Revisiting Quorum Sensing: Discovery of Additional Chemical and Biological Functions for 3-Oxo-N-Acylhomoserine Lactones

Revisiting quorum sensing: Discovery of additional chemical and biological functions for 3-oxo-N-acylhomoserine lactones Gunnar F. Kaufmann, Rafaella Sartorio, Sang-Hyeup Lee, Claude J. Rogers, Michael M. Meijler, Jason A. Moss, Bruce Clapham, Andrew P. Brogan, Tobin J. Dickerson, and Kim D. Janda* Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 Communicated by Sydney Brenner, The Salk Institute, La Jolla, CA, November 23, 2004 (received for review October 21, 2004) Bacteria use small diffusible molecules to exchange information in a process called quorum sensing. An important class of autoinducers used by Gram-negative bacteria is the family of N-acylhomoserine lactones. Here, we report the discovery of a previously undescribed nonenzymatically formed product from N-(3-oxododecanoyl)-L-homoserine lactone; both the N-acylhomoserine and its novel tetramic acid degradation product, 3-(1-hydroxydecylidene)-5-(2-hydroxyethyl)pyrrolidine-2,4-dione, are potent antibacterial agents. Bactericidal activity was observed against all tested Gram-positive bacterial strains, whereas no toxicity was seen against Gram-negative bacteria. We propose that Pseudomonas aeruginosa utilizes this tetramic acid as an interference strategy to preclude encroachment by competing bacteria. Additionally, we have discovered that this tetramic acid binds iron with comparable affinity to known bacterial siderophores, possibly providing an Fig. 1. AHLs used by P. aeruginosa in quorum sensing. unrecognized mechanism for iron solubilization. These findings merit new attention such that other previously identified autoinducers be reevaluated for additional biological functions. patients become infected with P. aeruginosa, and, of those, the majority die of septicemia. These infections are especially trou- tetramic acid ͉ bactericidal agents ͉ evolution blesome because P. aeruginosa continues to grow more resistant to many antibiotics. Over the last 10 years, significant progress has been made in he term ‘‘quorum sensing’’ has been coined to describe the elucidating the molecular mechanisms underlying P. aeruginosa Tability of a population of unicellular bacteria to act as a pathogenicity. Two different AHLs, N-(3-oxododecanoyl) ho- multicellular organism in a cell-density-dependent manner; that moserine lactone 1, synthesized by LasI, and N-butyrylhomo- is, a way to sense ‘‘how many are out there’’ (1–3). Bacteria use serine lactone 2, synthesized by RhlI, have been identified as the small diffusible molecules to exchange information among them- main quorum-sensing signaling molecules in P. aeruginosa (Fig. selves (2). An important class of ‘‘quormones,’’ or autoinducers, 1) (6). Genes regulated by this mechanism encode enzymes such is the family of N-acylhomoserine lactones (AHLs) used by as elastases A and B, catalase, and superoxide dismutase. Gram-negative bacteria. Variation in N-acyl chain length and the Furthermore, quorum sensing also has been demonstrated to BIOCHEMISTRY oxidation state of AHLs provide for bacterial strain specificity in control the expression of other virulence factors as well as the the signaling process and subsequent synchronization of gene formation of structures known as biofilms (7). Here, we dem- expression. Depending on their acylation pattern, most AHLs onstrate that N-(3-oxododecanoyl) homoserine lactone 1 per- diffuse freely across the bacterial cell membrane. Upon reaching forms a previously unrecognized role: The autoinducer itself and a critical threshold concentration, they bind to their cognate a corresponding degradation product derived from an unusual receptor proteins, triggering the expression of target genes; for Claisen-like condensation reaction function as innate bacteri- example, in the case of Vibrio fischeri, genes located on the lux cidal agents. Furthermore, the AHL degradation product tightly operon are transcribed that are responsible for the production of binds essential metals such as iron, possibly providing a previ- bioluminescence (4). Indeed, this account of V. fischeri was the ously unrecognized primordial siderophore. first description of a bacterial population acting in a concerted fashion to achieve a common goal: the simultaneous expression Materials and Methods of a particular set of genes. General Synthetic Methods. Unless otherwise stated, all reactions Pseudomonas aeruginosa is a common environmental micro- were performed under an inert atmosphere with dry reagents organism that has acquired the ability to take advantage of and solvents and flame-dried glassware. Analytical TLC was weaknesses in the host immune system to become an opportu- performed by using 0.25-mm precoated silica gel Kieselgel 60 nistic pathogen in humans (5). Most prominent is the role of P. F254 plates. Visualization of the chromatogram was by UV aeruginosa in patients suffering from cystic fibrosis because absorbance, iodine, dinitrophenylhydrazine, ceric ammonium lung-defense functions are severely impaired. As a result, infec- molybdate, ninhydrin, or potassium permanganate as appropri- tions with P. aeruginosa and the damage caused by the inflam- ate. All 1H NMR spectra were recorded on either a Bruker matory infection process increase the mortality rate in cystic fibrosis patients. Additionally, nosocomial infections by P. aeruginosa, particularly in burn victims, cause serious complica- Abbreviation: AHL, N-acylhomoserine lactone. tions because of the wide variety of virulence factors and *To whom correspondence should be addressed. E-mail: [email protected]. inherent antibiotic resistance. Significant percentages of burn © 2004 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0408639102 PNAS ͉ January 11, 2005 ͉ vol. 102 ͉ no. 2 ͉ 309–314 Downloaded by guest on September 30, 2021 AMX-500 or DRX-600 spectrometer (Billerica, MA) at 500 and of the extract was performed by using the described reverse- 600 MHz, respectively. All 13C NMR spectra were recorded on phase HPLC system (see above). The gradient used was as a Bruker AMX-500 spectrometer at 125 MHz. Optical rotations follows: t ϭ 0, 50% solvent B (0.1% trifluoroacetic acid in were determined at 598 nm in a conventional 10-cm cell by using acetonitrile) in solvent A (0.1% trifluoroacetic acid in water); t ϭ a PerkinElmer 241 MC polarimeter. MALDI-Fourier trans- 5, min 50% solvent B; t ϭ 25 min, 80% solvent B; t ϭ 35 min, form-MS experiments were performed on an IonSpec-Fourier 100% solvent B; t ϭ 50 min, 100% solvent B. Fractions were transform mass spectrometer (Lake Forest, CA). Electrospray collected every minute and analyzed by electrospray ionization- ionization-MS experiments were performed on an API 100 MS. Peaks with mass corresponding to tetramic acid 4 were PerkinElmer Sciex single-quadrupole mass spectrometer. Ana- found to elute at a retention time of 14.7 min and were confirmed lytical reverse-phase HPLC was performed on a Hitachi L-5000 by coinjection with authentic standard samples. series instrument equipped with a Vydac-C18 analytical column, a UV detector at 254 nm, and mobile phases composed of Kinetic Assays for the Formation of 3-(1-Hydroxydecylidene)-5-(2- mixtures of acetonitrile͞water (0.1% trifluoroacetic acid). Hydroxyethyl)Pyrrolidine-2,4-Dione 4. The assay was initiated by the addition of a solution of AHL (5 mM in DMSO) to phosphate General Procedure for Synthesis of Tetramic Acids. As a represen- buffer at 25°C (200 mM, pH 7.4͞10% DMSO cosolvent; 1 ml tative example, the synthesis of (S)-3-(1-hydroxydecylidene)-5- total volume). Continuous monitoring of the reaction was per- (2-hydroxyethyl)pyrrolidine-2,4-dione 4 is described. To a vial formed over 2,000 s by using a Hewlett Packard 8452A UV- containing (S)-3-oxo-C12-AHL 1 (111 mg, 0.374 mmol) in dry visible spectrophotometer. Tetramic acid product formation was MeOH (1 ml), NaOMe in MeOH (0.5 M, 0.747 ml, 0.374 mmol) determined spectrophotometrically by using an extinction coef- was added at room temperature under argon. After stirring at ficient of 13,900 MϪ1⅐cmϪ1 at ␭ ϭ 278 nm. The acquired data 55°C for 3 h, the reaction mixture was passed through acidic were fit to a two-state model in which competing reactions were ion-exchange resin (Dowex 50WX2–200, Ϸ2cm3) and further assumed to be nonequilibrating. Data analysis was performed by eluted with MeOH (20 ml). The combined filtrate was concen- nonlinear curve-fitting algorithms by using the program KALEI- trated under reduced pressure, and the resulting residue was DAGRAPH 3.6.2 (Synergy Software, Reading, PA). recrystallized (EtOAc͞hexanes) to give an off-white small crys- talline solid (80 mg). This product then was purified by prepar- Antibacterial Assays. The following bacterial strains were pur- ative HPLC to give tetramic acid 4 as white fluffy solid. Tetramic chased from the American Type Culture Collection (ATCC) acids were purified further by reverse-phase HPLC using a unless otherwise stated: Bacillus cereus ATCC 11778, B. cereus Vydac 214TP101522 column (Hesperia, CA) at a flow rate of 10 ATCC 13061, B. cereus ATCC 14579, B. cereus ATCC 27348, ml͞min with detection at 230 nm on a dual-pump Rainin Bacillus licheniformis 5A36 (from Bacillus Genetic Stock Center, Dynamax HPLC system (Rainin Instruments). In each prepar- Columbus, OH), Bacillus mycoides ATCC 6462, Bacillus subtilis ative separation, recrystallized material (40 mg) was dissolved in ATCC 6051, Enterococcus

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