Antibody Interference with N-Acyl Homoserine Lactone-Mediated Bacterial Quorum Sensing

Abstract

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 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 aeruginosa. 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 the role of P. aeruginosa in patients suffering from cystic fibrosis (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) homoserine lactone (3-oxo-C 12 -AHL) and N-butyryl homoserine lactone (C 4 -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. 2Interference 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 analogs 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 antibodies 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 analogs and their corresponding ring opened hydrolysis products, both of which contained a 4-methoxyphenyl amide group that allowed for a detailed investigation 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 hours. Interestingly, the oxidation state and chain length did not influence...

The Multiple Signaling Systems Regulating Virulence in Pseudomonas aeruginosa

“…In order to overcome the small size of AHLs and to potentiate the maximal response for antibody generation, Janda and coworkers (104) pioneered a study where AHLs and AHL analogues were chemically conjugated to keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA), leading to efficient production of AHL-sequestering antibodies. These antibodies have shown very promising efficacy in protecting mice against P. aeruginosa infections (103,104,135).…”

Exploiting Quorum Sensing To Confuse Bacterial Pathogens

“…The development of monoclonal antibodies (MAbs) against AHLs was first achieved using lactam-containing haptens whose linkers mimicked naturally occurring AHL acyl chains of various lengths and oxidation states (109). The highest-affinity MAbs were specific for 3OC12HSL, and investigators were able to demonstrate that one of these MAbs, RS2-1G9, inhibited QS signaling and QS-regulated pyocyanin production in P. aeruginosa in vitro, suggesting therapeutic potential of antibody-mediated QSI (109).…”

“…The development of monoclonal antibodies (MAbs) against AHLs was first achieved using lactam-containing haptens whose linkers mimicked naturally occurring AHL acyl chains of various lengths and oxidation states (109). The highest-affinity MAbs were specific for 3OC12HSL, and investigators were able to demonstrate that one of these MAbs, RS2-1G9, inhibited QS signaling and QS-regulated pyocyanin production in P. aeruginosa in vitro, suggesting therapeutic potential of antibody-mediated QSI (109). Shortly thereafter, Miyairi and colleagues demonstrated that active immunization with bovine serum albumin (BSA)-conjugated 3OC12HSL increased survival of mice following intranasal challenge with P. aeruginosa, despite having no effect on bacterial burdens in the lung (110).…”

High-Sensitivity Monoclonal Antibodies Specific for Homoserine Lactones Protect Mice from Lethal Pseudomonas aeruginosa Infections

“…Furthermore, existing monoclonal antibodies show little or no cross-reactivity with related HSL compounds (13). Antibodies that cross-react with C 4 -HSL would provide an added therapeutic advantage as they may also prevent the establishment of rhl QS system effects in the absence of the las system.…”