 over CNIR4 is most likely due to the bene-
ficial characteristics of the longer wavelength of excitation and
emission for Cy5.5 over Cy5, but the lower signals of CNIR9 and
CNIR10 are likely the result of differences in their membrane
localized β-lactamase. (A) The three common laboratory Mtb strains H37Rv,
CDC1551, and Erdman all produce similar levels of β-lactamase as measured by
the change in fluorescence of Fluorocillin Green (excitation: 485 nm; emission:
530 nm) in the presence of 107bacteria. In the case of CDC1551, the signal
produced is significantly lower (***P < 0.001) than Erdman and H37Rv, yet it
is at least 10-fold higher than background. The H37Rv blaCm strain with
a mutation in the blaC gene displays negligible β-lactamase activity. (B) Mem-
brane fractions of Mtb are responsible for the majority of the β-lactamase ac-
localized in Mtb. β-Lactamase levels in culture filtrates are significantly higher
than background (P < 0.001), indicating that BlaC is secreted at low levels.
Mycobacterium tuberculosis (Mtb) strains produce a membrane-
near-infrared (CNIR) fluorogenic substrates. The intact CNIR substrates do not
display fluorescence due to the close proximity of the fluorescent dye (Cy5 or
Cy5.5) and the quencher through fluorescence resonance energy transfer
(FRET), but become fluorescent after hydrolysis by β-lactamase, generating
the fluorescent product whose emission can be measured using a fluorome-
ter. The quencher groups used are QSY21 or derivatives and the CNIR dyes are
Cy5 (CNIR4) and Cy5.5 (CNIR5, CNIR9, and CNIR10). NIR, near-infrared.
Reporter enzyme fluorescence detection of M. tuberculosis with
| www.pnas.org/cgi/doi/10.1073/pnas.1000643107Kong et al.
permeability. This conclusion is supported by the lower incor-
poration of fluorescence into the bacteria over time observed for
both CNIR9 and CNIR10 compared with CNIR5 (Fig. S1C). Al-
though acetylated glucosamine improves uptake of these sub-
strates, the sulfates also play an unexpected role in improving the
observed imaging kinetics that cannot be compensated for by the
lower molecular weight of CNIR10.
Localization and Quantification of Pulmonary Infection. Pulmonary
infection is the natural route of infection by Mtb, but the greater
tissue depth and complexity of the lung make detection more diffi-
cult. We infected mouse lungs with bacillus Calmette–Guérin and
Mtb and imaged them by transillumination after administration of
as few as 104CFU could be detected in the lungs of living mice
(Fig. 5 A and C). Although the signal correlates well with bacterial
numbers at 24 h, the levels of signal equalize by 48 h post-
administration of CNIR5, suggesting that the fluorophore builds up
at the site of infection, consistent with pooling of fluorophore in
the cytoplasm of infected macrophages. Lung examined ex vivo at
uninfected animals, demonstrating that the source of the signal is
clearly within infected lungs (Fig. 5D). Localization of the source
of the signal from CNIR5 was confirmed by 3D fluorescence-
mediated molecular tomography (FMT) analyses in living mice
(Fig. S2). FMT of animals at 24 and 48 h consistently localizes the
the concentration of the fluorochrome, as shown by the nearly
linear correlation with the number of bacteria present (Fig. S2D;
r2= 0.863). We calculated the limit of detection for live animal
imaging using REF in mice after pulmonary infection to be 1.3 ±
0.6 × 104CFU. Because adult human lung attenuates near-
infrared signals approximately 10-fold for every 2.5 cm of depth
of low bacterial numbers (∼104CFU) within lung, enhanced ver-
sions of this approach may offer promise for imaging tuberculosis
in a clinical setting (30).
Evaluation of Therapeutic Efficacy with REF. After pulmonary in-
fection with 106CFU, we observed measurable fluorescent signal
M. tuberculosis (Mtb) in vitro and direct detection of
infected cells by confocal microscopy and fluorescence
activated cell sorting (FACS). (A) Correlation of fluo-
rescent signal and number of tubercle bacilli in culture
as determined by colony-forming units (CFU) in the
presence of CNIR5 for 24 h. (B) Fluorescence confocal
microscopy of Mtb coincubated with the CNIR sub-
strates for 24 h demonstrates fluorescence incor-
poration into the bacteria. (C) Correlation of fluo-
rescent signal and number of tubercle bacilli present
within J774A.1 murine macrophages after coincubat-
ing with CNIR5 for 24 h. (D) Fluorescence confocal
microscopy of J774A.1 murine macrophages infected
with GFP (green) expressing Mtb and coincubated
with CNIR substrates (red) for 24 h. Fixed cells were
stained with DAPI (blue) to visualize nuclei. Infected
cells can be identified by the presence of signal from
CNIR substrates whereas uninfected cells do not in-
corporate CNIR signal. (E) FACS allows separation of
infected J774A.1 murine macrophages labeled with
CNIR5 in a manner that correlates well with the mul-
tiplicity of infection (MOI) bacteria per cell. Separation
can be accomplished with GFP, GFP-expressing Mtb, or
CNIR or by using both fluorescent labels. ***P < 0.001:
significantly different from fluorescence of medium
alone (horizontal dashed lines in A and C) calculated
by ANOVA with the Bonferroni posttest.
Fluorogenic compounds allow detection of
terium tuberculosis (Mtb) and bacillus Calmette–Guérin
with similar sensitivity after s.c. infection. (A) Whole-body
imaging of mice s.c. infected with Mtb and administered
the indicated CNIR substrate. Images were taken 48 h
postinfection and administration of the substrate. (B) In-
oculation sites, number of bacteria present, and region of
interest used for each animal. The position of the refer-
ence measurement is indicated (R). (C) Comparison of the
signal as a function of time postinjection of each CNIR
substrate for 108Mtb or (D) bacillus Calmette–Guérin.
Fluorogenic probes allow detection of Mycobac-
Kong et al. PNAS
| July 6, 2010
| vol. 107
| no. 27
that normally increases and plateaus in signal intensity after the
first 48 h postinfection. When rifampin and isoniazid (10 μg/mL
each) are administered together either in vitro to bacteria alone
or to infected mice, we observed a significantly different (P < 0.01)
signal from the untreated controls within 24–48 h posttreatment
(Fig. S3). These observations confirm that REF allows rapid eval-
uation of therapeutic efficacy either under laboratory conditions
or during infections in mice.
Endogenous enzymes combined with custom substrates allow
sensitive detection of bacterial infection directly in living mice, as
demonstrated by our use of REF for Mtb. The high sensitivity of
this approach is due to a combination of the fact that the substrate
does not need to gain access to the bacterial cytoplasm—only to
the surface-localized BlaC enzyme—and the fluorescent product
can build up at the site of infection, enhancing signal. Previous
microarraystudiesindicatethat BlaC is expressed constitutivelyby
Mtb in vivo after more than 28 d postinfection in BALB/c and
of nonreplicating persistence (32), suggesting that BlaC will make
a goodreporterundermost in vivo-relevantconditions.Giventhat
fluorogenic substrates can be developed for numerous enzymes,
including proteases, kinases, ureases, β-galactosidases, and β-
lactamases, REF technology should have utility for detection and
imaging of many pathogens. The stability of the fluorescent pro-
duct ensures proper localization to infected tissue and the ability
to follow the site of infection in real time. This system displays
promise for use in detecting infection in patients where bacteria
cannot be tagged with conventional reporters before imaging
Probes for bacteria that use catalytic enzymes, as in the case of
REF,arelikely tobeverysensitive because they arenot limited by
infection as compared with background, but continuously pro-
duce more signal as long as substrate is available. Use of a natural
enzyme that is not expressed by the host ensures specificity and
prevents potential metabolic impacts due to heterologous gene
expression. The value of using endogenous systems for imaging is
supported by studies using thymidine kinase for SPECT (33), but
levels from mice infected with bacillus Calmette–Guérin by the pulmonary route of inoculation that were administered CNIR5 immediately after infection or
uninfected mice that were also administered CNIR5. The yellow boxes within the first two mouse image panels indicate the regions that were magnified
fourfold to produce the images above the mouse image panels. (B) Depiction of dorsal and ventral views of a mouse with organs to provide the anatomical
context for the observed signal. (C) Whole-body images using transillumination after pulmonary infection with Mtb in mice in the same manner as in A. The
mouse on the left is uninfected but was administered the CNIR5 substrate whereas the mouse on the right is infected with Mtb by aerosol and was ad-
ministered CNIR5 immediately after infection. Images were collected at 48 h postinfection with administration of CNIR5 at the same time as infection. (D)
Lungs were harvested from infected (lower two sets of lungs) and uninfected (top lungs) animals immediately after live animal images were collected. Lungs
were harvested postmortem from the same animals that were first imaged alive (C), and then the lungs themselves were imaged (D). The CFU means and SD
of four mice for each dose were (A) 1.3 ± 0.6 × 104, 1.1 ± 0.02 × 105, and 1.0 ± 0.2 × 106and (C and D) 1.0 ± 0.6 × 106.
Fluorogenic probes allow detection of bacillus Calmette–Guérin and Mycobacterium tuberculosis (Mtb) after pulmonary infection of mice. (A) Signal
| www.pnas.org/cgi/doi/10.1073/pnas.1000643107 Kong et al.
thymidine kinase-based imaging is not an option for clinical
mycobacteria because they do not produce this enzyme and a
recombinant strain is required (9, 10). An additional benefit of
BlaC as an endogenous reporter is the fact that it is available
outside the bacterial cell. The conclusion that the Mtb BlaC is
surface-localized after secretion is consistent with observations
that β-lactamase activity is associated with the surface in myco-
bacteria (15, 34, 35). The fact that BlaC is surface-localized and
expressed in all Mtb allows the fluorescent product to provide
bacterial location without the need for the substrate to cross the
bacterial membrane. Hence, the sensitivity of this strategy allows
quantitative detection of Mtb both under laboratory conditions
and during animal infections.
The potential uses of this technology to facilitate tuberculosis
research are nearly limitless. We demonstrate labeling of the
bacteria, quantitative assessment of host cell infection, identifi-
cation of infected cells by both microscopy and FACS, detection
of Mtb in animals, quantitative analysis of infection, and locali-
zation to specific tissues. The nearly linear correlation between
bacterial numbers and fluorescence signal indicates that REF is
well suited for quantitative analysis of bacterial load during
experiments where colony-forming units are commonly used and
in animal tissues during infection or challenge studies. Because
evaluation of Mtb bacterial numbers normally requires 3 wk or
more, an immediate readout will have a profound impact on the
Mtb field. Furthermore, considering the high sensitivity observed
for mice, this approach should be applicable to other animal
models, including rabbits and guinea pigs (36–40). Even if REF
were used in combination with colony-forming units, having in-
formation that would allow experiments to be repeated or revised
on the basis of the REF data before obtaining the CFU data
would greatly speed progress.
Our study demonstrates that Mtb can be detected and quanti-
fied in living mice using custom fluorescent substrates for en-
dogenous β-lactamase, which allows detailed real-time analysis
of pulmonary infections. Because BlaC is expressed by all Mtb
strains examined, it is likely that REF can be directly applied to
clinical strains and other pathogenic mycobacteria because it
utilizes a naturally produced enzyme for detection. Although the
half-life for β-lactamases can vary somewhat (41), it has been
estimated at around 206 min in mammalian cells (22), suggesting
that enzyme levels represent a reasonable surrogate for numbers
of viable bacteria. This assertion is supported by the strong cor-
relation that we observed between viable bacterial numbers and
REF signal in vitro and in animals (Fig. 3A and Fig. S2D). Thus,
REF allows real-time estimation of the bacterial load in tissues,
a key measure of virulence for attenuated mutants, efficacy of
vaccines, and antimicrobial therapeutic potential. Our observa-
tion that therapeutics can be evaluated within 24–48 h with REF
supportsthis conclusion.The presenceofβ-lactamasesin bacteria,
such as P. aeruginosa or methicillin-resistant S. aureus, that cause
community-acquired pneumonias (CAP) could potentially inter-
fere with REF, but the clinical presentation for tuberculosis is
frequently sufficiently different from CAP (42–46)that the typical
nodular lesions in the apical lobe of the lung found with tuber-
culosis are more likely to be misdiagnosed as carcinoma (46–48).
Carcinoma could be differentiated from tuberculosis using REF
imaging, making REF a potentially valuable tool in the diagnostic
arsenal. Because the Mtb BlaC enzyme has a relatively unique
active site pocket (21), studies are ongoing to further optimize the
structure of our custom probes to produce even more highly sen-
sitive and specific detection and imaging of Mtb in animals with
the hope of ultimately applying this technology directly to tuber-
culosis patients. This tool will also facilitate tuberculosis research
vaccines, and therapeutics.
Materials and Methods
Strains and Growth Conditions. With the exception of Fig. 1 where multiple
strains were used, Mtb strain Erdman was used for all experiments. All
strains were grown as described previously (49). Bacterial numbers were
confirmed in all experiments by plating dilutions for colony-forming units.
Additional details are included in SI Materials and Methods.
Cell Lines and Culture Conditions. The murine macrophage cell line J774A.1
(ATCC TIB67) was maintained at 37 °C and 5% CO2in high glucose Dulbecco’s
Modified Eagle Medium (DMEM; Gibco) supplemented with 10% heat-
inactivated FBS (Gibco) and 2 mM L-glutamine.
Macrophage Infection Assays. J774A.1 cells were infected as described pre-
viously (50). Additional details are included in SI Materials and Methods.
Flow Cytometry. Mtb strain CDC1551 was transformed with the vector pFJS8
that carries the L5 promoter expressing GFP (51). Flow cytometry was con-
ducted essentiallyas described previously (52). Additional detailsare included
in SI Materials and Methods.
Mouse Infections. Infections werebyintratrachealinstillationof104–106CFU to
the lungs (53) for bacillus Calmette–Guérin and aerosol using the Madison
chamber (52) for Mtb. Comparable imaging and bacterial viability data were
obtained with either route. Additional details are included in SI Materials and
Animal Care and Use Committee of Texas A&M University.
β-Lactamase Activity Assays and Fractionation. Cultures of mycobacteria
grown to early exponential phase (OD600= 0.4) and centrifuged for 10 min at
3,000 × g to separate the bacteria from the supernatant. The supernatant
was filtered through a 0.45-μm filter and retained as the culture filtrate. The
pellet was washed twice with sterile PBS and resuspended to the original
volume in PBS (whole-cell fraction). A portion of the whole-cell fraction was
lysed by sonication with 30-s pulses at 60% output over 6 min on ice (crude
lysate). The crude lysate was centrifuged for 30 min at 20,000 × g at 4 °C, and
the pellet was resuspended in an equal volume of PBS (membrane fraction).
Volumes of each fraction equivalent to 107bacteria were aliquoted into 96-
well plates for assays. Fluorocillin Green (Invitrogen) was added at 4.5 μM to
each well, and the plates were shaken gently for 5 min at room temperature
and incubated at 37 °C between 30 min and 20 h. The change in fluorescence
was measured by excitation at 485 nm and emission at 530 nm.
Design and Synthesis of β-Lactamase Imaging Probes. The general principle for
probe design is FRET. Each probe contains the lactam component that will be
recognized by β-lactamase, a fluorescent group, and a light absorber. Be-
cause of the FRET-based quenching effect, they are initially at the quenched
or dark state when the fluorescent group is excited. β-Lactamase cleavage of
the lactam ring triggers spontaneous fragmentation that releases the light
absorber connected to the 3′ position of the lactam from the fluorescent
group, thus breaking the FRET process and leading to fluorescence emission.
Two near-infrared cyanine dyes (Cy5, Cy5.5) were chosen due to their long
emission wavelength that is suitable for in vivo fluorescence imaging. Cor-
respondingly, we modified the nonemitting dye QSY21 as the quenching
group in the probe into a hydrophilic QSY21 disulfonate that absorbs
maximally at 649 nm and a hydrophobic QSY22 that absorbs maximally at
670 nm. Both were paired with Cy5 or Cy5.5 to make four probes, desig-
nated CNIR4, CNIR5, CNIR9, and CNIR10. CNIR5 is very stable in PBS, with
a spontaneous hydrolysis rate of 1.75 × 10−7·s−1. The enzymatic acceleration
is 3.36 × 106. All probes display good solubility in aqueous solutions due to
the sulfonate groups present. CNIR5 is soluble to at least 100 μM in water. All
probes were prepared through multiple-step organic syntheses, purified by
reverse-phase HPLC, and validated by matrix-assisted laser desorption/ioni-
zation time-of-flight mass spectrometry. Additional details for synthesis are
included in SI Materials and Methods.
Imaging Tuberculosis Infections. Mice were anesthetized with isofluorane and
imaged in an IVIS Spectrum (Caliper Life Sciences) with and without filters for
fluorescence. Images were analyzed with Living Image Software v3.1 using
spectral unmixing algorithms to remove autofluorescence, with one of the
resulting channels locked to fit the emission spectrum of the appropriate
fluorophore (Cy5 or Cy5.5). Representative images were randomly selected
from images of four mice for all figures. Additional details are included in SI
Materials and Methods.
Kong et al.PNAS
| July 6, 2010
| vol. 107
| no. 27
Confocal Fluorescence Microscopy. Cells were seeded in 8-well chamber slides Download full-text
with 1 × 105cells per well in 200 μL of DMEM plus 10% FBS overnight at 37 °C
in 5% CO2. The medium was removed, and GFP expressing Mtb was added to
each well at an MOI of 10 (bacteria per cell) in 200 μl of medium. The bacteria
were coincubated with the cells for 30 min and washed twice with PBS to
remove extracellular bacteria, and medium plus 200 μg/mL amikacin was
added for 2 h at 37 °C to select for intracellular bacteria. The cells were
washed twice with PBS and placed in medium plus 20 nM substrate for 24 h
at 37 °C in 5% CO2and washed twice with PBS. The cells were stained with
10 μg/mL DAPI, washed twice with PBS, and fixed in 4% paraformaldehyde in
PBS for 30 min at room temperature. After washing, the slides were dried and
mounted for confocal fluorescence microscopy.
Statistical Analyses. All experiments were carried out in triplicate and re-
peated at least three times. The significance of the results was determined
using the Student’s t test or ANOVA, as needed. P < 0.05 was considered
ACKNOWLEDGMENTS. This work was supported by Grant 48523 from the
Bill & Melinda Gates Foundation and by Grant AI47866 from the National
Institutes of Health.
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