Biochemical and Biophysical Research Communications
Switch of SpnR function from activating to inhibiting quorum sensing by its exogenous addition
Introduction
The virulence expression of some opportunistic pathogens can be regulated by quorum sensing (QS), wherein activation is dependent on the concentration of a signal around cells [1], [2]. QS is a cell-density-dependent system for the expression of target genes because the diffusible QS signal enables local accumulation through a cell membrane [3], [4], [5]. Various gram-negative bacteria produce N-acylhomoserine lactone (AHL) as the common QS signal, whereas QS activation for some gram-positive bacteria is triggered by increasing peptide concentrations [6], [7]. The AHL complex with its intracellular receptor is stabilized by high AHL concentrations, and the complex interacts with a promoter region to activate transcription of the target gene. In many cases, receptors in bacterial cells exhibiting AHL-mediated QS are homologous to LuxR in Vibrio fischeri, which specifically recognizes N-(3-oxohexanoyl) homoserine lactone (3oxoC6HSL) [8], [9], [10]. In this study, we constructed a QS regulatory system based on the AHL trap using a LuxR homologue. Genetically engineered AHL receptor was added outside of cells to remove AHLs and inhibit QS.
As a model bacterium, Serratia marcescens AS-1 exhibits N-hexanoylhomoserine lactone (C6HSL)-mediated QS for the production of antibacterial prodigiosin, which is visualized as a red color [11], [12]. The LuxR homologue SpnR, which possesses binding sites for C6HSL and the prodigiosin biosynthesis gene (pig) cluster promoter at the N- and C-termini, respectively, can form a complex with C6HSL to enhance QS. Intracellular SpnR natively operates as a transcriptional activator of pig clusters, leading to the biosynthesis of prodigiosin synthase [13] upon formation of a complex with AHL (Fig. 1). Extracellular addition of SpnR in the culture broth is thus expected to effectively remove diffusible AHL molecules from the surrounding cells based on the high-affinity interaction. Thus, we overexpressed maltose-binding protein (MBP)-tagged SpnR (MBP-SpnR) in Escherichia coli and purified the fusion protein for application as a matrix with molecular selectivity [14].
Artificial reduction of the AHL concentration allows maintenance of the inactive state of QS even after reaching a quorum with respect to cell number. Previous studies have demonstrated that the amphiphilic AHL could be collected on cyclodextrin (CD) with hydrophobic cavities [15], [16], [17], carboxylate polymers copolymerized with hydrophobic domains [18], [19], and self-assembled micelles of the block polymers containing hydrophilic and hydrophobic domains [20]. However, to the best of our knowledge, no report of the use of engineered natural products to inhibit QS by trapping AHL is available. Here, we demonstrated a method for changing the role of SpnR in AHL-mediated QS from activation to inhibition as a novel system for the artificial regulation of QS.
Section snippets
Materials
3,3′-Dithiopropionic acid (DPA) and N-hydroxysuccinimide (NHS) were purchased from Tokyo Chemical Industry. N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), isopropyl-β-d(−)-thiogalactopyranoside (IPTG), and ampicillin sodium were purchased from Wako Pure Chemical. All reagents for capillary gel electrophoresis including protein standards (SDS-MW analysis kit) were purchased from Sciex.
Purification of MBP-tagged SpnR
SpnR (28 kDa) derived from S. marcescens AS-1 was overexpressed as a fusion protein with an
Specific interaction of C6HSL with the added MBP-SpnR
Specific complex formation between immobilized C6HSL and added MBP-SpnR was studied by measuring ΔF2 with QCM analysis. The quartz plate coated with a gold electrode (4.9 mm2) was modified with C6HSL moieties (8.4 pmol; Fig. 2a). MBP-SpnR was purified by affinity chromatography with amylose resin, and its molecular weight, as determined by capillary gel electrophoresis, was consistent with the calculated value. The ΔF2 value clearly decreased, indicating MBP-SpnR uptake onto the electrode (
Acknowledgments
We are grateful to Prof. T. Morohoshi for helpful discussions. We thank C. Okano and S. Ito for technical assistance. This work was supported by the Core Research for Evolutional Science and Technology - Japan Science and Technology Agency (CREST-JST).
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