Characterizing PqsE’s enzymatic activity in Pseudomonas aeruginosa by Volatile Organic Compound analysis with GC×GC-TOFMS

Significance of the Topic

Pseudomonas aeruginosa is a major opportunistic pathogen in immunocompromised patients, where its ability to form biofilms and produce virulence factors is regulated by quorum sensing (QS). The hydrolase PqsE interacts with the QS receptor RhlR to control production of key virulence factors. Characterizing PqsE’s activity in vivo can guide antibiotic development by identifying inhibitors that disrupt this interaction.

Study Objectives and Overview

This study aimed to profile volatile organic compounds (VOCs) produced by wild-type and PqsE-deficient P. aeruginosa strains, then apply the small molecule inhibitor Vorinostat to measure dose-dependent suppression of PqsE activity. Comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry (GC×GC-TOFMS) and headspace solid-phase microextraction (HS-SPME) were leveraged to achieve high sensitivity and peak capacity in VOC detection.

Methodology

• Sampling Protocol:

• Overnight cultures diluted 1:100, grown 8 h at 37 °C, centrifuged, filtered supernatants transferred to headspace vials.

• VOC Extraction and Analysis:

• HS-SPME using a DVB/CWR/PDMS arrow fiber for 5 min at 50 °C, agitation at 1 000 rpm.
• GC×GC-TOFMS on Pegasus BTX system with Rxi-5MS (1D) and Rxi-17Sil MS (2D) columns; modulator period 4 s.
• Oven program: 40 °C (2 min), ramp 5 °C/min to 230 °C (2 min); He carrier gas at 0.5 mL/min (1D) and 30 mL/min (2D); MS detector operating in full-scan mode.

Used Instrumentation

• Pegasus BTX GC×GC-TOFMS system (LECO Corporation)
• LECO L-PAL3 autosampler equipped with SPME arrow fiber
• Rxi-5MS and Rxi-17Sil MS capillary columns (Restek Corporation)

Key Results and Discussion

• Eight VOCs showed significant concentration differences (F-ratio > 15) between wild-type and a catalytically inactive PqsE mutant (D73A):

• Acetophenone
• Benzaldehyde
• Methyl benzoate
• 2-Nonanone
• 2-Ethylbutanol
• 2,4-Dimethylfuran
• 2,4-Dimethylheptene
• 2-Methylbutanal

• Acetophenone emerged as the most responsive marker (F-ratio ~696) and was selected for inhibitor assays.
• In vivo HS-SPME GC×GC-TOFMS revealed a clear, dose-dependent decrease in acetophenone levels upon Vorinostat treatment, matching the inactive mutant baseline at 100 µM inhibitor.
• An in vitro esterase assay confirmed Vorinostat’s direct inhibition of PqsE.

Benefits and Practical Applications

• Provides a robust metabolomic assay to quantify PqsE activity in live bacterial cultures.
• Enables screening of potential quorum-sensing inhibitors for antibiotic development.
• Demonstrates the utility of GC×GC-TOFMS and HS-SPME for high-throughput VOC profiling.

Future Trends and Applications

• Shorten analysis time by optimizing GC×GC temperature programs and modulation periods.
• Enhance detection of low-abundance VOCs through improved sampling and data processing pipelines.
• Expand inhibitor screening to novel small molecules targeting the PqsE-RhlR interaction.
• Integrate machine learning‐based deconvolution of complex VOC data for rapid biomarker discovery.

Conclusion

This work establishes a sensitive, GC×GC-TOFMS‐based VOC profiling platform to characterize PqsE enzymatic activity and assess inhibitor efficacy in P. aeruginosa. Acetophenone serves as a reliable in vivo biomarker, and Vorinostat demonstrates dose‐dependent suppression of PqsE. The methodology lays a foundation for accelerated discovery of quorum‐sensing inhibitors and advances analytical approaches in microbial metabolomics.

Reference

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2. LECO Corporation. Pegasus BTX 4D System—GC×GC-TOFMS Configuration and Operation Manual. 2021.