Mining Public Mass Spectrometry Data to Characterize the Diversity and Ubiquity of P. aeruginosa Specialized Metabolites

Abstract

Pseudomonas aeruginosa is a ubiquitous environmental bacterium that causes chronic infections of burn wounds and in the lungs of cystic fibrosis (CF) patients. Vital to its infection is a myriad of specialized metabolites that serve a variety of biological roles including quorum sensing, metal chelation and inhibition of other competing bacteria. This study employed newly available algorithms for searching individual tandem mass (MS/MS) spectra against the publicly available Global Natural Product Social Molecular Networking (GNPS) database to identify the chemical diversity of these compounds and their presence in environmental, laboratory and clinical samples. For initial characterization, the metabolomes of eight clinical isolates of P. aeruginosa were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and uploaded to GNPS for spectral searching. Quinolones, rhamnolipids, phenazines and siderophores were identified and characterized; including the discovery of modified forms of the iron chelator pyochelin. Quinolones were highly diverse with the three base forms Pseudomonas quinolone signal 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS), 4-heptyl-4(1H)-quinolone (HHQ) and 2-heptyl-4-quinolone-N-oxide (HQNO) having extensive variation in the length of their acyl chain from as small as 3 carbons to as large as 17. Rhamnolipids were limited to either one or two sugars with a limited set of fatty acyl chains, but the base lipid form without the rhamnose was also detected. These specialized metabolites were identified from diverse sources including ant-fungal mutualist dens, soil, plants, human teeth, feces, various lung mucus samples and cultured laboratory isolates. Their prevalence in fecal samples was particularly notable as P. aeruginosa is not known as a common colonizer of the human gut. The chemical diversity of the compounds identified, particularly the quinolones, demonstrates a broad spectrum of chemical properties within these the metabolite groups with likely significant impacts on their biological functions. Mining public data with GNPS enables a new approach to characterize the chemical diversity of biological organisms, which includes enabling the discovery of new chemistry from pathogenic bacteria.

Metabolomic profiling of Burkholderia cenocepacia in synthetic cystic fibrosis sputum medium reveals nutrient environment-specific production of virulence factors

“…The feature with m/z 339.083 had a database annotation as a pyochelin methyl ester in GNPS (Supplementary Fig. S1 a) 60 . However, pyochelin methyl ester has only been reported as a synthetic product generated to facilitate NMR characterization, and there is no available biosynthetic evidence that would support methylation on the carboxylic acid to produce an ester 61 .…”

Characterization of Pseudomonas aeruginosa Quorum Sensing Inhibitors from the Endophyte Lasiodiplodia venezuelensis and Evaluation of Their Antivirulence Effects by Metabolomics

“…A large part of these differences was associated with known virulence-linked metabolites such as rhamnolipids, alkyl quinolones, siderophores (pyochelin) and phenazines (pyocyanin) [ 64 ]. Recent advances in the development of open access MS databases, such as GNPS [ 49 ], and available data on the specialized metabolome of P. aeruginosa , allow for confident identification of these key groups of metabolites directly in the P. aeruginosa PAO1 supernatant [ 65 ].…”

“…The rhamnolipid cluster consisted of the rhamnose-free precursors: 3-(3-hydroxyalkanoyloxy) alkanoic acids (HAAs), rhamnolipids containing one (Rha) or two (Rha-Rha) rhamnoses [ 65 ]. All these forms of rhamnolipids were impacted by the incubation of 2 or 4 ( Figure 7 C–E and Figure S55 ).…”

“…The pseudomonal iron siderophore pyochelin is known to exist in enantiomeric forms, cis and trans, and both could be annotated in the network ( Figure 7 C and Figure S56 ) [ 65 ]. Metabolomic data showed a 28-fold decrease in pyochelin relative intensity when incubated with 2 and 17-fold when incubated with 4.…”

Metabolic Footprints of Burkholderia Sensu Lato Rhizosphere Bacteria Active against Maize Fusarium Pathogens

“…Representative DIESI-MS spectra of the 200-800 m/z region showed the most intense ions corresponding to the exo-metabolites secreted to the culture medium by each strain (Figure S2). The identity of the most intense ions, as determined by the RF model (Figure 5), was obtained by their subsequent fragmentation (see Table 2 and Table S2 [54][55][56]). Remarkably, B. contaminans MSR2 generated ion signals with m/z values of 737.7, 759.7 and 781.6, whose maximal intensity was confirmed by an ANOVA test.…”