Metabolomics responses and tolerance of Pseudomonas aeruginosa under acoustic vibration stress

Abstract

Sound has been shown to impact microbial behaviors. However, our understanding of the chemical and molecular mechanisms underlying these microbial responses to acoustic vibration is limited. In this study, we used untargeted metabolomics analysis to investigate the effects of 100-Hz acoustic vibration on the intra- and extracellular hydrophobic metabolites of P. aeruginosa PAO1. Our findings revealed increased levels of fatty acids and their derivatives, quinolones, and N-acylethanolamines upon sound exposure, while rhamnolipids (RLs) showed decreased levels. Further quantitative real-time polymerase chain reaction experiments showed slight downregulation of the rhlA gene (1.3-fold) and upregulation of fabY (1.5-fold), fadE (1.7-fold), and pqsA (1.4-fold) genes, which are associated with RL, fatty acid, and quinolone biosynthesis. However, no alterations in the genes related to the rpoS regulators or quorum-sensing networks were observed. Supplementing sodium oleate to P. aeruginosa cultures to simulate the effects of sound resulted in increased tolerance of P. aeruginosa in the presence of sound at 48 h, suggesting a potential novel response-tolerance correlation. In contrast, adding RL, which went against the response direction, did not affect its growth. Overall, these findings provide potential implications for the control and manipulation of virulence and bacterial characteristics for medical and industrial applications.

Fig 1. Volcano plots of differential metabolites in Pseudomonas aeruginosa at 48 h after 100-Hz acoustic vibration exposure.

In the untargeted metabolomics analysis, the levels of each (a, b) extracellular and (c, d) intracellular metabolite ion in both the (a, c) positive (pos; blue triangles) and (b, d) negative (neg; green diamonds) ion modes were compared between sample groups with (100 Hz) or without (control) 100-Hz sound exposure. Each data point represents a metabolite ion with an average mass ion intensity (MSII) of at least 10,000 counts in the more abundant sample groups. Its p-value (p) from a Student’s t-test is plotted against the fold change of the 100-Hz-treated group over the control. Ions above the horizontal dash line have a p-value of less than 0.05. Those located outside the right and left vertical dash lines exhibit relative levels at least 2-fold higher in the 100-Hz group compared to the control and vice versa, respectively. These ions are considered changed metabolite ions. The changed metabolite ions, pos(ID) and neg(ID), represent the structurally characterized positive-mode (pos) and negative-mode (neg) ions in this study, depicted with red open triangles and orange open diamonds, respectively. Characterization was achieved through a combination of accurate masses, previously reported retention times, and tandem mass spectra.

Fig 2

Chemical structures with proposed MS/MS fragments (top), extracted ion chromatograms (middle), and MS/MS spectra (bottom) of representative differential metabolites. The [M–H]⁻ions of the two most abundant detected rhamnolipids, (a) monorhamnolipid (10:0/10:0) and (b) dirhamnolipid (10:0/10:0), were decreased in their levels in the extracellular samples upon exposure to 100-Hz sound (middle; black solid lines), compared to the controls (middle; red dashed lines), whereas the [M + H]⁺ ion of (c) 2-alkyl-4-hydroxyquinoline (9:0) was elevated in its level in the intracellular samples. The structures of all representative metabolites were confirmed by comparing both the retention times and MS/MS spectra of samples (bottom; top spectra with black lines) with those of commercial standards (bottom; bottom spectra with blue lines). The proposed fragments observed in the MS/MS spectra are indicated with green dashed lines, along with the corresponding m/z values in the chemical structures.

Fig 3. Biosynthetic pathways of differential and related metabolites in Pseudomonas aeruginosa and their interconnection.

The key intermediates and enzyme-encoding genes are shown for the de novo fatty acid synthase (FAS II), β-oxidation, quinolones, N-acyl homoserine lactones, and rhamnolipids pathways. Metabolites or genes with elevated, decreased, or unchanged levels after 100-Hz sound exposure in the metabolomics analysis or quantitative real-time polymerase chain reaction are indicated by red, green, and blue letters, respectively. Abbreviations: 2-ABA, 2’-aminobenzoylacetate; 2-HABA, 2’-hydroxylaminobenzoylacetate; 3-oxo-C₁₂-HSL, N-(3-oxododecanoyl)-L-homoserine lactone; ACP, acyl carrier protein; AHQ, 2-alkyl-4-hydroxyquinoline; AQNO, 2-alkyl-4-hydroxyquinoline N-oxide; C₄-HSL, N-butyl-L-homoserine lactone; CoA, coenzyme A; C_n-PQS, 2-alkyl-3-hydroxy-4-quinolone; DRL, dirhamnolipid; HAA, 3-(3-hydroxyalkanoyloxy)alkanoic acid; MRL, monorhamnolipid.

Fig 4. Relative transcript expression levels of genes involved in the biosynthesis of differential metabolites and related pathways in Pseudomonas aeruginosa.

mRNA expression levels were quantitated in Pseudomonas aeruginosa at (a) 12 h and (b) 48 h after exposure to 100-Hz sound (100 Hz; black bars) and are indicated in the graph by normalizing the level of each gene with that of the control group without sound exposure (control; white bars). Quantitative real-time polymerase chain reaction was performed using the 2^–ΔΔCt method and standardized against the rpoD and proC transcript expression levels as internal controls. Data from three independent experiments are shown as the average ± standard errors of the mean. Student’s t-test: ^*, p < 0.05.

Fig 5. Acoustic vibration tolerance of Pseudomonas aeruginosa in the presence of response metabolites.

Growth curves of Pseudomonas aeruginosa in the absence (control; Con; blue tone circle) or presence (red tone triangle) of (a) 2 mM sodium oleate (NaOA; 2 mM NaOA), (b) 10 mM NaOA, (c) 1.2 g L^–1 rhamnolipids (RLs; 1.2 g L^–1 RLs), or (d) 6.0 g L^–1 RLs with (-100Hz; open marker with dashed line) or without (closed marker with solid line) stimulation by 100 Hz sound. One mL of cultures was collected at 6, 12, 24, or 48 h of incubation, mixed vigorously to obtain homogeneous culture, and subjected to optical density measurement at 600 nm (OD₆₀₀). Data from three or four independent cultures are shown as the average ± standard errors of the mean. Student’s t-test for compound-treated samples with vs. without sound: ^*, p < 0.05. Student’s t-test for compound-treated vs. Con samples without sound: ^†, p < 0.05; ^††, p < 0.01; ^†††, p < 0.005; ^††††, p < 0.001. Student’s t-test for compound-treated vs. Con samples with sound: ^‡, p < 0.05; ^‡‡, p < 0.01; ^‡‡‡, p < 0.005; ^‡‡‡‡, p < 0.001.