Bioinspired, releasable quorum sensing modulators

Article abstract of DOI:10.1039/c2cc37287h

We demonstrate the synthesis and immobilization of natural product hybrids featuring an acyl-homoserine lactone and a nitrodopamine onto biocompatible TiO₂ surfaces through an operationally simple dip-and-rinse procedure. The resulting immobili

Full text of DOI:10.1039/c2cc37287h

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Bioinspired, releasable quorum sensing modulators†
a
b
c
b
´
Jose Gomes, Alexander Grunau, Adrien K. Lawrence, Leo Eberl* and
Karl Gademann*ac
Cite this: Chem. Commun.,
2013, 49, 155
Received 5th October 2012,
Accepted 2nd November 2012
DOI: 10.1039/c2cc37287h
www.rsc.org/chemcomm
We demonstrate the synthesis and immobilization of natural product
hybrids featuring an acyl-homoserine lactone and a nitrodopamine
onto biocompatible TiO2 surfaces through an operationally simple dip-
and-rinse procedure. The resulting immobilized hybrids were shown to
be powerful quorum sensing (QS) activators in Pseudomonas strains
acting by slow release from the surface.
Fig. 1 Conceptual design of a QS modulator attached to a TiO2 surface using a
molecular anchor connected by a linker.
Nosocomial infections constitute a major health care concern world-
wide and in particular bacterial biofilm formation on indwelling communication, we report the immobilization of QS activators
medical devices poses a significant challenge. Direct systemic treat- on TiO2 beads and their successful biological evaluation in
ment with antibiotics remains difficult both due to tissue encapsula- Pseudomonas strains.
tion, leading to an up to 1000-fold decrease in antibiotic efficacy,1,2
A first challenge consisted in finding suitable surface-active
and due to the rise of resistant bacterial strains.1–5 Several hybrids that can still be recognized by the bacteria as QS modulators.
approaches aimed at inhibiting bacterial biofilm formation via As anchor groups, we hoped to exploit catechols that have been
the generation of antimicrobial or antifouling surfaces have been shown to bind strongly to several metal oxides and can thus be used
documented6–10 that could, however, give rise to resistance upon as binding groups for generating self-assembled monolayers using
long term exposure. Another approach to prevent biofilm formation an operationally simple dip-and-rinse procedure.29 Therefore, we
involves the inhibition of quorum sensing (QS),11–15 a cell-to-cell envisaged the synthesis of hybrids as shown in Fig. 1. These
signalling system that is used by numerous bacteria to control compounds would consist of a bioactive moiety, which should
expression of certain genes in a cell-density dependent manner. interfere with QS (blue in Fig. 1), a catechol anchor able to bind to
Chemically, QS is based on the exchange of diffusible small- surfaces (red), and a suitable linker to combine these two fragments.
molecule signals (often also referred to as autoinducers (AIs)),16 such AHL was chosen as the bioactive moiety as it is frequently used for
as, for example, N-acyl-L-homoserine lactones (AHLs), between cells cell-to-cell communication by various gram-negative bacteria.
of many gram-negative bacteria.17–19 Interfering with QS with small Dopamine, nitro-dopamine and the anachelin chromophore7,8 were
molecules20–24 therefore represents
a fundamentally different selected as possible anchors to investigate the recognition impact of
approach for the prevention and eradication of bacterial bio- the anchor group in the hybrids. These two fragments were
films.25–27 Blackwell and coworkers recently reported the controlled
release of non-native QS modulators from thin PLG films.28 How-
ever, covalently bound QS modulators on metal oxide surfaces
have, to the best of our knowledge, not yet been reported. In this
a Department of Chemistry, NCCR Chemical Biology, University of Basel, St.
Johanns-Ring 19, 4056 Basel, Switzerland. E-mail: karl.gademann@unibas.ch
b Department of Microbiology, Institute of Plant Biology, University of Zurich,
Zollikerstrasse 107, 8008 Zurich, Switzerland
c Chemical Synthesis Laboratory, Swiss Federal Institute of Technology (EPFL), 1015
Lausanne, Switzerland
† Electronic supplementary information (ESI) available: Synthesis and analytical
data of all hybrids, bacteriological data, release quantification, and microscopy
Fig. 2 Natural product hybrids as QS modulators with different anchors.
Chem. Commun., 2013, 49, 155--157 155
results. See DOI: 10.1039/c2cc37287h
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Scheme 1 Synthesis of compound 2 and immobilization on TiO2 beads Ti-2.
connected by alkyl linkers of various lengths and preliminary screen-
ing showed that the hybrid featuring a C12 linker (Fig. 2) induced QS
in the AHL biosensor strain C. violaceum CV026.30 Other linker
lengths did not lead to QS induction, even at high concentrations.
The influence of the catechol anchor group on activity was studied
next, and no significant difference between compounds 1–3 was
observed. We therefore chose the nitro-dopamine derivative 2 for
further studies, due to the ease of accessibility, strong binding
properties towards metal oxides, and stability towards oxida-
tion.29c The synthesis of hybrid 2 is shown in Scheme 1 and was
achieved in 3 steps from commercial dodecanedioic acid in
72% yield.
Fig. 3 Phase contrast (A) and GFP fluorescence (B) of the sensor as a negative
control. Phase contrast (C) and GFP fluorescence (D) of the sensor incubated with
compound 2 as a positive control. Phase contrast (E) and GFP fluorescence (F) of
the sensor incubated with the functionalized beads Ti-2.
After having established that hybrid 2 is able to interfere with QS
in a liquid based assay (see ESI†), we wanted to evaluate its activity
after immobilization on surfaces. We decided to use titanium
dioxide as this metal oxide is widely used for orthopedic and dental
implants due to its biocompatibility. For ease of handling and functionalized beads Ti-2 were incubated with the P. putida
visualization, we decided to use TiO2 beads with an average F117 (pAS-C8) biosensor strain for 2 hours and the experiments
diameter of 1 mm. Therefore, a modified dip-and-rinse procedure29 were monitored by fluorescence microscopy (Fig. 3). In the
was applied: the beads were incubated in a dilute solution of 2 in absence of an inducer, no fluorescence could be observed in the
high salt buffer (0.1 M MOPS/0.6 M NaCl/0.6 M K2SO4) for 4 hours negative control (Fig. 3A and B). By contrast, in the presence of
at 50 1C to result in the functionalized beads Ti-2. In a control compound 2, which served as a positive control, the reporter
experiment, no hydrolysis products of 2 could be detected after strain produced GFP as shown in Fig. 3C and D. Similarly to
4 hours at 50 1C by UPLC-MS. After evaluating several bacterial AHL compound 2, the functionalized beads Ti-2 (visible in the phase
reporter strains, we observed that hybrid 2 and Ti-2 activated the contrast image Fig. 3E) induced the biosensor (Fig. 3F), suggesting
GFP-based biosensor P. putida F117 (pAS-C8) similarly to N-octanoyl- that hybrids are released from the surface.
L-homoserine lactone (C8-AHL), for which it was shown to be most
Several mechanisms can be formulated to explain these observa-
sensitive.31 GFP fluorescence is induced in this strain only when tions. On one hand, induction of QS by contact of a bacterium with
AHL molecules with C6 to C12 acyl side chains are provided the functionalized bead Ti-2 and concomitant capture of the QS
externally. The functionalized beads Ti-2 induced a similar fluores- modulator can be hypothesized (‘the billiards hypothesis’). As an
cence response as the natural agonist in this assay.
alternative, more feasible hypothesis, slow release of QS modulators
We then evaluated if the immobilized hybrid 2 on TiO2 beads from the surface and subsequent induction of QS in the reporter
could be removed by sequential washing operations (Fig. S1, ESI†) strains might also be operative. In order to address this question, we
thus potentially impacting the activity. Interestingly, washing the performed dialysis assays using cellulose ester membranes with 3.5–
beads up to ten times did not alter the biological activity of Ti-2 as 5 kDa molecular weight cut off. In this assay the beads are retained
judged by measured GFP levels in the pAS-C8 reporter strain. These in dialysis bags but released AHL mimics can diffuse into the
results clearly demonstrate that the immobilized hybrid Ti-2 is a surrounding medium and induce the biosensor. The functionalized
potent QS inducer which retains activity even upon repeated rinsing. beads Ti-2 were dialyzed overnight in buffer solution, and the
Next, we wanted to address the mode of action of the supernatant was then incubated with the sensor for 3 hours at
functionalized beads Ti-2. In particular, we were interested to 30 1C (see ESI†). Fluorescence was observed in the reporter strain,
determine if QS is induced exclusively at the surface of the which supports the mechanism that AHL mimics are released from
beads or in the entire bacterial population. Therefore, the the beads. In order to test the kinetics of release, samples were taken
c
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