2,4-Di-Tert-Butylphenol Isolated From an Endophytic Fungus, Daldinia eschscholtzii, Reduces Virulence and Quorum Sensing in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is among the top three gram-negative bacteria according to the WHO's critical priority list of pathogens against which newer antibiotics are urgently needed and considered a global threat due to multiple drug resistance. This situation demands unconventional antimicrobial strategies such as the inhibition of quorum sensing to alleviate the manifestation of classical resistance mechanisms. Here, we report that 2,4-di-tert-butylphenol (2,4-DBP), isolated from an endophytic fungus, Daldinia eschscholtzii, inhibits the quorum-sensing properties of P. aeruginosa. We have found that treating P. aeruginosa with 2,4-DBP substantially reduced the secretion of virulence factors as well as biofilm, and its associated factors that are controlled by quorum sensing, in a dose-dependent manner. Concomitantly, 2,4-DBP also significantly reduced the expression of quorum sensing-related genes, i.e., lasI, lasR, rhlI, and rhlR significantly. Importantly, 2,4-DBP restricted the adhesion and invasion of P. aeruginosa to the A549 lung alveolar carcinoma cells. In addition, bactericidal assay with 2,4-DBP exhibited synergism with ampicillin to kill P. aeruginosa. Furthermore, our computational studies predicted that 2,4-DBP could bind to the P. aeruginosa quorum-sensing receptors LasR and RhlR. Collectively, these data suggest that 2,4-DBP can be exploited as a standalone drug or in combination with antibiotic(s) as an anti-virulence and anti-biofilm agent to combat the multidrug resistant P. aeruginosa infection.

Interaction Analysis of T7 RNA Polymerase with Heparin and Its Low Molecular Weight Derivatives - An In Silico Approach.

The single subunit T7 RNA polymerase (T7RNAP) is a model enzyme for studying the transcription process and for various biochemical and biophysical studies. Heparin is a commonly used inhibitor against T7RNAP and other RNA polymerases. However, exact interaction between heparin and T7RNAP is still not completely understood. In this work, we analyzed the binding pattern of heparin by docking heparin and few of its low molecular weight derivatives to T7RNAP, which helps in better understanding of T7RNAP inhibition mechanism. The efficiency of the compounds was calculated by docking the selected compounds and post-docking molecular mechanics/generalized Born surface area analysis. Evaluation of the simulation trajectories and binding free energies of the complexes after simulation showed enoxaparin to be the best among low molecular weight heparins. Binding free energy analysis revealed that van der Waals interactions and polar solvation energy provided the substantial driving force for the binding process. Furthermore, per-residue free energy decomposition analysis revealed that the residues Asp 471, Asp 506, Asp 537, Tyr 571, Met 635, Asp 653, Pro 780, and Asp 812 are important for heparin interaction. Apart from these residues, most favorable contribution in all the three complexes came from Asp 506, Tyr 571, Met 635, Glu 652, and Asp 653, which can be essential for binding of heparin-like structures with T7RNAP. The results obtained from this study will be valuable for the future rational design of novel and potent inhibitors against T7RNAP and related proteins.

Insight into virus encapsulation mechanism through in silico interaction study between coat protein and RNA operator loops of Sesbania mosaic virus.

Viruses are parasite by nature and they are responsible for many diseases. Inhibitor development is very difficult for viruses due to their rapid mutative nature. A common approach for treating virus infection is targeting them at the genomic level and an encapsulation mechanism can be one of the targets. Sesbania mosaic virus (SeMV) is a spherical virus and its capsid is formed by a coat protein, which contains the Arginine Rich Motif (ARM). This ARM interacts with RNA operator loops present in their genome and starts encapsulation. Though the structure of SeMV was already solved by crystallography, it lacks the critical ARM domain. We predicted the full-length three-dimensional structure of this protein by using crystal structure (lacking ARM) as a template along with tertiary structure of RNA operator loops. Docking studies were performed to discover the interacting residues of protein and RNA which are driving protein and RNA to interact with each other. We observed that these interactions lead to conformation changes in the coat protein structure, which starts genome encapsulation process. The ARM region is found to be crucial for these interactions. Molecular dynamics simulation studies were performed to check the conformational changes and free energy landscapes were generated to check the viability of these changes in terms of energy. In this work we proposed one RNA operator loop that is responsible for noticeable conformational changes in the SeMV structure and might be involved in the activation of the viral protein. The results of this in silico study can be tested further through in vitro studies and can be used to stop encapsulation.

Quercetin Influences Quorum Sensing in Food Borne Bacteria: In-Vitro and In-Silico Evidence.

Quorum sensing (QS) plays a vital role in regulating the virulence factor of many food borne pathogens, which causes severe public health risk. Therefore, interrupting the QS signaling pathway may be an attractive strategy to combat microbial infections. In the current study QS inhibitory activity of quercetin and its anti-biofilm property was assessed against food-borne pathogens using a bio-sensor strain. In addition in-silico techniques like molecular docking and molecular dynamics simulation studies were applied to screen the quercetin's potentiality as QS inhibitor. Quercetin (80 µg/ml) showed the significant reduction in QS-dependent phenotypes like violacein production, biofilm formation, exopolysaccharide (EPS) production, motility and alginate production in a concentration-dependent manner. Synergistic activity of conventional antibiotics with quercetin enhanced the susceptibility of all tested pathogens. Furthermore, Molecular docking analysis revealed that quercetin binds more rigidly with LasR receptor protein than the signaling compound with docking score of -9.17 Kcal/mol. Molecular dynamics simulation predicted that QS inhibitory activity of quercetin occurs through the conformational changes between the receptor and quercetin complex. Above findings suggest that quercetin can act as a competitive inhibitor for signaling compound towards LasR receptor pathway and can serve as a novel QS-based antibacterial/anti-biofilm drug to manage food-borne pathogens.

Three dimensional electron microscopy and in silico tools for macromolecular structure determination.

Recently, structural biology witnessed a major tool - electron microscopy - in solving the structures of macromolecules in addition to the conventional techniques, X-ray crystallography and nuclear magnetic resonance (NMR). Three dimensional transmission electron microscopy (3DTEM) is one of the most sophisticated techniques for structure determination of molecular machines. Known to give the 3-dimensional structures in its native form with literally no upper limit on size of the macromolecule, this tool does not need the crystallization of the protein. Combining the 3DTEM data with in silico tools, one can have better refined structure of a desired complex. In this review we are discussing about the recent advancements in three dimensional electron microscopy and tools associated with it.