Enzyme-responsive reporter molecules for selective localization and fluorescence imaging of pathogenic biofilms

Article abstract of DOI:10.1039/c6cc09296a

Pathogenic bacteria and their biofilm formation are responsible for a broad spectrum of microbial infections. A novel enzyme-responsive reporter molecule (ERM-1), which can specifically recognize AmpC β-lactamase (Bla) in drug resistant bacteria, has been developed to enable the selective localization of biofilms.

Full text of DOI:10.1039/c6cc09296a

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Enzyme-Responsive Reporter Molecules for Selective Localization
and Fluorescent Imaging of Pathogenic Biofilms
Junxin Awa, Frances Widjajaa, Yichen Dingc,d, Jing Mua, Yang Liangc, Bengang Xing*a,b
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Pathogenic bacteria and their biofilm formation are responsible extensive investigations still need to be further performed.
for a broad spectrum of microbial infections. A novel enzyme-
Moreover, apart from the important roles of Blas expression in
responsive reporter molecule (ERM-1), which can specifically
antimicrobial resistance, another typical self-defence strategy for
recognize AmpC, β-lactamase (Bla) in drug resistant bacteria, has
the bacterial persistence and survival from antibiotic treatment will
been developed to enable the selective localization of biofilms.
be their modes of growth. Different from the planktonic way in
Currently, the emergence of antibiotics resistant bacteria has been most laboratory culturing conditions, bacteria can easily grow as
a serious medical concern in healthcare and community1. One main biofilms on surfaces, a type of highly populated multicellular
cause of such rapid increasing of resistance is the high-level communities embedded in a biopolymer matrix, which provide
expression of β-lactamases (Blas), a family of bacterial enzymes bacteria additional protection against immune defenses and
produced as a means of self-defence against β-lactam antibiotics antibiotic treatment.5 Bacterial populations in biofilms usually
including penicillins and cephalosporins, and thus leading to become more resistant and thus give rise to various chronic
therapeutic failure2,3
.
As such, conducting the specific Blas infections that are notoriously hard to eradicate5. Therefore,
measurements and a better understanding of their molecular establishment of effective strategies to identify biofilm-associated
mechanisms in bacterial pathogens before prescription of antibiotic bacterial infections will be imperative to decipher their structure
therapy will be of paramount clinical importance. Among the and formation, as well as to facilitate the development of novel
different varieties of Blas, Class A and C Blas are known as the most modalities for unique antimicrobial treatment.
significant members responsible for β-lactam antibiotics resistance
Generally, traditional methods for biofilms identification focus
in bacteria. By right, Class A β-lactamase, such as, TEM-1, have been
on the direct visualization of their growing in the culture medium,
well studied for resistance inactivation and for imaging of biological
processes in vitro and in vivo4. Yet, as compared to this well-
which are usually labour intensive, time-consuming and lack of
detailed intrinsic studies for individual cells6. To date, various
exploited Blas counterpart, Class C Blas with the similar serine
laboratory-based methods to detect biofilm samples have been well
established7. Amongst them, optical imaging for effectively
hydroxyl group in the active site, have been far less investigated.
Currently, Class C β-lactamase genes have been found to spread
monitoring of biofilm functions and biological processes has shown
worldwide and their presence leads to extensive resistance, thus
great potentials and been widely utilized in biomedical applications.
posing a remarkable clinical threat. Unfortunately, unlike the case
For example, the incorporation of green fluorescent protein (GFP)
in class A Blas, lack of unique and selective recognition of Class C
or its color variants in bacteria has been employed to study the
Blas in vitro and in living systems remains a technical concern and
formation of biofilms7a-c. However, the tested strains that express
foreign genes may not be identical to the original bacterial
pathogens. And the large size of GFP tag (~ 27 kDa) may normally
a
Division of Chemistry and Biological Chemistry, School of Physical &
lack signal amplification, which could potentially affect the
Mathematical Sciences, Nanyang Technological University, Singapore, 637371,
Singapore. E-mail: bengang@ntu.edu.sg
bInstitute of Materials Research and Engineering (IMRE), Agency for Science,
Technology and Research (A*STAR), Singapore, 117602, Singapore.
cCentre for Environmental Life Sciences Engineering (SCELSE),
School of Biological Sciences, Nanyang Technological University, Singapore,
637551, Singapore
d Interdisciplinary Graduate School (IGS), Nanyang Technological University,
639798, Singapore
dynamics and efficiency of the whole imaging process8. Moreover,
several standard imaging methods based on organic fluorochromes
or quantum dot nanocrystals (QD) have also been utilized for
visualization of the overall structure of biofilm and description of
their entire expanse7d-g. However, their intrinsic affinity to the
bacterial biofilms may present the concerns of specificity, and
meanwhile, usage of fluorescent particles may also suffer from the
potential issue of diffusion and toxicity7,9. As such, development of
simple and specific strategies which can target biofilm structures,
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and more importantly, can selectively report different resistant
enzymes expressed by pathogens in biofilms will be highly
desirable. Unfortunately, so far such relevant studies have not been
fully exploited yet.
The enzyme activity of reporter molecules ERM-1 and ERM-2
were first studied by measuring the fluorescent emission in
phosphate buffered saline (PBS) solution (0.1 M, pH = 7.4). In the
absence of Blas, there was only little fluorescent signal observed.
In this work, we present a unique class C AmpC Bla enzyme However, after treatment of the probes with TEM-1 and AmpC Blas
sensitive reporter molecule (ERM-1) that can selectively localize separately at 37 °C for 1h, the obvious fluorescence change was
drug resistant pathogens in biofilms. As proof of concept, we chose detected at wavelength of 478 nm. As shown in Figure 1A, the
a
typical tetraphenylethylene (TPE) moiety as our target maximum fluorescence enhancement in ERM-1 was ~ 120 folds
fluorophore, which was covalently linked to the cephalosporin after reaction with AmpC, whereas, a decreased activity was found
structure. The major reason we used TPE in this molecular design is when ERM-1 reacted with TEM-1 and there was only ~ 40 folds
mainly attributed to its promising aggregation induced emission fluorescence observed after enzymatic reaction (Figure 1A). These
(AIE) property at 478 nm9. Unlike the most commonly used results demonstrated that enzyme hydrolysis could break the linker
fluorophores that may suffer from the aggregation caused at the 3’-position of cephalosporins, thus resulting in the effective
fluorescence quench, these TPE based dye molecules exhibit strong release of the TPE moiety. Then, the subsequent aggregation of TPE
emission in aggregated state and have thus been extensively linker leads to an enhancement in fluorescence mostly owing to the
applied for biosensing and imaging in living systems10. More restriction of intramolecular rotation of TPE. 10
importantly, the aggregated TPE products after enzyme interactions
Similar enzyme analysis was also investigated by using one
typical AmpC inhibitor, Aztreonam (AZT)12. The enzyme inhibition
could overcome the common issues over most existing probes that
may have problems of random diffusion, and can thus serve as
results clearly showed that AZT can greatly suppress AmpC activity.
robust fluorogenic probes to real-time image biofilms with different
In the presence of AZT, there was little fluorescence observed after
bacterial pathogens.
enzyme treatment (Fig. S1A, ESI†), clearly suggesting that the
Scheme 1 illustrates the rational design and synthesis of such developed ERM-1 can specifically recognize AmpC enzyme. As a
unique enzyme responsive reporter molecules. Typically, a 4- control, the further enzymatic activity was also carried out on the
aminothiophenol linker was covalently introduced at the 3’-position basis of ERM-2 with the less bulky group at 7’-position of
of cephalosporin structure, which was further conjugated with a cephalosporin (Fig 1B and Fig. S1B ESI†). There was ~ 120 folds
TPE fluorophore. In order to achieve the selectivity towards fluorescence enhancement detected after the ERM-2 incubated
different Blas, one bulky methoxyimino group was connected to the with both TEM-1 and AmpC, indicating that reporter molecule,
7’-amino of β-lactam ring. Such bulky moiety allows the ERM-2 exhibited the same enzyme activity and it could not reflect
cephalosporin based molecule (ERM-1) more susceptible to AmpC the different enzyme recognition between Class A and C Blas.
enzyme, but resistant to its Class A counterparts, mostly owing to During the enzyme reaction, although some non-specific
its steric hindrance to block the active site in class A enzyme fluorescence change observed when ERM-1 was incubated with
pocket11. As contrast, one simple acetyl group with less steric TEM-1 enzyme. The more significant fluorescence enhancement
hindrance was also introduced at the 7’-position of cephalosporin based on ERM-1 interactions with AmpC revealed that the bulky
structure to afford the controlled reporter molecule (ERM-2). Upon methoxyimino group at the 7th position of the cephalosporin
the successful synthesis of the developed substrates, the final structure could greatly increase the selectivity with class C Bla and
products were purified by reverse HPLC and finally characterized by thus result in a higher enzymatic reactivity.
NMR and mass spectrometry (ESI-MS, ESI†). These well-designed
probe molecules will be used to react with TEM-1 and AmpC Blas
enzymes, and their capability to achieve
Fig. 1 (A) Emission Spectra of ERM-1 and (B) ERM-2 (10 μM) before and after
incubation with TEM-1 and AmpC Blas in PBS (pH = 7.4).
The further enzyme kinetics analysis for ERM-1 and ERM-2 was
carried out in PBS (pH 7.4) at 37 °C and the fluorescent changes
were measured over a period of 1 hr (Fig. S2, ESI†).13 The results
demonstrated that ERM-1 could be hydrolysed by AmpC and TEM-1
respectively with the reasonable catalytic constants (Kcat = 14.2,
3.01 min-1) and Michaelis constants (KM = 11.8, 14.1 μM). As
Scheme 1: Enzyme-responsive fluorescent change upon the reaction of
reporter molecule of ERM-1 with TEM-1 and AmpC Bla.
contrast, the relevant studies were also evaluated for the controlled
different enzyme recognition will be systematically investigated.
molecule, ERM-2, and the kinetic constants for TEM-1 and AmpC
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were determined to be KM = 10.3, 11.4 μM and Kcat = 16.8, 15.9 min- incubated with 20 μM of ERM-1 or 2 for 1 hr at 37°C, and
1
,
subsequently subjected to the confocal microscope for fluorescence
imaging. As shown in Fig. 2, strong fluorescence emission was
observed after incubation of ERM-1 with AmpC expressed E.
cloacae, whereas, the similar bacterial incubation in TEM-1
expressed E.coli BL-21 only led to weak fluorescence. Importantly,
there was no obvious fluorescence detected in the control E. coli
DH5α bacteria and E. cloacae strains pretreated with AmpC
inhibitors, AZT (Fig. 2A and Fig. S5, ESI†). Moreover, similar bacterial
imaging experiments based on ERM-2 demonstrated the obvious
fluorescence in both E. cloacae and E.coli BL-21 samples. There was
no fluorescent difference detected within these two strains (Fig. S6,
ESI†). These results unequivocally indicated the intrinsic capability
of the rationally developed enzyme responsive ERM-1 molecule to
selectively report AmpC Bla activity and label the resistant bacteria
pathogens. Furthermore, we explored the feasibility to quantify the
specific labelling of AmpC expressed resistant bacteria with flow
cytometer (FCM). In this experiment, three different bacteria
strains: E. cloacae and E. coli BL-21, and E. coli DH5α were used to
respectively, suggesting the promising capability of ERM-1 toward
the selective recognition to AmpC enzyme reaction.
The enzyme-triggered TPE formation was further verified by
HPLC and dynamic light scattering (DLS) analysis (Fig. S3 and S4, ESI
†). In the presence of AmpC Bla, ERM-1 showed the complete
hydrolysis of TPE with the retention time at 35 mins. DLS
measurements showed that the average hydrous dynamics
diameters of aggregated TPE was 200 nm. Whereas, when ERM-1
reacted with TEM-1, only partial hydrolysis was found in the
solution with the size distribution of the aggregated products
around ~ 100 nm (Fig. S4A, ESI†). Similarly, the controlled studies
through the incubation of ERM-2 with AmpC and TEM-1 Blas led to
the complete hydrolysis of the reporter molecule as the reaction
between ERM-1 and AmpC. These results further confirmed the
selective reaction of ERM-1 to AmpC enzyme, which was consistent
with the observation in the fluorescence detection.
Inspired by the results for enzyme activity in PBS solution, we incubate with ERM-1 and ERM-2 separately (10 μM) at 37 °C for 1
investigated the applicability of ERM-1 and 2 for live cell imaging. In hr. The fluorescence signals from individual bacteria were collected
this study, two different Gram negative penicillin resistant bacteria at 478 nm. Fig. 2B demonstrated
a strong fluorescence
strains: Enterobacter cloacae (E. cloacae) and E. coli BL-21, were enhancement (~ 10 folds) for ERM-1 after incubation with AmpC
chosen due to their high expression levels of AmpC and TEM-1 Blas expressed E. cloacae and a weaker fluorescent change (~ 3 folds) for
respectively11. Additionally, an antibiotic susceptible E. coli DH5α TEM-1 expressed E. Coli BL-21 as compared to the control E. coli
strain (ATCC 53868) without Bla expression was used as a negative DH5α strain. Similarly, FCM studies based on ERM-2 were also
control. All these strains have been encoded with green fluorescent carried out and the results indicated no obvious fluorescence
protein (GFP) plasmid, which can efficiently express GFP and can
difference between E. cloacae and E.coli BL-21 strains (Fig. 2C).
These data clearly indicated that ERM-1 could serve as a reliable
reporter molecule for quantifying the AmpC activity in antibiotic
resistant bacteria.
Fig. 3. Confocal imaging of penicillin resistant E. cloacae, E. coli BL-21, and
antibiotic susceptible E. coli DH5α bacteria biofilms with 20 μM of ERM-1 in
0.1M PBS, pH = 7.4. Ex = 350/50 nm; Em = 450/50 nm. Scale bar: 5 μm.
Importantly, we further investigated the capability of enzyme
responsive reporter molecules to selectively localize and monitor
the formation of bacteria biofilm with the different pathogens as
models. Basically, we applied the static biofilm as target for our
study. The bacterial biofilms were cultured onto coverslips in LB
Broth for 24 h at 37 °C according to the protocol reported
previously7. During the process, the GFP expressed in different
Fig. 2 (A) Confocal imaging of penicillin resistant E. cloacae and E. coli BL-21
bacteria, and antibiotic susceptible E. coli DH5α strains with 20 μM of ERM-
1 in 0.1M PBS, pH = 7.4. Scale bar: 5 μm. (B) Flow cytometry analysis of
ERM-1 (10 μM) with three different bacteria. (C) FCM analysis of ERM-2 (10
μM) with different bacteria.
serve as standard for visualizing the distribution of individual bacteria were first used to observe the formation and distribution
bacteria pathogens. Typically, the bacterial strains were separately of individual strains within the biofilms. The biofilm structures
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formed by different bacterial cells were then treated with
molecules, ERM-1 and 2, (20 μM) respectively for 1 hr and
subsequent biofilm imaging was carried out under microscopy with
the excitation at 350 nm. As shown In Fig. 3, the biofilm treated
with ERM-1 showed the different imaging staining. There was
significant fluorescence readout observed in the biofilm consisting
of AmpC expressed E. cloacae strains, whereas, only the weak signal
was found in the biofilms formed by E. coli BL-21, which expressed
TEM-1 Bla. There was no fluorescence in E. cloacae biofilm in the
presence of AZT inhibitor and in the controlled biofilm with E. coli
DH5α (Fig. 3 and Fig. S7, ESI†). Although the similar biofilm imaging
analysis was also conducted by using ERM-2, there was no
difference observed in the imaging results between two biofilm
structures with E. cloacae and E. coli, which expressed different
types of bacterial antibiotic degrading enzymes (Fig. S8, ESI†). All
these results clearly suggested that rational design of enzyme-
responsive reporter molecule structures can facilitate specific
targeting of biofilm components, which may thus greatly benefit
the biofilm formation and controlled bacterial resistant inactivation
studies.
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approach towards the effective fluorescent imaging and
localization of drug resistant AmpC β-lactamase producing
bacterial strains in biofilms. By taking advantage of the bulky
methoxyimino group on the 7’-position of cephalosporin ring,
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achieved. Such enzyme responsive reporter molecules could
serve as promising fluorescent probes to effectively image
AmpC producing bacteria in biofilms. Importantly, this
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Enzyme-Responsive Reporter Molecules for Selective Localization and
Fluorescent Imaging of Pathogenic Biofilms
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