Intervene effect on Streptococcus suis biofilm formation of emodin extracted from Rheum officinale Baill

Background Streptococcus suis is a zoonotic pathogen that causes serious systemic infections in pigs and humans. It is a great threat to pig breeding and public health safety. The formation of biofilm was one of the main reasons that it’s difficult to cure. Rhubarb water extract can inhibit the formation of biofilm of Streptococcus suis, but the main functional ingredient was not clear. And what’s the potential mechanism of emodin intervene the biofilm formation? Importantly, the gene expression of luxS of Streptococcus suis was significantly decreased with emodin treatment, if there is a possibility that emodin could combine with LuxS protein which in turn inhibit the formation of biofilm? Methods Optimization of green ultrasonic extraction of emodin from R. officinale by Response Surface Methodology. The activity of antibacterial was evaluated by MIC assay. And the ability that intervene biofilm formation was evaluated through Crystal violet staining and SEM. The combination mode between emodin and LuxS protein was identified through molecular docking. Results The optimum extraction conditions (ethanol concentration of 80%, extraction time of 21 min, and liquid to solid ratio of 12.5:1 mL/g) were determined through the interaction analysis of different influencing factors and model verification experiment. The minimal inhibitory concentration (MIC) of RUEP against S.suis was 15.625 µg·mL-1, and sub-inhibitory concentrations of RUEP (1/2, 1/4, 1/8 and 1/16 MIC) significantly inhibited the biofilm formation of S. suis. Moreover, scanning electron microscopy analysis confirmed that RUEP can damage the layered colony community structure in biofilm and effectively intervene in the biofilm formation of Streptococcus suis. Through molecular docking analyzed, the inhibition of biofilm formation could be realized through the formation of hydrogen bond with residues His14 and Glu 60, π-π staking with His61、Phe80 and hydrophobic interaction with Cys127. Conclusion Through our study, we found emodin have good antibacterial and antibiofilm formation properties and the potential mechanism of emodin intervention on biofilm formation of Streptococcus suis was inferred.

Impact of Citral and Phloretin, Alone and in Combination, on Major Virulence Traits of Streptococcus pyogenes

Streptococcus pyogenes is well documented as a multi-virulent and exclusively human pathogen. The LuxS-based signaling in these bacteria has a crucial role in causing several infections through pathways that are pathogenic. This study evaluated the individual and synergistic effects of citral and phloretin against S. pyogenes in relation to major virulence traits. The in vitro synergy of citral and phloretin was evaluated by the checkerboard method. The fractional inhibitory concentration (FIC) values were calculated to determine the interactions between the inhibitors. The bacteria’s virulence properties were tested in the presence of the molecules, individually as well as in combination. Molecules’ cytotoxicity was tested using human tonsil epithelial cells. The synergistic effects of the molecules on the expression of biofilm and quorum sensing genes were tested using quantitative real-time polymerase chain reaction (qRT-PCR). The molecules were also tested for their impact on LuxS protein by molecular docking, modeling, and free-energy calculations. When the two molecules were assessed in combination (synergistic effect, FIC Index of 0.5), a stronger growth inhibitory activity was exhibited than the individual molecules. The cell surface hydrophobicity, as well as genes involved in quorum sensing and biofilm formation, showed greater suppression when the molecules were tested in combination. The in silico findings also suggest the inhibitory potential of the two molecules against LuxS protein. The binding orientation and the binding affinity of citral and phloretin well support the notion that there is a synergistic effect of citral and phloretin. The data reveal the combination of citral and phloretin as a potent antibacterial agent to combat the virulence of S. pyogenes.

In-Silico Prediction and Modeling of the Quorum Sensing LuxS Protein and Inhibition of AI-2 Biosynthesis in Aeromonas hydrophila

luxS is conserved in several bacterial species, including A. hydrophila, which causes infections in prawn, fish, and shrimp, and is consequently a great risk to the aquaculture industry and public health. luxS plays a critical role in the biosynthesis of the autoinducer-2 (AI-2), which performs wide-ranging functions in bacterial communication, and especially in quorum sensing (QS). The prediction of a 3D structure of the QS-associated LuxS protein is thus essential to better understand and control A. hydrophila pathogenecity. Here, we predicted the structure of A. hydrophila LuxS and characterized it structurally and functionally with in silico methods. The predicted structure of LuxS provides a framework to develop more complete structural and functional insights and will aid the mitigation of A. hydrophila infection, and the development of novel drugs to control infections. In addition to modeling, the suitable inhibitor was identified by high through put screening (HTS) against drug like subset of ZINC database and inhibitor ((−)-Dimethyl 2,3-O-isopropylidene-l-tartrate) molecule was selected based on the best drug score. Molecular docking studies were performed to find out the best binding affinity between LuxS homologous or predicted model of LuxS protein for the ligand selection. Remarkably, this inhibitor molecule establishes agreeable interfaces with amino acid residues LYS 23, VAL 35, ILE76, and SER 90, which are found to play an essential role in inhibition mechanism. These predictions were suggesting that the proposed inhibitor molecule may be considered as drug candidates against AI-2 biosynthesis of A. hydrophila. Therefore, (−)-Dimethyl 2,3-O-isopropylidene-l-tartrate inhibitor molecule was studied to confirm its potency of AI-2 biosynthesis inhibition. The results shows that the inhibitor molecule had a better efficacy in AI-2 inhibition at 40 μM concentration, which was further validated using Western blotting at a protein expression level. The AI-2 bioluminescence assay showed that the decreased amount of AI-2 biosynthesis and downregulation of LuxS protein play an important role in the AI-2 inhibition. Lastly, these experiments were conducted with the supplementation of antibiotics via cocktail therapy of AI-2 inhibitor plus OXY antibiotics, in order to determine the possibility of novel cocktail drug treatments of A. hydrophila infection.

Signaling System in Porphyromonas gingivalis Based on a LuxS Protein

ABSTRACT The luxS gene of quorum-sensing Vibrio harveyi is required for type 2 autoinducer production. We identified a Porphyromonas gingivalis open reading frame encoding a predicted peptide of 161 aa that shares 29% identity with the amino acid sequence of the LuxS protein of V. harveyi. Conditioned medium from a late-log-phase P. gingivalis culture induced the luciferase operon of V. harveyi, but that from a luxS insertional mutant did not. In P. gingivalis, the expression ofluxS mRNA was environmentally controlled and varied according to the cell density and the osmolarity of the culture medium. In addition, differential display PCR showed that the inactivation ofP. gingivalis luxS resulted in up-regulation of a hemin acquisition protein and an arginine-specific protease and reduced expression of a hemin-regulated protein, a TonB homologue, and an excinuclease. The data suggest that the luxS gene inP. gingivalis may function to control the expression of genes involved in the acquisition of hemin.