Quorum Sensing Inhibition and Metabolic Intervention of 4-Hydroxycinnamic Acid Against Agrobacterium tumefaciens

Abstract

The natural product 4-hydroxycinnamic acid (HA) was firstly isolated from the metabolites of Phomopsis liquidambari, one endophytic fungus from Punica granatum leaves. The anti-QS potential of HA was evaluated by β-galactosidase assay and acylated homoserine lactones (AHL) analysis. The MIC of HA was > 1.20 mM. Exposure to HA at sub-MIC concentrations (0.30-0.60 mM) remarkably reduced the β-galactosidase activity and AHL secretion. Transcriptional analysis by qRT-PCR and docking simulation indicated that HA functions as an anti-QS agent by inhibiting the transcriptional levels of traI and traR rather than signal mimicry. The blocked QS lead to suppressed biofilm formation, motilities, and flagella formation after exposure to HA at concentrations ranging from 0.30 to 0.80 mM. The dysfunctional QS also resulted in repressed antioxidant enzymes and intensified oxidative stress. The intensified oxidative stress destroyed membrane integrity, induced energy supply deficiency, resulted in disorder of protein and nuclear acid metabolism, and ultimately weakened pathogenicity of A. tumefaciens. HA may have promising potential for controlling A. tumefaciens.

Keywords: 4-hydroxycinnamic acid; Agrobacterium tumefaciens; metabolism; pathogenicity; quorum sensing.

FIGURE 1

Growth profile of A. tumefaciens treated with HA. Growth was evaluated at various concentrations of HA (0.30, 0.45, 0.60, and 0.80 mM) for 24 h. DMSO was used as the negative control. Error bars represent standard deviations of three measurements.

FIGURE 2

Effect of HA on β-galactosidase activity and biofilm formation of A. tumefaciens. (A) Effect of HA (0.30, 0.45, 0.60, and 0.80 mM) on β-galactosidase activity using A. tumefaciens A136 as the AHL-responsive reporter strain. Salicylic acid (SA, 0.10 mM) and DMSO were used as positive and negative control, respectively. (B) Effect of HA (0.30, 0.60, and 0.80 mM) on biofilm formation. (C) SEM images of A. tumefaciens treated with (a) DMSO, (b) 0.30 mM, (c) 0.60 mM, and (d) 0.80 mM of HA. Statistical differences were determined by Student’s t-test. ***p < 0.001 vs. the DMSO control.

FIGURE 3

Relative quantification of 3-oxo-C8-HSL by LC-MS/MS chromatograms. (A) HPLC chromatograms of 3-oxo-C8-HSL produced by A. tumefaciens C58 treated with (c) DMSO and (d–f) HA (0.30, 0.45, and 0.60 mM, respectively). (a) and (b) represented the standard chemicals of 3-oxo-C8-HSL. (B) MS/MS spectra of 3-oxo-C8-HSL. (C) Quantitative analysis of 3-oxo-C8-HSL treated with 0.30, 0.45, and 0.60 mM of HA, respectively. Statistical differences were determined by Student’s t-test. ***p < 0.001 vs. the DMSO control.

FIGURE 4

Effect of HA on swimming motility (A), chemotaxis (B), and flagella formation (C). Images of (a–f) represented DMSO, salicylic acid (SA, 0.10 mM), 0.30, 0.45, 0.60, and 0.80 mM of HA treatment, respectively. The images of swimming motility and chemotaxis were obtained by a common camera. The flagella formation was observed using a light microscope.

FIGURE 5

¹H NMR spectra of A. tumefaciens extracts from AHE-treated (red line) and control group (black line). Labeled metabolites: cholate (1), isoleucine (2), leucine (3), valine (4), ethanol (5), lactate (6), alanine (7), lysine (8), putrescine (9), acetate (10), glutamate (11), glutathione (12), succinate (13), β-alanine (14), dimethylamine (15), sarcosine (16), N, N- dimethylglycine (17), ethanolamine (18), choline (19), arginine (20), betaine (21), methanol (22), glycine (23), sucrose (24), uracil (25), UDP-galactose (26), NAD⁺ (27), NADP⁺ (28), adenosine (29), AMP (30), phenylalanine (31), tryptophan (32), xanthine (33), formate (34), and ATP (35).

FIGURE 6

OSC-PLS-DA of metabolomics profiles from HA-treated and control groups. (A) PCA score plot. (B) S-plot points represent different variables (metabolites). (C,D) Color-coded loading plot after removal of water signals and affected regions. The color bar was applied in which red and blue represented metabolites that statistically significantly or indistinctively contributed to the separation of groups, respectively. Peaks in positive and negative status revealed decreased and increased metabolites relative to score plot in the HA-treated group.

FIGURE 7

Effect of HA at 0.60 mM on oxidative stress (A) and expressions of genes involved in QS, virulence, antioxidant enzyme, and flagella formation (B). Student’s t-test was used to calculate p-values (two-tailed). *p < 0.05 vs. the DMSO control; **p < 0.01 vs. the DMSO control; ***p < 0.001 vs. the DMSO control.

FIGURE 8

2D (top) and 3D (bottom) schematics of receptor-ligand interactions of TraR with 3-oxo-C8-HSL (A) and HA (B), respectively, using the Discovery Studio 4.0 program.

FIGURE 9

Effect of HA on tobacco plant infections. (A) Inoculation with A. tumefaciens C58 treated with (a) DMSO, (b) salicylic acid (SA, 0.10 mM), (c) 0.30 mM of HA, and (d) 0.60 mM of HA, respectively. (B) Quantification of crown gall weight treated with or without HA. Statistical differences were determined by Student’s t-test. The F values of SA and HA (0.30 and 0.60 mM) analysis were 1.000, 1.318, and 0.028, respectively. The df values of all treatments were 4. ***p < 0.001 vs. DMSO-treated control.

FIGURE 10

Schematic diagram of the metabolic pathways disturbed by HA. The red font indicated promoted pathways while blue font indicated inhibited pathways.