Targeted and untargeted quantification of quorum sensing signalling molecules in bacterial cultures and biological samples via HPLC-TQ MS techniques

Abstract

Quorum sensing (QS) is the ability of some bacteria to detect and to respond to population density through signalling molecules. QS molecules are involved in motility and cell aggregation mechanisms in diseases such as sepsis. Few biomarkers are currently available to diagnose sepsis, especially in high-risk conditions. The aim of this study was the development of new analytical methods based on liquid chromatography-mass spectrometry for the detection and quantification of QS signalling molecules, including N-acyl homoserine lactones (AHL) and hydroxyquinolones (HQ), in biofluids. Biological samples used in the study were Pseudomonas aeruginosa bacterial cultures and plasma from patients with sepsis. We developed two MS analytical methods, based on neutral loss (NL) and product ion (PI) experiments, to identify and characterize unknown AHL and HQ molecules. We then established a multiple-reaction-monitoring (MRM) method to quantify specific QS compounds. We validated the HPLC-MS-based approaches (MRM-NL-PI), and data were in accord with the validation guidelines. With the NL and PI MS-based methods, we identified and characterized 3 and 13 unknown AHL and HQ compounds, respectively, in biological samples. One of the newly found AHL molecules was C12-AHL, first quantified in Pseudomonas aeruginosa bacterial cultures. The MRM quantitation of analytes in plasma from patients with sepsis confirmed the analytical ability of MRM for the quantification of virulence factors during sepsis. Graphical abstract.

Keywords: Homoserine lactones; Hydroxyquinolones; Mass spectrometry; Pseudomonas aeruginosa; Quorum sensing molecules; Triple quadrupole.

Graphical abstract

Fig. 1

N-Acyl homoserine lactone generic structure. a Non-substituted N-acyl homoserine-L-lactone (Cn-HSL) acyl chain; b N-(3-hydroxyacylhomoserine)-L-lactone (3-OH-Cn-HSL) acyl chain; c N-(3-oxoacylhomoserine)-L-lactone (3-oxo-Cn-HSL) acyl chain. The length is variable, generally n = 4–14

Fig. 2

Generic structure of quinolone signalling molecules. a 2-Alkyl-4-hydroxyquinolone N-oxide with a variable chain length (C7–C9); b 2-alkyl-4(1H)-quinolone alkyl chain (the chain length is variable, C7–C11); c 2-alkyl-3-hydroxy-4(1H)-quinolone

Fig. 3

TQ MS/MS selective acquisition modes. M1 is a generic structural formula of a protonated molecule belonging to the N-acyl homoserine lactones (AHLs) family. R varies between 4 and 16 carbon atoms. M2 is the product ion formed after the neutral loss of a 2-amino-gamma butyrolactone molecule (MW 101 Da). M2′ is the product ion with m/z 102. Analogously, N1 is a general structural formula of compounds belonging to the hydroxyquinolone signalling molecule (HQ) family. R varies between 7 and 11 carbon atoms. N2′ is the product ion with m/z 175

Fig. 4

MS² fragmentation pathways of N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-AHL), N-butanoyl-L-homoserine lactone (C4-AHL), 2-heptyl-3-hydroxy-4(1H)-quinolone (C7 HQ) and N-hexanoyl-L-homoserine lactone-D3 (ND3). Product ion m/z 102 and product radical ion m/z 175 are the characteristic fragmentation products of molecules belonging to AHL and HQ families, respectively