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Alginate Oligosaccharide-Induced Modification of the lasI-lasR and rhlI-rhlR Quorum-Sensing Systems in Pseudomonas Aeruginosa

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Alginate Oligosaccharide-Induced Modification of the lasI-lasR and rhlI-rhlR Quorum-Sensing Systems in Pseudomonas Aeruginosa

Alison A Jack et al. Antimicrob Agents Chemother.

Abstract

Pseudomonas aeruginosa plays a major role in many chronic infections. Its ability to readily form biofilms contributes to its success as an opportunistic pathogen and its resistance/tolerance to antimicrobial/antibiotic therapy. A low-molecular-weight alginate oligomer (OligoG CF-5/20) derived from marine algae has previously been shown to impair motility in P. aeruginosa biofilms and disrupt pseudomonal biofilm assembly. As these bacterial phenotypes are regulated by quorum sensing (QS), we hypothesized that OligoG CF-5/20 may induce alterations in QS signaling in P. aeruginosa QS regulation was studied by using Chromobacterium violaceum CV026 biosensor assays that showed a significant reduction in acyl homoserine lactone (AHL) production following OligoG CF-5/20 treatment (≥2%; P < 0.05). This effect was confirmed by liquid chromatography-mass spectrometry analysis of C4-AHL and 3-oxo-C12-AHL production (≥2%; P < 0.05). Moreover, quantitative PCR showed that reduced expression of both the las and rhl systems was induced following 24 h of treatment with OligoG CF-5/20 (≥0.2%; P < 0.05). Circular dichroism spectroscopy indicated that these alterations were not due to steric interaction between the AHL and OligoG CF-5/20. Confocal laser scanning microscopy (CLSM) and COMSTAT image analysis demonstrated that OligoG CF-5/20-treated biofilms had a dose-dependent decrease in biomass that was associated with inhibition of extracellular DNA synthesis (≥0.5%; P < 0.05). These changes correlated with alterations in the extracellular production of the pseudomonal virulence factors pyocyanin, rhamnolipids, elastase, and total protease (P < 0.05). The ability of OligoG CF-5/20 to modify QS signaling in P. aeruginosa PAO1 may influence critical downstream functions such as virulence factor production and biofilm formation.

Keywords: OligoG CF-5/20; Pseudomonas aeruginosa; alginate; biofilms; homoserine lactone; natural antimicrobial products; quorum-sensing inhibitor.

Figures

FIG 1
FIG 1
Schematic diagram of the P. aeruginosa virulence regulatory network showing the three major QS signaling pathways, namely, the AHL Las and Rhl operons and the 2-heptyl-3-hydroxy-4-quinolone PQS operon.
FIG 2
FIG 2
Effect of OligoG CF-5/20 on the growth of P. aeruginosa PAO1 and the production of signaling molecules with the biosensor C. violaceum CV026. (A) Growth curves of P. aeruginosa PAO1 treated with OligoG CF-5/20 showing four specific sampling times (12, 18, 24, and 30 h) for AHL extractions (arrows). (B to D) Well diffusion time course assays detecting AHLs from 24-h (B) or 12-, 18-, 24-, and 30-h (C, D) extracts of P. aeruginosa cultures treated with OligoG CF-5/20 (0.2, 2, or 10%). Induction (zone of coloration) (B, C) and inhibition (zone of clearing) (B, D) of violacein synthesis in C. violaceum CV026 showing changes in C4- and 3-oxo-C12-AHL production following OligoG CF-5/20 treatment. The average from three replicates ± the standard deviation is shown. *, P < 0.05. Differences in culture biomass (at ≥2% OligoG CF-5/20) were corrected according to dry weight.
FIG 3
FIG 3
Method development for detection and quantification of AHLs. Panels: A, structure of C4-AHL; B, structure of 3-oxo-C12-AHL; C, C4-AHL and 3-oxo-C12-AHL LC-MS peaks; D, initial time course showing LC-MS quantification of AHL concentrations (μg/liter) from P. aeruginosa PAO1 grown in MH broth at different time points (18, 24, and 30 h). The average from three replicates ± the standard deviation is shown. *, P < 0.05. Differences in culture biomass (at ≥2% OligoG CF-5/20) were corrected according to dry weight.
FIG 4
FIG 4
Effect of OligoG CF-5/20 on AHL concentrations (μg/liter) determined by LC-MS at different time points (12, 18, 24, and 30 h) in P. aeruginosa PAO1 grown in MH broth with or without OligoG CF-5/20 (0.2, 2, or 10%). Panels: A, C4-AHL; B, 3-Oxo-C12-AHL. The average from three replicates ± the standard deviation is shown. *, P < 0.05. Differences in culture biomass (at ≥2% OligoG CF-5/20) were corrected according to dry weight.
FIG 5
FIG 5
Extracellular virulence factor production by P. aeruginosa from 12-, 18-, 24-, and 30-h cell-free culture supernatants treated with OligoG CF-5/20 (0.2, 2, or 10%). Panels: A, pyocyanin; B, rhamnolipids; C, total protease; D, elastase. The average from four replicates ± the standard deviation is shown. *, P < 0.05. Differences in culture biomass (at ≥2% OligoG CF-5/20) were corrected according to dry weight.
FIG 6
FIG 6
Relative fold change in gene expression compared to untreated control by qPCR of the lasI-lasR and rhlI-rhlR genes from 12-, 18-, 24-, and 30-h cultures of P. aeruginosa PAO1 treated with OligoG CF-5/20 (0.2, 2, or 10%). Panels: A, 12 h; B, 18 h; C, 24 h; D, 30 h. The average from three replicates ± the standard deviation is shown. *, P < 0.05. Differences in culture biomass (at ≥2% OligoG CF-5/20) were corrected according to dry weight.
FIG 7
FIG 7
CLSM of P. aeruginosa PAO1 biofilms treated with OligoG CF-5/20 (0.5, 2, or 10%) and stained with nucleic acid-specific TOTO-1 (green). (A) Biofilm formation assay. Biofilms were grown for 24 h in the presence of OligoG CF-5/20. (B) Biofilm disruption assay. (C, D) Twenty-four-hour established biofilms subsequently treated for 24 h with OligoG CF-5/20 are shown with corresponding fluorescence intensities. The average from three replicates ± the standard deviation is shown. *, P < 0.05.
FIG 8
FIG 8
Determination of eDNA concentration. The effect of OligoG CF-5/20 (0.2, 2, or 10%) on the relative eDNA concentration in P. aeruginosa biofilms was measured. In a biofilm formation assay, biofilms were grown for 24 h in the presence of OligoG CF-5/20. In a biofilm disruption assay, 24-h established biofilms were subsequently treated for 24 h with OligoG CF-5/20. The average from three replicates ± the standard deviation is shown. *, P < 0.05. Differences in culture biomass (at ≥2% OligoG CF-5/20) were corrected according to dry weight. RFU, relative fluorescence units.

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