Antibody interference with N-Acyl homoserine lactone-mediated bacterial quorum sensing

Article abstract of DOI:10.1021/ja0578698

Many bacterial pathogens coordinate their virulence factor expression in a cell density-dependent manner. This population-dependent coordination of gene expression in bacteria has been termed "quorum sensing" (QS). N-Acyl homoserine lactones (AHLs) are us

Full text of DOI:10.1021/ja0578698

Published on Web 02/09/2006
Antibody Interference with N-Acyl Homoserine Lactone-Mediated Bacterial
Quorum Sensing
Gunnar F. Kaufmann, Rafaella Sartorio, Sang-Hyeup Lee, Jenny M. Mee, Laurence J. Altobell, III,
David P. Kujawa, Emily Jeffries, Bruce Clapham, Michael M. Meijler, and Kim D. Janda*
The Skaggs Institute for Chemical Biology and Departments of Chemistry and Immunology,
The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037
Received November 20, 2005; E-mail: kdjanda@scripps.edu
Cell density-dependent coordination of gene expression in
bacteria has been termed “quorum sensing” (QS).1 N-Acyl L-
homoserine lactones (AHLs) are produced by over 70 species of
Gram-negative bacteria, and structural differences within AHLs
occur in the length and oxidation state of the acyl side chain (Figure
1). Upon reaching a critical threshold concentration, AHLs bind to
Figure 1. Examples of N-acyl homoserine lactones employed by bacteria
as quorum sensing molecules.
their cognate receptor proteins, triggering the expression of target
genes. AHLs have been shown to play an important role in the
establishment and course of bacterial infections. One example of a
Gram-negative pathogen that employs AHL-based QS to regulate
the expression of its pathogenicity factors is Pseudomonas aerugi-
nosa. This common environmental microorganism has acquired the
ability to take advantage of weaknesses in the host defenses to
become an opportunistic pathogen in humans. Most prominent is
Figure 2. 4-Methoxyphenyl amide AHL analogues.
the role of P. aeruginosa in patients suffering from cystic fibrosis
h. Interestingly, the oxidation state and chain length did not
influence significantly the hydrolytic rate of these molecules.5 While
the expected ring-opened hydrolysis product was produced, a
previously undetected side product, a tetramic acid, arising from
an intramolecular Claisen-like condensation reaction was also
uncovered.5 On the basis of these results and the insufficient long-
term stability of compounds 1-4, our choice was to replace the
lactone moiety with a lactam functionality. Thus, the lactam ring
would grant more chemical stability toward ring opening/hydrolysis
under basic conditions, as well as stability toward the intramolecular
Claisen condensation (vide supra).
The amino lactam 6 surrogate was synthesized from com-
mercially available (S)-2,4-diaminobutyric acid 5 according to
literature precedent.6 To enhance our chances for the elicitation of
antibodies that could differentiate AHL chain length and oxidation
state, different linker moieties were designed in recognition of AHLs
employed by different bacteria; thus, the linkers of 7-9 that mimic
the acyl chains of three types of autoinducers were prepared and
coupled to lactam 6, yielding haptens 7-9 (Scheme 1). These three
haptens were conjugated to carrier proteins KLH and BSA, resulting
in 18-23 haptens per carrier protein. Balb/c mice were immunized
with the KLH immunoconjugates for hybridoma production using
standard protocols. All immunizations resulted in excellent titers
(25600-12800), and based on ELISA results, 20 monoclonal
antibodies (mAbs) for RS1 (7), 21 for RS2 (8), and 27 for RS3 (9)
were selected and purified. The affinities for all mAbs against a
series of AHLs (C4, 3-oxo-C6, and 3-oxo-C12) as well as the 3-oxo-
C12-lactam were determined using competition ELISA (Supporting
Information). None of the assayed RS1 antibodies possessed a Kd
lower than 100 µM for the 3-oxo-AHLs, nor for the lactam
analogue. However, several of the RS2 mAbs generated against
the 3-oxo-hapten demonstrated good to excellent affinity (Kd )
150 nM to 5 µM) for 3-oxo-C12-AHL and the lactam analogue
and exhibited high specificity as they did not show any recognition
of the short chain 3-oxo-AHLs. Finally, the RS3 mAbs exhibited
(CF). Over the last 15 years, much progress has been made in
elucidating the molecular mechanisms underlying P. aeruginosa
pathogenicity.2 Two different AHLs, N-(3-oxododecanoyl) ho-
moserine lactone (3-oxo-C12-AHL) and N-butyryl homoserine
lactone (C4-AHL), have been identified as QS signaling molecules
in P. aeruginosa. Genes regulated by this QS mechanism encode
enzymes such as elastases A and B, catalase, and superoxide
dismutase as well as exotoxins.2
Interference with QS signaling has been suggested as a new
approach for anti-infective therapy.3 In fact, this strategy has yielded
interesting results using AHL analogues as QS antagonists in P.
aeruginosa.4 Alternatively, we have embarked on a program
utilizing antibodies to inhibit AHL-mediated quorum sensing
signaling in P. aeruginosa. AHL-based QS systems represent an
ideal target for antibody-based anti-infective therapy given the
highly conserved molecular scaffold and extracellular distribution
of AHLs.
Our initial hapten design for the elicitation of anti-AHL antibod-
ies focused on synthesizing a set of molecules highly congruent in
structure to AHLs while also possessing a pendant carboxylic acid
functionality that would enable carrier proteins BSA or KLH
conjugates to be easily accessed. However, we were aware that
such molecules might also be prone to hydrolysis (ring-opened
products) under conditions required for chemical coupling and also
immunization. Thus, to guide our hapten design, the stability of
several AHLs under physiological conditions was investigated; we
synthesized a number of AHL analogues and their corresponding
ring-opened hydrolysis products, both of which contained a
4-methoxyphenyl amide group that allowed for a detailed investiga-
tion into the rate of hydrolysis of the AHL analogues using HPLC
with UV detection (Figure 2).
Each compound was assayed for its hydrolysis rate in phosphate
buffer saline (PBS), pH 7.2, at a concentration of 200 µM at 37
°C. The half-lives of each of the lactones varied from 13.7 to 18.1
9
2802
J. AM. CHEM. SOC. 2006, 128, 2802-2803
10.1021/ja0578698 CCC: $33.50 © 2006 American Chemical Society

C O M M U N I C A T I O N S
Scheme 1. Synthesis of Lactam-Containing Haptens
Figure 3. Inhibition of 3-oxo-C12-AHL-mediated QS signaling by RS2
mAbs and control mAb in P. aeruginosa PAO (wild-type, filled columns)
and in PAO-JM2 (open columns) in a GFP reporter assay (A). Inhibition
of pyocyanin production in P. aeruginosa PAO (wild-type, filled columns)
and in PAO-JM2 (open columns) by mAb RS2-1G9 (B). Anti-nicotine
and anti-stilbene mAbs (NIC9D9 and EP2-19G2) and BSA were used as
additional controls.
low to moderate affinity (Kd ) 10-50 µM) to the short-chain 3-oxo-
AHLs and virtually no recognition of 3-oxo-C12-AHL nor the
lactam.
In total, the overall immune response seen for haptens 7-9 might
be construed as only moderately effective. However, we note that
this is one of the few demonstrations of the generation of tight
binding antibodies to structures of basically aliphatic composition
with no aromaticity, charge, and few hydrogen bond donor/acceptor
opportunities.7 All of these interactions are normally considered
critical for the generation of antibodies with high affinity and
specificity.
To investigate potential disruption of bacterial cell-cell com-
munication, five RS2 mAbs and one unrelated control mAb were
selected and evaluated for their ability to inhibit 3-oxo-C12-AHL-
mediated QS signaling in a green fluorescent protein (GFP) reporter
assay using the wild-type P. aeruginosa PAO strain and the double
knockout PAO-JM2 (∆rhlI, ∆lasI) strain. 3-oxo-C12-AHL (either
endogenously (PAO) produced or exogenously (PAO-JM2) added)
is required for reporter gene transcription activation, which in turn
can be measured as the level of GFP fluorescence.
The mAb RS2-1G9 exhibited excellent inhibition of QS signaling
in both P. aeruginosa strains, while all other tested mAbs show
little or no inhibition (Figure 3A). Excitingly, RS2-1G9 is the first
reported antibody to show an inhibitory effect on QS signaling in
both wild-type and mutant P. aeruginosa PAO cells. Furthermore,
in our evaluation of mAb RS2-1G9 for its ability to prevent
virulence factor expression, we observed a clear inhibitory effect
in both PAO and PAO-JM2 cultures on the production of pyocya-
nin, a QS-controlled virulence factor in P. aeruginosa (Figure 3B).4
In summary, we have demonstrated the validity of our hapten
design strategy to obtain specific mAbs to AHLs. One mAb, RS2-
1G9, possesses the ability to inhibit 3-oxo-C12-AHL-based QS
signaling. This antibody may be envisioned as a tool for future
investigations into 3-oxo-C12-AHL-based QS, which may aid in
gaining new insights into the pathogenesis of P. aeruginosa and
may ultimately lead to the development of new strategies to combat
bacterial diseases.
Acknowledgment. We thank Dr. Barbara H. Iglewski and Jane
Malone of the University of Rochester for the generous gift of
bacteria and plasmids used in our study. This work was supported
by the National Institutes of Health (AI055781) and The Skaggs
Institute for Chemical Biology.
Supporting Information Available: Experimental procedures for
the syntheses of compounds 1-9 and bioassays. This material is
available free of charge via the Internet at http://pubs.acs.org.
References
(1) (a) Waters, C. M.; Bassler, B. L. Annu. ReV. Cell DeV. Biol. 2005, 21,
319-346. (b) de Kievit, T. R.; Iglewski, B. H. Infect. Immun. 2000, 68,
4839-4849. (c) Fuqua, W. C.; Winans, S. C.; Greenberg, E. P. J.
Bacteriol. 1994, 176, 269-275.
(2) (a) Chun, C. K.; Ozer, E. A.; Welsh, M. J.; Zabner, J.; Greenberg, E. P.
Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 3587-3590. (b) Pearson, J. P.;
Gray, K. M.; Passador, L.; Tucker, K. D.; Eberhard, A.; Iglewski, B. H.;
Greenberg, E. P. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 197-201. (c)
Pearson, J. P.; Passador, L.; Iglewski, B. H.; Greenberg, E. P. Proc. Natl.
Acad. Sci. U.S.A. 1995, 92, 1490-1494. (d) Wagner, V. E.; Bushnell,
D.; Passador, L.; Brooks, A. I.; Iglewski, B. H. J. Bacteriol. 2003, 185,
2080-2095. (e) Juhas, M.; Eberl, L.; Tummler, B. EnViron. Microbiol.
2005, 7, 459-471.
(3) (a) Alksne, L. E.; Projan, S. J. Curr. Opin. Biotechnol. 2000, 11, 625-
636. (b) Lyon, G. J.; Muir, T. W. Chem. Biol. 2003, 10, 1007-1021. (c)
Finch, R. G.; Pritchard, D. I.; Bycroft, B. W.; Williams, P.; Stewart, G.
S. J. Antimicrob. Chemother. 1998, 42, 569-571.
(4) (a) Smith, K. M.; Bu, Y. G.; Suga, H. Chem. Biol. 2003, 10, 563-571.
(b) Geske, G. D.; Wezeman, R. J.; Siegel, A. P.; Blackwell, H. E. J. Am.
Chem. Soc. 2005, 127, 12762-12763. (c) Smith, K. M.; Bu, Y.; Suga, H.
Chem. Biol. 2003, 10, 81-89. (d) Manefield, M.; Rasmussen, T. B.;
Henzter, M.; Andersen, J. B.; Steinberg, P.; Kjelleberg, S.; Givskov, M.
Microbiology 2002, 148, 1119-1127.
(5) Kaufmann, G. F.; Sartorio, R.; Lee, S. H.; Rogers, C. J.; Meijler, M. M.;
Moss, J. A.; Clapham, B.; Brogan, A. P.; Dickerson, T. J.; Janda, K. D.
Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 309-314.
(6) Pellegata, R.; Pinza, M.; Pifferi, G. Synthesis 1978, 614-616.
(7) Gargir, A.; Ofek, I.; Meron-Sudai, S.; Tanamy, M. G.; Kabouridis, P. S.;
Nissim, A. Biochim. Biophys. Acta 2002, 1569, 167-173.
JA0578698
9
J. AM. CHEM. SOC. VOL. 128, NO. 9, 2006 2803