Bioorthogonal chemistry for selective recognition, separation and killing bacteria over mammalian cells

Article abstract of DOI:10.1039/c5cc10625g

By taking advantage of metabolic engineering and bioorthogonal click chemistry, we report a new strategy for selective recognition, separation and killing bacteria over mammalian cells.

Full text of DOI:10.1039/c5cc10625g

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DOI: 10.1039/C5CC10625G
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Bioorthogonal Chemistry for Selective Recognition, Separation
and Killing Bacteria over Mammalian Cells
Zhenhua Li,a,b Zhen Liu, a Zhaowei Chen,a,c Enguo Ju,a,c Wei Li,a,c Jinsong Ren*,a and Xiaogang Qua
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
By taking advantage of metabolic engineering and bioorthogonal identifying novel bacterial targets are time‐intensive, laborious,
click chemistry, we report a new strategy for selective recognition, and usually require sophisticated experimental techniques.
separation and killing bacteria over mammalian cells.
Hence, the design of an ideal system that can overcome the
above‐mentioned limits remains a big challenge in this field.
Over the last decade, the design of nanoparticle‐based drug
delivery systems (nano‐DDS) to overcome a variety of
biopharmaceutical drawbacks in the treatment of bacterial
infections has attracted wide attention. For instance,
liposomes and mesoporous silica nanoparticles have been
explored for antimicrobial drug delivery.1,2 Despite their
promise, however, these antibiotic‐loaded nanoparticles
would also enter host cells through endocytosis and release
the payloads, resulting in undesired systemic side effects by
nonspecific drug distributions in many tissues and organs.
Therefore, developing new antibiotic delivery systems that
provide greater selectivity for the bacteria over host cells
becomes highly desirable.3,4 To achieve very high selectivity,
nanomaterials have been functionalized with targeting groups
like antibodies, aptamers, ligands, etc. to recognize antigens or Scheme 1 a) Functionalization protocol of FMSN and drug
other epitopes on the exterior of a pathogen.5 The use of these
loading. b) Schematic representation of FMSN‐TTDDA‐N3
nanoparticles for the selective recognition, separation, and
killing of bacteria over mammalian cells by bioorthogonal click
chemistry.
targeting agents have improved treatment outcomes with
fewer side effects and overwhelm drug resistance mechanisms
with high sustained local drug concentrations. Despite these
burgeoning developments, limitations still exist: 1) unlike live
cells, bacteria in different growth states may preferentially
express different sets of molecules to escape from antibody;6 2)
antibodies and aptamers are not stable in harsh biological
environments, resulting in low targeting efficiency; 3) tedious
efforts are needed to preserve the binding site and maintain
the bioactivity of these targeting moieties during synthesis;7 4)
more importantly, the traditional protocols of discovering and
Owing to its extremely selective, versatile and
biocompatible properties, bioorthogonal click reaction has
been widely used as a rapid conjugation method for a variety
of biological applications, such as labeling of biomolecules and
imaging, cell surface modifications, protein engineering, and
drug development.8‐10 Toward these recent achievements,
bioorthogonal click reactions are now seeing widespread use
in metabolic glycoengineering.11,12 This technique relies on the
cellular biosynthetic machinery assimilating sugar derivatives
that contains a bioorthogonal chemical reporter. Subsequently,
the metabolic incorporation of this reporter into glycans can
be further detected with a reaction partner. This metabolic
glycan labeling technique has recently emerged as an
appealing approach for detecting and imaging glycans in live
cells and animals.13,14 Furthermore, this strategy has expanded
its application to investigate glycoconjugates in living
bacteria.15 For example, Bertozzi and co‐workers have
a. Laboratory of Chemical Biology and State Key laboratory of Rare Earth Resources
Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of
Sciences, Changchun 130022, China.
b. College of Chemistry & Environmental Science, Chemical Biology Key Laboratory
of Hebei Province, Hebei University, Baoding, 071002, China.
c. University of Chinese Academy of Sciences, Beijing 100039, China.
E‐mail: jren@ciac.ac.cn; xqu@ciac.ac.cn
Electronic
Supplementary
Information
(ESI)
available:
See
DOI: 10.1039/x0xx00000x
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exploited sugar metabolic pathways to label glycan with azide‐ was first reacted with carboxyl‐modified FMSN. Then the
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DOI: 10.1039/C5CC10625G
modified sugar analogues and subsequently detected and activated 2‐azidoacetic acid was conjugated to amine‐
visualized of bacteria by bioorthogonal ligation with alkyne‐ functionalized FMSN‐TTDDA to obtain FMSN‐TTDDA‐N3. The
functionalized probes.16 However, the method is not suitable characterizations of modified nanoparticles were given in
for labeling of Gram‐positive bacteria because they are supporting information (Figures S3).
surrounded by a peptidoglycan (PG) cell wall and lack of an
outer membrane.
Recently, D‐amino acids were found to be incorporated
into both Gram‐positive and Gram‐negative bacteria rather
than mammalian cells, and subsequently their derivatives were
designed as a new reporter for highly selective bacteria
labeling. For example, both Michael S. VanNieuwenhze’s group
and Bertozzi’s group have exploited PG’s unique D‐amino acid
constituents to label PG in both Gram‐positive bacteria and
Gram‐negative bacteria.17,18 However, the use of nano‐DDS for
selective binding to bacteria with D‐amino acid‐directed
bioorthogonal ligation is still an incipient area of research.
Since metabolic incorporation of D‐alanine derivatives is
specific to bacteria, we expect that bioorthogonal chemistry
together with metabolic engineering would greatly enhance
the bacteria targeting ability of nanoparticles. Furthermore,
bioorthogonal click chemistry would be more effectively
applied to nanoparticles than single molecules because of the
multivalent effect. Thus, we envision that artificially
introducing targetable groups in bacteria may offer the
possibility of improving the recognition ability of nano‐DDS.
In this work, we develop a new strategy for selective
recognition, separation and killing bacteria over mammalian
cells through metabolic engineering‐directed bioorthogonal
click chemistry (Scheme 1). Firstly, targeting groups, alkynyl
derived D‐alanine, are artificially generated in bacteria without
affecting mammalian cells. This technique is expected to
enable temporal generation of chemical groups on the surface
of bacteria just like biological receptors, controlling the
Fig. 1 Incubation of bacteria in D or L‐alanine analogues and
then captured by fluorescence‐labeled FMSN‐TTDDA‐N3
nanoparticles: Binding of FITC‐labeled FMSN‐TTDDA‐N3 to
EDA‐treated B. subtilis (a) and S. aureus (c); ELA‐treated B.
subtilis (b) and S. aureus (d) through CuAAC reaction.
We first verified that D‐amino acids analogues would be
well‐tolerated by the bacterial biosynthetic machinery,
allowing the generation of chemical reporters on cell surface.
Since ethynyl‐D‐alanine (EDA) could easily incorporate into the
stem peptide of the PG subunit, it was used as the precursor
targetability of nanoparticles. Then, antibiotic‐loaded and
azide conjugated magnetic mesoporous silica nanoparticles
(FMSN) could selectively bind to these alkynyl groups on the
for metabolic engineering. Bacteria were first grown in media
surface of target bacteria by bioorthogonal click reactions.
containing EDA for one or more generations. Generated
Finally, the antibiotics would be released from the
alkynyl groups on the cell surface were first visualized in green
accumulated nanocarriers. In this way, this targeted antibiotics
fluorescence after treatment with FAM (fluorescein) azide. As
expected, EDA‐treated Bacillus subtilis (B. subtilis), Escherichia
coli (E. coli) and Staphylococcus aureus (S. aureus) successfully
produced unnatural D‐amino acids on their surface (Fig. S4).
Neither of the ethynyl‐L‐alanine (ELA) resulted in significant
labeling, thus indicating that the labeling was specific to the D‐
delivery strategy would enhance the therapeutic index of
antimicrobials in the intracellular environment, while
minimizing the side effects associated with the systemic
administration of the antibiotic. To the best of our knowledge,
no attempt has been made using bioorthogonal chemical
reporters as targeting groups to realize selective treatment of
enantiomers (Fig. S5). Incorporation of EDA was catalyzed by
bacterial infections with less harmful effects on mammalian
transpeptidases which cross‐linked peptidoglycan chains to
form rigid cell walls.
cells.
In our experiments, FMSN were synthesized according to
To investigate the targeting ability of nanoparticles,
our previous report in which the fabrication process had been
described in detail.19 The characterizations of the as‐
fluorescein isothiocyanate (FITC)‐derivatized FMSN‐TTDDA‐N3
were then incubated with EDA‐treated or ELA‐treated bacteria.
The binding of FMSN‐TTDDA‐N3 to bacteria was analyzed by
fluorescence microscopy. Fig. 1 demonstrated that FMSN‐
TTDDA‐N3 bound efficiently to EDA‐treated Gram‐positive
synthesized nanomaterials were given in supporting
information (Figures S1 and S2). To bind of chemical reporters
on the surface of bacteria, FMSN was modified with azide
groups because of its high reactivity to alkynyl groups through
copper‐catalyzed azide‐alkyne cycloaddition (CuAAC). A short‐
N3 showed minimal binding to the ELA‐treated bacteria. This
chain PEG linker 4,7,10‐Trioxa‐1,13‐tridecanediamine (TTDDA)
bacteria (B. subtilis and S. aureus). As a control, FMSN‐TTDDA‐
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result clearly confirmed that the artificial targetable groups with an excellent ability to selectively recognize and image
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DOI: 10.1039/C5CC10625G
generated by metabolic EDAs could be efficiently captured by bacteria over live cells.
FMSN‐TTDDA‐N3 through bioorthogonal click reaction.
After having demonstrated success in selectively binding
However, unlike fluorescent molecules, FITC‐labeled FMSN‐ to bacteria against mammalian cells, the magnetic separation
TTDDA‐N3 failed to bind to E. coli (date not shown). This notion efficiency of the FMSN‐TTDDA‐N3 nanoparticles was then
might be attributed to the fact that the outer membrane of investigated. EDA‐treated mixtures were incubated with
Gram‐negative bacteria blocked the binding of FMSN‐TTDDA‐ FMSN‐TTDDA‐N3 at 37°C. After CuAAC reaction, we used a
N3. To acquire further evidence, field‐emission SEM was magnet to capture the “magnetized” bacteria. As shown in Fig.
employed to visualize the capturing capability of azide‐ 3, the “magnetized” B. subtilis were attracted to a magnet and
modified nanoparticles. As shown in Fig. S6, SEM images separated from live cells. These results clearly demonstrated
revealed that both EDA‐treated B. subtilis and S. aureus were that FMSN‐TTDDA‐N3 nanoparticles have the ability to
indeed covered with FMSN‐TTDDA‐N3. However, in a control selectively recognize and separate bacteria from live cells
experiment, FMSN‐TTDDA‐N3 failed to cover the surface of through metabolic engineering‐directed bioorthogonal click
bacteria treated with ELA. Meanwhile, the lower adhesion of chemistry.
FMSN‐TTDDA‐N3 on Gram‐negative bacteria could be
attributed to the presence of the outer membrane (Fig. S6c
and 6d). The direct information from SEM images was
consistent with the results of fluorescence microscopy
experiments.
Fig. 3 (a) The scheme shows the separation of “magnetized” B.
Fig. 2 The scheme shows FMSN‐TTDDA‐N3 nanoparticles
selectively bind to bacteria over live cells (top). The enlarged
images of HeLa cells and Gram‐positive B. Subtilis (bottom).
The co‐culture system were treated with EDA (a) and ELA (b)
and then captured by FMSN‐TTDDA‐N3 nanoparticles through
CuAAC reaction.
subtilis by a small magnet. (b) A digital photo of the cell culture
dish after magnetic separation. (c‐e) Optical microscopy
images of “magnetized” B. subtilis and HeLa cells after
magnetic separation. Images were taken at different locations
in the culture dish: 1) close to the magnet (c), 2) in the middle
(d), and 3) far from magnet (e). (f‐h) Fluorescence microscopy
images of “magnetized” B. subtilis and HeLa cells after
magnetic separation. Images were taken at different locations
in the culture dish: (f) close to the magnet, (g) in the middle
and (h) far from magnet.
Because D‐alanine is an essential component of their cell
wall, all bacteria are expected to utilize D‐alanine as substrate.
Plants and mammals appear to lack such capacity and, as a
result, D‐alanine is specific to bacteria.20 We then investigated
the selective association of FMSN‐TTDDA‐N3 with bacteria in a
mixture of bacteria and live cells. B. subtilis were used as
model bacteria owing to that they were big enough to be
visualized. EDA or ELA was added into the mixtures of HeLa
cells and B. subtilis. As shown in Fig. 2a and Fig. S7a, only
bacteria were found to be stained in green fluorescence after
CuAAC reaction and almost no fluorescence signals were
detected for HeLa cells. In contrast, most of FMSN‐TTDDA‐N3
nanoparticles were found to be internalized by HeLa cells
when the mixtures were treated with ELA (Fig. 2b and Fig. S7b).
This result was consistent with the obtained result when HeLa
cells alone were treated with FMSN‐TTDDA‐N3 nanoparticles
(Fig. S7c). These images demonstrated that FMSN‐TTDDA‐N3
can selectively identify bacteria in the mixtures of mammalian
cells and bacteria. Furthermore, colocalization assays in ELA‐
treated HeLa cells showed that the nanoparticles entered the
lysosomes (Fig. S8). Thus, the results endow FMSN‐TTDDA‐N3
Ciprofloxacin (Cip),
a
powerful broad‐spectrum
fluoroquinolone antibiotic, had been chosen as a model drug
for incorporation in FMSN‐TTDDA‐N3 nanoparticles. The size of
the Cip molecule was small enough to fit into the pores of the
FMSN. Regarding the loading efficacy of guest molecules,
FMSN‐TTDDA‐N3 nanoparticles exhibited a loading capacity of
16 wt% for Cip (Fig. S9a). Sustained‐release pharmaceutical
dosage forms are very important and they enable an active
principle to be released gradually into the body. We then
studied the drug release properties of nanocarriers. As shown
in Figure S9b, the sustained release of antimicrobial drugs
from nanoparticles was detected during the first 3 days, and
then the release rate reached a platform gradually. We further
carried out a bacteria viability assay to verify the bacteria
survival rate using Cip‐loaded FMSN‐TTDDA‐N3 nanocarriers
(Fig. S10). As for EDA‐treated Gram‐positive bacteria (B.
subtilis and S. aureus), similar antimicrobial activity was
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observed between FMSN@Cip‐TTDDA‐N3 and Cip alone within binding of bacteria surface, the “magnetized” bacteria could
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DOI: 10.1039/C5CC10625G
the initial 6 h. However, compared to that without targeted be separated from the mixture of bacteria and live cells and
nanocarriers, higher antibacterial efficacy was observed after killed efficiently by the released drugs with less harmful effects
treatment with FMSN@Cip‐TTDDA‐N3 as time prolonging. This on mammalian cells. Ongoing study is to investigate this
higher antibacterial activity of Cip‐loaded nanoparticles was targeting and killing bacteria strategy to in vivo system by
mainly attributed to the sustained release of drug from designing bioorthogonal copper‐free click reactions.
targeted nanoparticles. More targeted FMSN@Cip‐TTDDA‐N3 Collectively, these findings suggest that the novel targeted
nanoparticles were covered the surface of bacteria, leading to drug delivery platform should show great potential for the
the increase of drug concentration around bacteria. However, treatment of bacterial infections.
for EDA‐treated Gram‐negative bacteria, there was no
Financial support was provided National Basic Research
significant difference in antibacterial efficiencies between Program of China (Grant 2012CB720602), the National Natural
FMSN@Cip‐TTDDA‐N3 and Cip alone. One possible reason Science Foundation of China (21210002, 21431007, 21403209,
might be that the outer membrane of Gram‐negative bacteria 21533008) and Natural Science Foundation of Hebei Province
limited the binding of FMSN@Cip‐TTDDA‐N3 to bacteria. (B2015201097).
Further tests of the viability were conducted by observing the
number of colony‐forming units on an LB agar plate (Fig. S11).
The colony count and the killing efficiency have an inversely
Notes and references
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assess the selective antibacterial effects of the FMSN@Cip‐
TTDDA‐N3 when both mammalian and bacterial cells were
introduced simultaneously. For group of control, most of the
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Conclusions
In summary, we have developed a two‐step strategy for
selective recognition, separation and killing of bacteria over
mammalian cells based on metabolic engineering and
bioorthogonal click chemistry. Bacteria surface was first
labeled with bioorthogonal chemical reporters through
metabolic engineering. Then, these unnatural groups could
effectively enhance the accumulation of azide‐modified
nanoparticles around the surface of bacteria through
bioorthogonal click chemistry. Importantly, the absence of D‐
amino acid metabolism in mammals invites the application of
this strategy to treat mycobacterial infection in host cells. After
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