Furanone and phytol influence metabolic phenotypes regulated by acyl-homoserine lactone in Salmonella

Abstract

Salmonella is an important foodborne pathogen, and it is unable to produce the quorum sensing signaling molecules called acyl-homoserine lactones (AHLs). However, it synthesizes the SdiA protein, detecting AHL molecules, also known as autoinducer-1 (AI-1), in the external environment. Exogenous AHLs can regulate specific genes related to virulence and stress response in Salmonella. Thus, interfering with quorum sensing can be a strategy to reduce virulence and help elucidate the cell-to-cell communication role in the pathogens' response to extracellular signals. This study aimed to evaluate the influence of the quorum sensing inhibitors furanone and phytol on phenotypes regulated by N-dodecanoyl homoserine lactone (C12-HSL) in Salmonella enterica serovar Enteritidis. The furanone C30 at 50 nM and phytol at 2 mM canceled the alterations promoted by C12-HSL on glucose consumption and the levels of free cellular thiol in Salmonella Enteritidis PT4 578 under anaerobic conditions. In silico analysis suggests that these compounds can bind to the SdiA protein of Salmonella Enteritidis and accommodate in the AHL binding pocket. Thus, furanone C30 and phytol act as antagonists of AI-1 and are likely inhibitors of the quorum sensing mechanism mediated by AHL in Salmonella.

Keywords: Furanone C30; Glucose consumption; Phytol; QS-inhibitor; Quorum sensing; Thiol.

Fig. 1

Growth of Salmonella Enteritidis PT4 578 in anaerobic TSB at 37 °C during 24 h, in the presence of 50 nM of C12-HSL, 50 nM of furanone C30, 2 mM of phytol, 50 nM of C12-HSL plus 50 nM of furanone C30, and 50 nM of C12-HSL plus 2 mM of phytol. The control experiments were also performed using 0.5% (v/v) of acetonitrile (acetonitrile), 1.0% (v/v) of acetonitrile (acetonitrile 2X), 0.05% (v/v) of ethanol (ethanol), and 0.5% (v/v) of acetonitrile plus 0.05% (v/v) of ethanol (acetonitrile + ethanol). The vertical dashed lines indicate the times of collection of supernatant and cells for the evaluation of the concentration of glucose in the extracellular medium and the levels of free cellular thiol, respectively. There was no statistical difference between the treatment and controls (p > 0.05). Error bars indicate standard error

Fig. 2

Concentration of glucose in the extracellular medium after 6 and 7 h of cultivation of Salmonella Enteritidis PT4 578 in anaerobic TSB at 37 °C, in the presence of 50 nM of C12-HSL, 50 nM of furanone C30, 2 mM of phytol, 50 nM of C12-HSL plus 50 nM of furanone C30, and 50 nM of C12-HSL plus 2 mM of phytol. The control experiments were also performed using 0.5% (v/v) of acetonitrile (acetonitrile), 1.0% (v/v) of acetonitrile (acetonitrile 2X), 0.05% (v/v) of ethanol (ethanol), and 0.5% (v/v) of acetonitrile plus 0.05% (v/v) of ethanol (acetonitrile + ethanol). Averages followed by different letters (between treatments at the same time) mean that there was a difference at 5% probability (p < 0.05) between them by the Tukey's test. Where the same letter or no letter is shown, no statistical difference between treatments was observed (p > 0.05). Error bars indicate standard error

Fig. 3

Levels of free cellular thiol after 6 and 7 h of cultivation of Salmonella Enteritidis PT4 578 in anaerobic TSB at 37 °C in the presence of 50 nM of C12-HSL, 50 nM of furanone C30, 2 mM of phytol, 50 nM of C12-HSL plus 50 nM of furanone C30, and 50 nM of C12-HSL plus 2 mM of phytol. The control experiments were also performed using 0.5% (v/v) of acetonitrile (acetonitrile), 1.0% (v/v) of acetonitrile (acetonitrile 2X), 0.05% (v/v) of ethanol (ethanol), and 0.5% (v/v) of acetonitrile plus 0.05% (v/v) of ethanol (acetonitrile + ethanol). Averages followed by different letters (between treatments at the same time) mean that there was a difference at 5% probability (p < 0.05) between them by the Tukey's test. Where the same letter or no letter is shown, no statistical difference between treatments was observed (p > 0.05). Error bars indicate standard error

Fig. 4

Molecular docking of the 4Y17-S structure of SdiA protein of Salmonella Enteritidis PT4 578 with C12-HSL, furanone C30, E-phytol, Z-phytol, and OCL. (A-E) Structures of C12-HSL, furanone C30, E-phytol, Z-phytol, and OCL, (F-J) backbone representation of the 4Y17-S structure of SdiA protein of Salmonella Enteritidis PT4 578 with hydrogen bonds between the amino acid residues and compounds evaluated, (K–O) surface and backbone representations and (P–T) surface representation. Gray surface representation, SdiA protein; Red surface representation, C12-HSL; Orange surface representation, furanone C30; Pink surface representation, E-phytol; Yellow surface representation, Z-phytol; Purple surface representation, OCL; Gray backbone representation, SdiA protein; Black arrow indicates the binding site; Yellow arrow, C12-HSL or furanone C30 or E-phytol or Z-phytol or OCL; Blue dashed line, hydrogen bond