Simultaneous quantitative profiling of N-acyl-l-homoserine lactone and 2-alkyl-4(1H)-quinolone families of quorum-sensing signaling molecules using LC-MS/MS

Abstract

An LC-MS/MS method, using positive mode electrospray ionization, for the simultaneous, quantitative and targeted profiling of the N-acyl-L-homoserine lactone (AHL) and 2-alkyl 4-(1H)-quinolone (AQ) families of bacterial quorum-sensing signaling molecules (QSSMs) is presented. This LC-MS/MS technique was applied to determine the relative molar ratios of AHLs and AQs produced by Pseudomonas aeruginosa and the consequences of mutating individual or multiple QSSM synthase genes (lasI, rhlI, pqsA) on AHL and AQ profiles and concentrations. The AHL profile of P. aeruginosa was dominated by N-butanoyl-L-homoserine lactone (C4-HSL) with lesser concentrations of N-hexanoyl-L-homoserine lactone (C6-HSL) and 3-oxo-substituted longer chain AHLs including N-(3-oxodecanoyl)-L-homoserine lactone (3-oxo-C10-HSL) and N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL). The AQ profile of P. aeruginosa comprised the C7 and C9 long alkyl chain AQs including 2-heptyl-4-hydroxyquinoline (HHQ), 2-nonyl-4-hydroxyquinoline, the "pseudomonas quinolone signal" (2-heptyl-3-hydroxy-4-quinolone) and the N-oxides, 2-heptyl-4-hydroxyquinoline N-oxide and 2-nonyl-4-hydroxyquinoline N-oxide. Application of the method showed significant effects of growth medium type on the ratio and the nature of the QSSMs synthesized and the dramatic effect of single, double and triple mutations in the P. aeruginosa QS synthase genes. The LC-MS/MS methodology is applicable in organisms where either or both AHL and AQ QSSMs are produced and can provide comprehensive profiles and concentrations from a single sample.

Regulon Studies and In Planta Role of the BraI/R Quorum-Sensing System in the Plant-Beneficial Burkholderia Cluster

“…The total run time was 20 min for each sample. MS detection was operated in a multiple reaction monitoring (MRM) mode, screening for all unsubstituted AHLs, 3-oxo-AHLs, and 3-OH-AHLs with acyl chain lengths of 4,6,8,10,12, and 14 carbon atoms (18). All AHL standards used in this experiment were chemically synthesized, purified, and characterized in the School of Molecular Medical Science, Centre for Biomolecular Science, University of Nottingham.…”

Synthesis and analysis of stable isotope-labelled N-acyl homoserine lactones

“…24,25 Several groups have reported the synthesis of isotope-labelled AHLs 1-10 ( Figure 1) which can be used as internal standards. 13,[26][27][28][29][30][31][32][33] In the case of isotope dilution mass spectrometry (IDMS), the spike compound should be chemically identical with the analyte. As the isotope-labelled compound acts as an internal standard, compensating for losses during sample isolation and analysis, the mass difference between the isotope and the analyte should not be too high, to avoid possible isotope effects.…”

An investigation into the effect of erythromycin on Pseudomonas aeruginosa quorum sensing in non-cystic fibrosis bronchiectasis

“…Newer biosensor techniques have been able to detect quorum sensing signal molecules directly from sputum [91], bronchial alveolar lavage fluid [96], tracheal aspirates [81] and tissue specimen culture medium [90], which is likely more representative of what is occurring at that point within the host. Other techniques include liquid chromatography [96], mass spectrometry [97,98] and radiometric analysis [99] of quorum sensing molecules, directly in bodily fluids and lung tissue. This has improved both sensitivity and specificity for the detection of these molecules.…”

“…Whilst biosensors are useful for the detection of quorum sensing molecules, mass spectrometry gives sensitive, specific, validated values for both the quantity and presence of specific quorum sensing molecules [97,98]. The detection range of quorum sensing molecules in clinical samples with biosensors is in the nanomolar range for C4-HSL (2500 -0.0025 nM) and in the picomolar range for 3-oxo-C12-HSL (20 nM-0.002 pM) [93].…”

“…The detection range of quorum sensing molecules in clinical samples with biosensors is in the nanomolar range for C4-HSL (2500 -0.0025 nM) and in the picomolar range for 3-oxo-C12-HSL (20 nM-0.002 pM) [93]. In mass spectrometry, sensitivity (to date) is not as good, with clinical detection in the micromolar range (0.25 -1.4 µM) dependent upon the culture conditions [98]. However, specificity is much better allowing simultaneous, quantitative profiling of all quorum sensing molecules in a sample.…”