The Skaggs Institute
for Chemical Biology

Scientific Report 2008

Bacterial Quorum Sensing

K.D. Janda, J. Ashley, K. Capková, S. De Lamo Marin, J. Denery, T.J. Dickerson, A. Di Mola, B. Ellis, L. Eubanks, K. Fukuchi, C. Hernandez, G. Kaufmann, C. Lowery, S. Mahajan, A. Mayorov, G. McElhaney, J. Mee, A. Moreno, Y. Nakai, A. Nguyen, A. Nunes, J. Park, A. Rohrbach, C. Saccavini, N. Salzameda, S. Steiniger, J.B. Treweek, A. Willis, Y. Xu, Y. Yoneda, B. Zhou, H. Zhou

Originally described as a method of cell-to-cell signaling through which bacterial populations engaged in coordinated behavior, quorum sensing has since been shown to mediate several microbial processes, from formation of biofilms and bioluminescence to expression of virulence factors, interspecies competition, and infectivity. Thus, the study of quorum sensing has both general relevance to the field of microbiology and medical implications in relation to combating bacterial infectivity. With respect to bacterial infectivity, interference of quorum sensing has been approached experimentally by designing antagonists against autoinducers, the signaling molecules secreted by bacteria, and by using immunopharmacotherapeutic agents to bind secreted quorum-sensing compounds, thereby preventing signal transmission between cells.

Modulation Of Quorum Sensing Through Synthetic 4,5-Dihydroxy-2,3-Pentanedione Analogs

The capacity for autoinducers to mediate a variety of interspecies and interkingdom interactions is achieved through the specificity of different bacterial species in secreting and recognizing quorum-sensing molecules within distinct structural classes. Traditionally, autoinducers have been classified within 2 major groups: acyl homoserine lactones (AHLs), used by gram-negative bacteria, and oligopeptides, used by gram-positive bacteria. It has since been realized that the signaling molecules used by bacteria span a much larger chemical space; more notable autoinducers include the Pseudomonas quinolone signal, bradyoxetin, and AI-2, a class of autoinducers derived from the precursor 4,5-dihydroxy-2,3-pentanedione (DPD). The AI-2 quorum-sensing system is used by both gram-negative and gram-positive bacteria, and the DPD synthase is expressed by more than 70 bacterial species.

The prevalence of AI-2 was suggestive of its role in interspecies signaling, and indeed the hypothesis that a common biosynthetic pathway exists among bacteria has been confirmed chemically. We chose Vibrio harveyi and Salmonella typhimurium as bacterial models for our investigations of AI-2–based quorum sensing because these are the only species in which the discrete structures of DPD-based autoinducers and their respective receptor proteins have been identified. We hypothesized that upon DPD secretion by one species, many DPD-based signaling molecules would be generated because of the reactivity and variable stereochemistry of the parent DPD compound.

To probe the specificity of AI-2–based quorum sensing, we synthesized a panel of C1-substituted DPD analogs and evaluated them in 2 biological assays (Fig. 1). A goal of this experimental plan was to identify DPD-based agonists or antagonists that could be used in investigations of unknown AI-2 receptor proteins and the modulation of AI-2–based signaling. In the first assay, the effect of all DPD analogs on β-galactosidase production in S typhimurium was measured in the absence (agonist screen) and presence (antagonist screen) of DPD. Excitingly, all test compounds antagonized AI-2–based quorum sensing. In particular, the propyl- and butyl-substituted analogs caused potent inhibition of quorum sensing, with IC₅₀ values 10-fold lower than the concentration of the natural DPD signal (Fig. 1B).

Fig. 1. DPD analogs. A, Synthetic route for construction of DPD with alkyl groups introduced at the C-1 position. B, Effect of synthesized DPD-related compounds 5a–5g on quorum sensing as assessed in 2 biological assays: induction of β-galactosidase activity in S typhimurium and bioluminescence of V harveyi.

Like S typhimurium, V harveyi is responsive to the AI-2 class of autoinducers. The second screen capitalized on the inability of V harveyi cells to luminesce through either the AHL pathway or the AI-2 pathway in the absence of exogenous DPD. Application of the DPD analogs to V harveyi cultures resulted in only mild agonist activity. However, addition of 1 μ M DPD to these cultures had a dramatic synergistic effect: activation of bioluminescence with the DPD analogs was several-fold greater than that caused by 1 μ M DPD alone (Fig. 1B).

Impeding Bacterial Infection Through Antibodies Against Quorum-Sensing Compounds

We have pioneered an antibody-based strategy to combat bacterial infectivity by disrupting transmission of quorum-sensing signals. Recently, we applied our antibody-based technology to disruption of the quorum-sensing circuits of Pseudomonas aeruginosa. This gram-negative bacterium uses various quorum-sensing systems for more nefarious purposes with respect to interspecies communication. Namely, the 2-alkyl-4-quinolones and related AHLs (Fig. 2) secreted by P aeruginosa have antibacterial activity against gram-positive bacteria, allowing P aeruginosa to outcompete other bacterial species within a shared environment. The clinical relevance of quorum sensing by P aeruginosa includes the displacement of Staphylococcus aureus in the lungs of patients with cystic fibrosis and detrimental AHL-mediated immunomodulation in host cells, including an altered inflammatory response, a weakened host defense system, and induction of apoptosis. Therefore, removal of AHLs, which are thought to mediate the cytotoxicity in mammalian macrophages and neutrophils, may be advantageous to controlling this aspect of quorum sensing–related pathogenicity.

Fig. 2. General chemical structure of ALHs (left), a major class of autoinducers, and a representative member of this class, 3-oxo-C12-HSL (right).

To this end, we engineered monoclonal antibodies against AHL targets, and most notably, we showed that the monoclonal antibody RS2-1G9 had inhibitory activity in vitro against quorum sensing of P aeruginosa based on 3-oxo-C₁₂-homoserine lactone (3-oxo-C₁₂-HSL; Fig. 2). RS2-1G9 not only protected murine macrophages exposed to 3-oxo-C₁₂-HSL in a concentration-dependent manner but also prevented the downstream activation of cellular stress kinase pathways, indicating complete sequestration of 3-oxo-C₁₂-HSL. Thus, using this immunopharmacotherapy to quench expression of bacterial virulence factors and quorum sensing holds promise both in preventing infection and AHL-associated cytotoxicity and in developing therapies that will not promote the evolution of methicillin-resistant S aureus and future "superbugs."

Publications

Brogan, A.P., Dickerson, T.J., Janda, K.D. Nornicotine-organocatalyzed aqueous reduction of α,β-unsaturated aldehydes. Chem. Commun. (Camb.) Issue 46:4952, 2007.

Capková, K., Hixon, M.S., McAllister, L.A., Janda, K.D. Toward the discovery of potent inhibitors of botulinum neurotoxin A: development of a robust LC MS based assay operational from low to subnanomolar enzyme concentrations. Chem. Commun. (Camb.) Issue 30:3525, 2008.

Capková, K., Yoneda, Y., Dickerson, T.J., Janda, K.D. Synthesis and structure-activity relationships of second-generation hydroxamate botulinum neurotoxin A protease inhibitors. Bioorg. Med. Chem. Lett. 17:6463, 2007.

Debler, E.W., Kaufmann, G.F., Meijler, M.M., Heine, A., Mee, J.M., Pljevaljcic, G., Di Bilio, A.J., Schultz, P.G., Millar, D.P., Janda, K.D., Wilson, I.A., Gray, H.B., Lerner, R.A. Deeply inverted electron-hole recombination in a luminescent antibody-stilbene complex. Science 319:1232, 2008.

Kaufmann, G.F., Park, J., Janda, K.D. Bacterial quorum sensing: a new target for anti-infective immunotherapy. Expert Opin. Biol. Ther. 8:719, 2008.

Kaufmann, G.F., Park, J., Mee, J.M., Ulevitch, R.J., Janda, K.D. The quorum quenching antibody RS2-1G9 protects macrophages from the cytotoxic effects of the Pseudomonas aeruginosa quorum sensing signalling molecule N-3-oxo-dodecanoyl-homoserine lactone. Mol. Immunol. 45:2710, 2008.

Kravchenko, V.V., Kaufmann, G.F., Mathison, J.C., Scott, D.A., Katz, A.Z., Grauer D.C., Lehmann, M., Meijler, M.M., Janda, K.D., Ulevitch, R.J. Modulation of gene expression via disruption of NF-κB signaling by a bacterial small molecule. Science 321:259, 2008.

Lowery, C.A., Dickerson, T.J., Janda, K.D. Interspecies and interkingdom communication mediated by bacterial quorum sensing. Chem. Soc. Rev. 37:1337, 2008.

Lowery, C.A., Park, J., Kaufmann, G.F., Janda, K.D. An unexpected switch in the modulation of AI-2-based quorum sensing discovered through synthetic 4,5-dihydroxy-2,3-pentanedione analogues. J. Am. Chem. Soc. 130:9200, 2008.

Park, J., Dickerson, T.J., Janda, K.D. Major sperm protein as a diagnostic antigen for onchocerciasis. Bioorg. Med. Chem. 16:7206, 2008.

Park, J., Jagasia, R., Kaufmann, G.F., Mathison, J.C., Ruiz, D.I., Moss, J.A., Meijler, M.M., Ulevitch, R.J., Janda, K.D. Infection control by antibody disruption of bacterial quorum sensing signaling. Chem. Biol. 14:1119, 2007.

Park, J., Kaufmann, G.F., Bowen, J.P., Arbiser, J.L., Janda K.D. Solenopsin A, a venom alkaloid from the fire ant Solenopsis invicta, inhibits quorum sensing signaling in Pseudomonas aeruginosa. J. Infect. Dis. 198:198, 2008.

Richardson, H.N., Zhao, Y., Fekete, E.M., Funk, C.K., Wirsching, P., Janda, K.D., Zorrilla, E.P., Koob, G.F. MPZP: a novel small molecule corticotropin-releasing factor type 1 receptor (CRF₁) antagonist. Pharmacol. Biochem. Behav. 88:497, 2008.

Willis, B., Eubanks, L.M., Dickerson, T.J., Janda, K.D. The strange case of the botulinum neurotoxin: using chemistry and biology to modulate the most deadly poison. Angew. Chem. Int. Ed.47:8360, 2008.

Willis, B., Eubanks, L.M., Wood, M.R., Janda, K.D., Dickerson, T.J., Lerner, R.A. Biologically templated organic polymers with nanoscale order. Proc. Natl. Acad. Sci. U. S. A. 105:1416, 2008.

Xu, Y., Hixon, M.S., Dawson, P.E., Janda, K.D. Development of a FRET assay for monitoring of HIV gp41 core disruption. J. Org. Chem. 72:6700, 2007.

Yoneda, Y., Steiniger, S.C., Capková, K., Mee, J.M., Liu, Y., Kaufmann, G.F., Janda, K.D. A cell-penetrating peptidic GRP78 ligand for tumor cell-specific prodrug therapy. Bioorg. Med. Chem. Lett. 18:1632, 2008.

Zarebski, L.M., Vaughan, K., Sidney, J., Peters, B., Grey, H., Janda, K.D., Casadevall, A., Sette, A. Analysis of epitope information related to Bacillus anthracis and Clostridium botulinum. Expert Rev. Vaccines 7:55, 2008.

Zhou, B., Carney, C., Janda, K.D. Selection and characterization of human antibodies neutralizing Bacillus anthracis toxin. Bioorg. Med. Chem. 16:1903, 2008.

Zhou, B., Pellett, S., Tepp, W.H., Zhou, H., Johnson, E.A., Janda, K.D. Delineating the susceptibility of botulinum neurotoxins to denaturation through thermal effects. FEBS Lett. 582:1526, 2008.

Zhou, H., Zhou, B., Ma, H., Carney, C., Janda, K.D. Selection and characterization of human monoclonal antibodies against Abrin by phage display. Bioorg. Med. Chem. Lett. 17:5690, 2007.