Probing Interkingdom Signaling Molecules via Liquid Extraction Surface Analysis–Mass Spectrometry

Previously, metabolites diffused or secreted from microbial samples have been analyzed via liquid chromatography–mass spectrometry (LC–MS) approaches following lengthy extraction protocols. Here, we present a model system for growing biofilms on discs before utilizing rapid and direct surface sampling MS, namely, liquid extraction surface analysis, to study the microbial exometabolome. One of the benefits of this approach is its surface-specific nature, enabling mimicking biofilm formation in a way that the study of planktonic liquid cultures cannot imitate. Even though Pseudomonas aeruginosa (P. aeruginosa), Staphylococcus aureus (S. aureus), and Candida albicans (C. albicans) have been studied previously in isolation, very few studies consider the complexity of the interplay between these pathogens, which are commonly combined causative agents of infection. Our model system provides a route to investigate changes in the exometabolome, such as metabolites that become circulatory in the presence of multiple pathogens. Our results agree with previous reports showing that 2-alkyl-4(1H)-quinolone signal molecules produced by P. aeruginosa are important markers of infection and suggest that methods for monitoring levels of 2-heptyl-4-hydroxyquinoline and 2,4-dihydroxyquinoline, as well as pyocyanin, could be beneficial in the determination of causative agents in interkingdom infection including P. aeruginosa. Furthermore, studying changes in exometabolome metabolites between pqs quorum sensing antagonists in treated and nontreated samples suggests suppression of phenazine production by P. aeruginosa. Hence, our model provides a rapid analytical approach to gaining a mechanistic understanding of bacterial signaling.


Strains and colony biofilms culture conditions
Inocula for each microbial species were prepared independently for single-species and polymicrobial biofilm growth. SH1000 and PAO1-L strains were propagated in LB broth for 16 h at 37°C with 200 rpm shaking. C. albicans SC5314 was grown in yeast peptone dextrose (YPD [Oxiod, Cambridge, UK) for 16 h at 30°C in a shaking incubator at 200 rpm. Cells were washed (x3) in phosphate-buffered saline (PBS), and inocula for each specific microbe were prepared at 1x104, 1x106 and 1x105 CFU/mL for PAO1-L, SH1000 and SC5314, respectively. UVC-sterilised polycarbonate (PC) discs (0.2 µm pore size and 13 mm diameter, Sigma) deposited on wells of 6-well plates filled with 5 mL of Roswell Park Memorial Institute (RPMI) -1640 medium (without sodium bicarbonate and phenol red, Sigma-Aldrich) supplemented with 1.5% agar (w/v) and 165 mM MOPs (Sigma-Aldrich).
For treatment with the QS inhibitors, SEN19 and SEN89 compounds were supplemented at 10 µM in the final stock inoculum of PAO1-L. Treatment of 18 h PAO1-L colony biofilms with ciprofloxacin was performed by adding 20 µL of 64 µg/mL in H2O pipetted gently on top of the preformed colony biofilm. At endpoint, PC discs with attached colony biofilms were aseptically removed and agar plugs were used for QS signal detection. In parallel RPMI-1640 agar samples without microbial inoculation were used as background control for LESA mass spectrometry analysis. Triplicate biological and technical repeats were conducted for all experiments presented.

Colony-forming unit (CFU) counting 1
PC discs were removed from agar and transferred to separate 1 mL microcentrifuge tubes containing 1 mL of 1x PBS and 5x 2.6 mm zirconium ceramic oxide beads (Fisherbrand, Loughborough, UK). The samples were then vortexed to remove the biofilm from the PC discs. The PC discs were removed and the biofilm disaggregated by bead beating for 30 sec using a FastPrep-24™ 5G Homogenizer (MP Biomedicals, Loughborough, UK). The 1 mL volume was then transferred to a 5 mL Bijou containing 4 mL 1x PBS. Bijous were then sonicated, in a sonicating water bath for 15 min at 37 kHz. Ten-fold serial dilutions were then performed in 1x PBS from the sonicated samples and 20 µL of each dilution were plated in triplicate on the appropriate selection agar.

Pyocyanin assay
Pyocyanin colorimetric quantification assay was adapted from Essar and colleagues 2 . Briefly, PC discs with biofilms were removed and the agar was excised using a sterile scalpel and placed in a 15 mL falcon tube. To each falcon tube 7.5 mL of dH2O and 4.5 mL of Chloroform was added under fume cabinet and shaken vigorously, then centrifuged at 10,000 x g at 4°C for 10 min. The yellow top layer was then removed by pipette and 3 mL of the chloroform layer was transferred to a new falcon containing 1.5 mL 0.2M HCL and vortexed for 10 s. The falcons were then centrifuged at 10,000 x g at 4°C for 2 min. Avoiding transfer of residual chloroform, 1 mL of the pink top layer was transferred by pipette to cuvettes and the OD520 nm obtained. The pyocyanin concentration can then be determined by multiplying the OD520 nm value by 17.072 and then 1.5.

Tandem (MSMS) experiments were conducted via high-energy collision-induced dissociation (HCD).
For MSMS experiments, AGC was used with a target of 1 × 10 6 charges and a maximum injection time of 500 ms. HCD was performed in the ion trap at a resolution of 70000 and a normalised collision energy between 20 and 25%, and fragments were detected in the Orbitrap. The isolation width was 1.0 Th.

MSMS
High-energy collision-induced dissociation (HCD) of m/z 244.17 confirmed that this is HHQ; the MSMS spectrum in Supplemental Figure 1A confirms fragments at m/z 159.07 and 172.08 that typify HHQ derived compounds 10,11 . HCD fragmentation of m/z 260.16 shows characteristic fragments of HQNO at m/z 159.07, 172.08, and 186.09, alongside characteristic fragments of PQS at m/z 188.10, 175.06 and 162.06 in lower abundance confirming that both C7-PQS and C7-HQNO isomers are present in the sample, see Supplemental Figure 1B. Numerous C7 AQs have been shown to be elevated in virulent strains of P. aeruginosa previously 12 . Upon dissociation of m/z 272.20 the following product ions were detected; m/z 159.08, 172.07 however there was no fragment at m/z 188.10 or 175.06 suggesting that this species is NHQ rather than C9-PQS.

Supplemental Figures
Supplemental Figure 1