Development and characterisation of an immunochemical test system for the determination of bacterial signal molecules (N-acylated homoserine lactones)

Quorum Sensing (QS) is a process that enables bacteria to communicate via chemical signalling molecules, which are called autoinducers (AI). When a threshold concentration of QS molecules is reached, the bacteria start their QS regulated gene expression e.g. bioluminescence (Vibrio fischeri), virulence factor secretion (Pseudomonas aeruginosa), biofilm formation (Burkholderia cepacia), sporulation, and mating. It was found that many Gram-negative bacteria use acylated homoserine lactones (AHLs or HSLs) as autoinducers. Due to the broad biological functions of HSLs, the interest in detection and analysis of HSLs is increasing for medical, biotechnological and agricultural applications. In the past years, numerous analytical methods have been developed for HSLs. Conventional analysis, which usually combines chromatography, mass spectrometry (MS), and nuclear magnetic resonance (NMR) has been very successfully applied for identification and quantification of HSLs. But normally, conventional analysis requires many steps of sample preparation, e.g. extraction, pre-concentration and optimisation of conditions to separate individual HSL molecules. In addition, many sensitive bioreporter assays have been developed using different LuxR responsive promoters, which contain LuxR family functional proteins but lack the HSL synthase. A combination of different bioassays is strongly recommended, since no bioreporter is sensitive to all HSLs. Alternatively, in this study, an anti-HSL antibody based immunochemical detection method has been successfully developed and established. HSL molecules consist of a homoserine lactone ring and an acyl side chain (4-18 carbon atoms), and they differ only from side chain length and substitution at C3 atom. Regarding the variation of the molecule structures, four HSL haptens, named HSL1, HSL2, HSL3 and HSL4, were designed for antibody and assay development. HSL1 and HSL3 have a long chain (C11-COOH), but HSL1 has an -oxo and HSL3 has an –OH functional group at the C3 position. In comparison, HSL2 (C5-COOH) and HSL4 (C9-COOH) have shorter side chains and no substitution on the C3 atom. The haptens were synthesised and were covalently coupled to the C-terminal COOH-group of the NH2-residues (lysines) of the carrier proteins (BSA/OVA). Using these HSL hapten-conjugates, rat and mouse anti-HSL monoclonal antibodies (mAbs) were produced, screened and further characterised with enzyme-linked immunosorbent assays (ELISAs). Corresponding to hapten structures, the antibodies showed different selectivities to HSLs with different substitution on C3 position and chain length. Eight mAbs (HSL1-1A5, HSL1-8E1, HSL1/2-2C10, HSL1/2-4H5, HSL4-4C9, HSL4-5H3, HSL4-5E12 and HSL4-6D3) were selected from about 200 mAbs and characterised in detail using coating antigen and enzyme tracer formats. It was demonstrated that the new assays have HSL detection ranges from nM to low µM, which is sensitive enough for detection of HSLs in natural samples according to literature. Interestingly but not surprisingly, AHLs mAbs have at least 20 times higher sensitivity against hydrolysed HSLs (named HSs) than original HSLs, because the conjugation and immunisation conditions, e.g. pH and temperature, for mAb development resulted in HSL hydrolysis. This property of antibodies additionally offers a new sensitive method to detect quorum quenching (QQ) relevant homoserines (HSs), which are important degradation products of HSLs. Comparable results have been obtained by Biacore and Aqua-Optosensor biosensors for HS (L) characterisation. Based on these properties of the mAbs, a detection method of HSLs and HSs in biological samples has been developed and optimised. With the comparison of the real samples before and after hydrolysis treatment, the assays could simply present the relative HSL- and HS- contents in the samples. Similar to bio-reporters, the identification or quantitation of single HSL molecules is not possible only using immunoassay due to the broad recognition of HSLs and HSs. For this purpose, a combination with conventional chemical analysis is a must. However, as a novel sensitive HSL and HS detection method, the developed immunoassays have the advantages of being fast, cost effective and having low sample volume requirement. Using the direct or indirect fluorescence signals of fluorophore labelled anti-HSL mAbs, the in situ experiments with modified Burkholderia cepacia biofilm on ibidi slide (plastic flow chamber from ibidi GmbH) and Pseudomonas putida inoculated barley root have been carried out. Unfortunately, the in situ tests were not successful, mainly due to remaining unspecific background signals. Nevertheless, a few steps, e.g. fluorophore labelling, biofilm formation, and surface blocking have been optimised. The door of HSL in situ tests with the antibodies is still open, if a suitable specific visualising detection method could be found in the future. Certainly, the antibodies can also be broadly applied for many other immunochemical techniques, such as immunosensors or immunoaffinity columns for characterisation or pre-screening of HSLs/HSs, as have been demonstrated successfully.