Synthesis and validation of a probe to identify quorum sensing receptors

Article abstract of DOI:10.1039/b917507e

The synthesis and evaluation of a 'tag-free' probe to isolate and identify receptors for N-acyl homoserine lactones is described.

Full text of DOI:10.1039/b917507e

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Synthesis and validation of a probe to identify quorum sensing receptorsw
a
b
Luba Dubinsky,z Lucja M. Jarosz,z Neri Amara,a Pnina Krief,a Vladimir V. Kravchenko,c
Bastiaan P. Krom*b and Michael M. Meijler*a
Received (in Cambridge, UK) 25th August 2009, Accepted 28th September 2009
First published as an Advance Article on the web 28th October 2009
DOI: 10.1039/b917507e
The synthesis and evaluation of a ‘tag-free’ probe to isolate and
institute countermeasures.12 The resulting response in
identify receptors for N-acyl homoserine lactones is described.
eukaryotes seems to point to a conserved downregulation of
the immune response, which is beneficial to the bacteria and
may or may not be beneficial to the host. In another example
of eukaryotic response to bacterial crosstalk, studies have
shown that the nematode Caenorhabditis elegans senses the
presence and possibly also the population size of P. aeruginosa
by recognizing and responding to C12.13,14
The term ‘‘quorum sensing’’ (QS) is used to describe the
mechanism that bacteria use to coordinate their behavior in
a cell-density dependent manner.1 Using this mechanism,
which is based on the exchange of small diffusible signaling
molecules between cells, bacterial cultures can essentially
function as multicellular organisms. Examples of QS-controlled
behaviors are production of light (bioluminescence), virulence
factor expression and biofilm formation. These processes are
advantageous to a bacterial population only when they are
carried out simultaneously by all its members.2
Recently, it has been shown that in many cases of
Candida albicans infection, biofilm formation is involved.
For both the infection process and biofilm formation, a
morphological transition from yeast cells to filamentous cells
(hyphae) is essential.15 Because of its importance, this
morphological switch is tightly regulated. Recently, C12 was
shown to repress hyphal formation.16 The mechanisms
underlying the interactions between C12 and putative receptors
in the mentioned eukaryotes are completely unknown. Jahoor
et al. recently proposed that PPARg is a mammalian
C12 receptor, although no binding studies were reported;11
their observed effects of C12 on PPARg activity could be the
result of C12 binding to a different protein, ultimately resulting
in downstream activity. We set out to isolate, identify and
characterize eukaryotic receptors for C12, through a ‘tag-free’
activity-based protein profiling approach based on a highly
selective copper(I) mediated azide–alkyne cycloaddition
reaction.17–19 Our design scheme was based on the following
findings: (1) it was shown previously that any modification in
the lactone ring leads to diminished activity in mammalian
cells;4,10 (2) small modifications in the alkyl chain did not
cause a significant loss of activity.
During the past two decades a wealth of information
regarding intercellular communication among bacteria have
been uncovered. QS is mediated by secretion and recognition
of small diffusible molecules, of which an example is the class
of N-acyl homoserine lactones (AHLs), used by many known
Gram-negative bacteria, such as the opportunistic pathogen
Pseudomonas aeruginosa. The QS molecule N-(3-oxododecanoyl)
homoserine lactone (3-oxo-C12-HSL, C12, Fig. 1, 1) has been
identified as the primary signal to enable QS in P. aeruginosa
and this system involves the LasI (synthase) and LasR
(receptor) proteins. This is part of a sophisticated strategy to
survive under conditions that do not favor bacteria living
solely as individual cells.
A growing number of reports indicate that bacterial AHL
autoinducers can influence gene expression in eukaryotic cells.
Most of these studies focus on the effects of the P. aeruginosa
autoinducer C12 on the production of a number of cytokines,
such as IFN-g, by immune cells in vitro and in vivo.3–11
A recent study by Kravchenko et al. has uncovered a mechanism
in which C12 selectively disrupts NF-kB signaling in activated
mammalian cells.9,10 These findings indicate that P. aeruginosa
has developed means to detect host immune activation and to
We therefore chose to design AHL analogs with only
minimal structural deviations in the alkyl chain so as to
increase the chances of recognition. In this approach, the
probe is first conjugated to its receptor through irradiation
of a functional moiety, such as diazirine. This smallest possible
photoactive group, that can be activated by light irradiation to
generate a highly active carbene, has been used successfully by
others to label proteins.20–22 Then, following the methodology
a Department of Chemistry, Ben-Gurion University of the Negev,
Be’er-Sheva 84105, Israel. E-mail: meijler@bgu.ac.il;
Fax: +972-8-6472751; Tel: +972-8-6472751
b Department of Biomedical Engineering, The Kolff Institute,
University Medical Center Groningen and the University of
Groningen, Antonius Deusinglaan 1, 9713 AV Groningen,
The Netherlands. E-mail: b.p.krom@med.umcg.nl;
Fax: +31-50-3633159; Tel: +31-50-3633160
c Department of Immunology and Microbial Sciences,
The Scripps Research Institute, 10550 North Torrey Pines Road,
La Jolla, CA 92037, USA
w Electronic supplementary information (ESI) available: Synthetic
procedures and characterization of compounds. Experimental details
of biological evaluations and analysis by mass spectrometry. See DOI:
10.1039/b917507e
Fig. 1 Structures of 3-oxo-C12-HSL (C12, 1) and diazirine alkynyl
probe 2.
z These authors contributed equally to the project.
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Scheme 1 Labeling of unknown receptors with use of activatable
bifunctional AHL-based probes. Addition of two minimally perturbing
moieties to the homoserine lactone will allow for specific binding to an
AHL receptor amidst a wide variety of proteins. Irradiation with UV
light (365 nm) will result in a covalent bond between the probe and the
receptor. A highly specific 1,3-dipolar cycloaddition reaction (‘click’)
with a tag (or fluorophore) will then allow for isolation and identification
of the receptor.
Fig. 2 (A) Induction of bioluminescence by 5 mM C12 (lane 4,
triplicates) and 5 mM probe 2 (lane 3) in a lasR-based AHL biosensor
(E. coli pSB1075) strain. Luminescence was measured after 3 h growth.
Lane 1 contains 0.5% DMSO in LB medium, lane 2 only LB medium.
All wells were inoculated with a 1/100 diluted overnight culture;
(B) biological activity of C12 and its diazirine alkynyl analog 2 in
bone marrow-derived macrophages (BMDM). BMDM were treated
with C12 or 2 for 30 min, and the cellular extracts were analyzed by
Western blot for the phosphorylated form of eIF2a (p-eIF2a), as a
biochemical marker of C12-mediated activation of mammalian cells.
Western blot for actin was used as a control. Relative fold of p-eIF2a
induction was estimated by densitometry. See ref. 9 for experimental
details; (C) effects of C12 and probe 2 on germ tube formation in
C. albicans. Calculated IC50: 15.3 mM (C12), 21.8 mM (2).
developed by Cravatt and co-workers for the identification of
unannotated enzymes,17 the probes, while attached to their
target proteins, will undergo reaction with their cycloaddition
partner, which itself is attached to a tag/reporter group such as
biotin or rhodamine (Scheme 1). This will then enable isolation
of the probe-bound protein, followed by identification by mass
spectrometry. By applying this general scheme, we aim to
develop a method to characterize the interaction between
AHLs and unknown proteins that bind the AHLs.
Probe 2 was prepared from 6-chlorohexyne as shown in
Scheme 2. Following procedures described by Hodgson
et al.,23 4-oxononynoic acid 5 was prepared, and upon
treatment with liquid ammonia the aziridine intermediate
was obtained. Oxidation with iodine resulted in the formation
of diazirine 6, followed by homologation with mono tert-butyl
malonate. Deprotection of 7 and coupling with homoserine
lactone yielded diazirine alkynyl probe 2.
was induced by 2, albeit slightly less than by C12 (Fig. 2B).
Inhibition of germ tube (GT) formation in C. albicans was
inhibited by C12 and probe 2 similarly (Fig. 2C). These
experiments indicate that probe 2 is capable of mimicking
C12 in diverse microbial and eukaryotic systems.
In order to determine accurate and optimized conditions for
protein labeling by this probe and other diazirine-based
probes, we focused on labeling of LasR. Bottomley et al.
published the first crystal structure of the ligand binding
domain (LBD) of LasR,24 bound to C12, and following their
protocol we overexpressed this protein in E. coli.
Probe 2 was first tested for its ability to mimic C12 using
Escherichia coli pSB1075 as the reporter strain. Induction of
luminescence, indicative of activation of the LasI–LasR QS
system was observed (Fig. 2A). In addition, the induction of
elastase, a virulence factor under control of the Las system,
was analyzed in a P. aeruginosa LasI mutant unable to
synthesize C12. IC50 values ranged from 1 mM for C12 to
0.03 mM for probe 2 (ESIw, Fig. S1). We then verified whether
2 would mimic the activity of C12 in two eukaryotic systems.
Phosphorylation of the initiator factor eIF2a in macrophages
During expression of the protein in the presence of 2, cells
were irradiated for 15 min with a UV-lamp (365 nm), after
which cell membranes were lysed and LasR-LBD was isolated
through affinity purification. Proteins were analyzed by
LC/MS as described by us previously,25 and while extracted
protein that was expressed in the presence of C12 alone
Scheme 2 Synthesis of ‘tag-free’ diazirine alkynyl AHL probe 2.
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(20 mM) yielded a single peak (22 427 Da, Fig. 3A) corresponding
to the expected mass of LasR-LBD (22 430 Da), the experiment
in which LasR was expressed in the presence of probe 2 (20 mM)
resulted in an additional peak with a mass difference of 292 Da
(Fig. 3B, expected difference: 290 Da). In a third experiment in
which LasR was expressed in the presence of both C12 and 2
(20 mM each) the additional peak had disappeared (Fig. 3C),
showing that 2 competes with C12 for the same binding site,
albeit with lower affinity. From these experiments it follows that
under the crosslinking conditions a significant fraction of LasR is
labeled covalently and specifically.
This work was supported by the Human Frontier Science
Program (Young Investigator Grant RGY0072/2007, BPK and
MMM), the National Institutes of Health (AI079436, VVK), and
by the US–Israel Binational Science Foundation (Startup Grant
2006287, MMM). We thank Dr R. A. Ulevitch for valuable
insights and support, Dr K. D. Janda and Dr G. F. Kaufmann
for helpful discussions, and Dr M. J. Bottomley, Dr D. A. Hogan
and Dr W. J. Quax for bacterial strains and plasmids.
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specific labeling of LasR by probe 2 as observed in our
experiments is encouraging in our attempts to isolate and
identify putative receptors in eukaryotes that bind C12. We
are currently using probe 2 in pursuing this target. As the exact
location of the diazirine moiety may influence specific binding
to C12-binding proteins in different organisms we are also
currently expanding our toolset with C12-based probes with
diazirine moieties at various locations in the alkyl chain.
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Fig.
3 Deconvoluted ESI mass spectra of crude LasR-LBD:
(A) overexpressed in E. coli in the presence of C12 (20 mM). Calculated
mass: 22 430 Da. Observed: 22 428 Da, and several adducts—possibly
the result of phosphorylation and/or methionine oxidation; (B) over-
expressed in the presence of diazirine probe 2 (20 mM). Calculated mass
increase upon labeling with 2: 290 Da. Observed increase: 292 Da;
(C) overexpressed in the presence of both probe 2 (20 mM) and C12
(20 mM). 22 484 Da is an unknown (possibly oxidized) adduct. All three
cultures were irradiated with a UV lamp (365 nm) for 15 min to effect
crosslinking of the probe, before lysis and affinity purification.
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This journal is The Royal Society of Chemistry 2009
7380 | Chem. Commun., 2009, 7378–7380