Chemical methods to interrogate bacterial quorum sensing pathways

Abstract

Bacteria frequently manifest distinct phenotypes as a function of cell density in a phenomenon known as quorum sensing (QS). This intercellular signalling process is mediated by "chemical languages" comprised of low-molecular weight signals, known as autoinducers, and their cognate receptor proteins. As many of the phenotypes regulated by QS can have a significant impact on the success of pathogenic or mutualistic prokaryotic-eukaryotic interactions, there is considerable interest in methods to probe and modulate QS pathways with temporal and spatial control. Such methods would be valuable for both basic research in bacterial ecology and in practical medicinal, agricultural, and industrial applications. Toward this goal, considerable recent research has been focused on the development of chemical approaches to study bacterial QS pathways. In this Perspective, we provide an overview of the use of chemical probes and techniques in QS research. Specifically, we focus on: (1) combinatorial approaches for the discovery of small molecule QS modulators, (2) affinity chromatography for the isolation of QS receptors, (3) reactive and fluorescent probes for QS receptors, (4) antibodies as quorum "quenchers," (5) abiotic polymeric "sinks" and "pools" for QS signals, and (6) the electrochemical sensing of QS signals. The application of such chemical methods can offer unique advantages for both elucidating and manipulating QS pathways in culture and under native conditions.

Fig. 1

General schematic of the QS process in bacteria. Pentagons represent autoinducer (AI) signals. A. LuxR/LuxI-type QS in Gram-negative bacteria. B. QS system in certain Gram-positive bacteria.

Fig. 2

Examples of AIs used by bacteria for QS. A. N-acylated L-homoserine lactones (AHLs) found in Gram-negative bacteria. B. Autoinducer-2 (AI-2) found in both Gram-positive and Gram-negative bacteria. C. Group I autoinducing peptide (AIP-I) used by Staphylococcus aureus. D. The Pseudomonas Quinolone Signal (PQS) used by Pseudomonas aeruginosa.

Fig. 3

Top: Polystyrene-based solid-phase synthesis of AHLs, as described by Geske et al.^, DIC = N,N′-diisopropylcarbodiimide; HOBt = 1-hydroxybenzotriazole; Fmoc = 9-fluorenylmethoxycarbonyl group; Met = methionine; CNBr = cyanogen bromide; TFA = trifluoroacetic acid; μW = microwave irradiation; rt = room temperature. Bottom: Generic structures of QS modulators generated utilizing this synthetic methodology. From left to right: phenylacetyl homoserine lactone, phenoxyacetyl homoserine lactone, and phenylpropionyl homoserine lactone.

Fig. 4

Combinatorial approaches to the synthesis QS modulators. A. Macroarray synthesis of AHLs by Praneenararat et al. B. Macroarray synthesis of diketopiperazines (DKPs) by Campbell et al. C. 3D microarrays devised by Spring and co-workers.^– Top: individual amino-containing compounds can be printed on an N-hydroxysuccinimide activated slide. This array could be probed with fluorescently-labelled CarR (a LuxR-type protein in P. carotovora) for binding assays. Bottom: catch-and-release 3D microarrays allow the synthesis of amides that were non-covalently deposited onto the support. Reagents and abbreviations: CDI = 1,1′-carbonyldiimidazole; NMI = N-methylimidazole; Trt = trityl group; DBU = 1,8-diazabicyclo-[5.4.0]undec-7-ene; Boc = tert-butyloxycarbonyl group; Cy3 = a cyanine fluorescent dye.

Fig. 5

Various AHL-based chemical tools. A. Biotin-tagged AHL described by Spandl et al. B. Piperazine-modified AHLs synthesized by Seabra et al. C. Alkynyl and azido AHLs synthesized by Garner et al. D. Amino AHL for affinity pull-down assays by Praneenararat et al. E. Diazirine and alkynyl-tagged AHL reported Dubinsky et al. F. Isothiocyanate AHL reported by Amara et al. G. BODIPY-tagged AHL reported by Rayo et al.^.

Fig. 6

Haptens used to prepare quorum quenching antibodies. A. RS2 hapten reported by Kaufmann et al. with a modified atom (in red) from the original lactone ring. B. AIP-IV analog with an ester group (in red) instead of the natural thioester group reported by Park et al. C. Squaric monoester monoamide hapten reported by De Lamo Marin et al. originally utilized to generate catalytic antibodies that hydrolyze paraoxon. D. A sulfone transition state mimic for AHL hydrolysis as reported by Kapadnis et al.

Fig. 7

Abiotic polymers used to study QS. A. Poly-itaconic acid used to sequester OHHL or OdDHL. B. 2-Hydroxypropyl β-cyclodextrin used to trap AHLs by interacting with the acyl tail.^, C. Poly(N-dopamine methacrylamide-co-N-[3-(dimethylamino)propyl]methacrylamide) designed to capture the boron-containing AI-2 signal and promote cell adhesion. D. PLG used for the controlled release of AHLs.

Fig. 8

Redox-active molecules used for the electrochemical analysis of QS. A. Pyocyanin used by Bukelman et al. and Sharp et al. B. PQS used by Zhou et al. C. p-Aminophenol (PAP) used by Baldrich et al.