Identification of Unanticipated and Novel N-Acyl L-Homoserine Lactones (AHLs) Using a Sensitive Non-Targeted LC-MS/MS Method

Abstract

N-acyl L-homoserine lactones (AHLs) constitute a predominant class of quorum-sensing signaling molecules used by Gram-negative bacteria. Here, we report a sensitive and non-targeted HPLC-MS/MS method based on parallel reaction monitoring (PRM) to identify and quantitate known, unanticipated, and novel AHLs in microbial samples. Using a hybrid quadrupole-high resolution mass analyzer, this method integrates MS scans and all-ion fragmentation MS/MS scans to allow simultaneous detection of AHL parent-ion masses and generation of full mass spectra at high resolution and high mass accuracy in a single chromatographic run. We applied this method to screen for AHL production in a variety of Gram-negative bacteria (i.e. B. cepacia, E. tarda, E. carotovora, E. herbicola, P. stewartii, P. aeruginosa, P. aureofaciens, and R. sphaeroides) and discovered that nearly all of them produce a larger set of AHLs than previously reported. Furthermore, we identified production of an uncommon AHL (i.e. 3-oxo-C7-HL) in E. carotovora and P. stewartii, whose production has only been previously observed within the genera Serratia and Yersinia. Finally, we used our method to quantitate AHL degradation in B. cepacia, E. carotovora, E. herbicola, P. stewartii, P. aeruginosa, P. aureofaciens, the non-AHL producer E. coli, and the Gram-positive bacterium B. subtilis. We found that AHL degradation ability varies widely across these microbes, of which B. subtilis and E. carotovora are the best degraders, and observed that there is a general trend for AHLs containing long acyl chains (≥10 carbons) to be degraded at faster rates than AHLs with short acyl chains (≤6 carbons).

Quorum Sensing and Quorum Quenching in the Mediterranean Seagrass Posidonia oceanica Microbiota

“…As previous studies reported (Morin et al, 2003;Patel et al, 2016), we observed some characteristic fragments arising from fragmentation of the lactone ring. A daughter ion with m/z 102.055 is always observed, and three others ions with m/z 56.050, 74.061, and 84.045 are usually detected.…”

“…The analytical method includes full-scan MS with successive all-ion fragmentation (AIF) MS/MS scan allowing the acquisition high resolution data of the parent ion and daughter ion masses in a single chromatographic run. As described by Patel et al (2016), four characteristic fragments originating from the lactone ring in AHLs were detected in almost all of the MS/MS spectra of the AHL standards (Girard et al, 2017). One daughter ion was abundant and always present in fragmentation patterns: the 102.055 m/z ion, which corresponds to the lactone ring itself; three other ions that arose from the fragmentation of the lactone ring (the 56.050, 74.061, and 84.045 m/z fragments) were usually observed.…”

Large Diversity and Original Structures of Acyl-Homoserine Lactones in Strain MOLA 401, a Marine Rhodobacteraceae Bacterium

“…The confirmation of the presence of the HSL subunit was obtained by the method described by Patel et al (Patel et al, 2016). In our case, all the AHLs had all 4 diagnostic fragments at m/z 102.055, 84.045, 74.061, and 56.050 in MS/MS.…”

Peptides as Quorum Sensing Molecules: Measurement Techniques and Obtained Levels In vitro and In vivo

“…Generally, these methods are capable of generating limits of detection (LOD) in the broad nM-range (median LOD of 2.3 nM for 19 N -acyl homoserine lactone standards; range: 0.6 nM–2838.9 nM), with some compounds owing a limit of detection around 0.6 nM (for C10-HSL) (Patel et al, 2016). …”