Broad-spectrum quorum sensing and biofilm inhibition by green tea against gram-negative pathogenic bacteria: Deciphering the role of phytocompounds through molecular modelling

Highlights

• This is the first report of broad-spectrum QS and biofilm inhibition by green tea against Gram -ve bacterial pathogens.

• Further dissection crude methanolic extract was performed for enrichment to obtain better activity than previous findings.

• The in vivo validation of therapeutic efficacy of extract was also confirmed.

• In silico studies gave the closer insights regarding its mode of action.

Abstract

The emerging prevalence of multidrug-resistance in Gram-negative pathogens, due to conventional antimicrobial therapeutics, has led the researchers to emphasize on development of alternative novel strategies to suppress the bacterial virulence and pathogenicity through inhibition of quorum sensing (QS) and biofilms. QS is a bacterial communication system to produce density-dependent response via chemical signalling that controls pathogenesis and biofilms formation. Leaves of green tea are used worldwide as beverage which is also known for its broad-spectrum therapeutic efficacy. In this work, we have identified and characterized the most bioactive faction of green tea extract and evaluated the anti-QS and antibiofilm activity of green tea ethyl acetate fraction (GTEF) i.e. most active fraction, on three different Gram-negative bacterial pathogens. GTEF inhibited the violacein production by >75% in C. violaceum 12472. Many virulence factors of P. aeruginosa PAO1 viz. pyocyanin, pyoverdin, exoprotease, elastase, rhamnolipid production, and swimming motility were remarkably reduced in presence of sub-MICs of GTEF. Moreover, prodigiosin, protease activity, cell surface hydrophobicity, and swimming of S. marcescens MTCC 97 were also decreased significantly by the supplementation of GTEF in culture media. GTEF exhibited broad-spectrum antibiofilm action with >80% reduction in biofilm formation of test pathogens. In silico studies gave a mechanistic insight of action of GTEF. Molecular modelling revealed that phytoconstituents detected by GC/MS exhibited affinity (in order of 10⁴ M⁻¹) towards AHL synthases (LasI and EsaI). The molecular binding between phytocompounds and receptor proteins (LasR, RhlR, and PqsR) of QS circuit was also energetically favourable (ΔG°≥ 5.0 kcal mol⁻¹) and supported by hydrogen bonds and hydrophobic interactions. These compounds were found to be docked in ligand binding domain of CviR and occupied same cavity as that of its antagonist. Squalene and thunbergol interacted with LasA at tartaric acid binding pocket and the complex was strengthened with binding energy −5.9 kcal mol⁻¹. Moreover, interaction of thunbergol with biofilm-associated proteins viz. PilT and PilY1, might be disabling the pilus assembly and consequently inhibiting biofilm formation. In vivo validation of results suggested the protective role GTEF against QS-mediated pathogenicity and it might become a novel non-antibiotic QS inhibitor to control bacterial infection.

Introduction

The worldwide emergence of multidrug resistance in bacterial pathogens has created a global health issue. The demand can only be met by the development of novel alternative strategies to overcome microbial infections. The antibiotics can no longer be entrusted for long-term therapeutic applications either due the development of resistance against them or detrimental effects of on host's microbiome or both [1]. In recent past, research on discovery or synthesis of anti-infective agents have focussed on selective intervention of virulence pathways to manage or cure microbial infections as such drug targets do not affect the survival of pathogens making them less prone to develop resistance compared to conventional antimicrobials [2]. To be developed as next generation anti-infective drugs, bioactive compounds derived from natural sources, such as medicinal plants, are exhibiting promising therapeutic properties in management of evolving resistance in pathogens [3].

Quorum sensing (QS) is a communication system in bacteria to give density dependent response via chemical signalling that includes pathogenesis and biofilms formation in many pathogenic bacteria [4]. The concentration of autoinducers (AIs) or chemical signal molecules increases with increase in population density and govern the expression of QS-regulated genes by transcriptional regulation [5]. Most common AIs in Gram-negative bacteria are acyl-homoserine lactones (AHLs). There are some common features that are present in most of Gram-negative QS systems that include synthesis and diffusion of AHLs or the signal molecules; binding of AIs to specific receptors present either in inner membrane or cytoplasm; expression of QS-mediated genes; and increased synthesis of AIs to establish a feed-forward loop which is supposed to foster the synchronous gene expression [6]. There are three major QS systems in P. aeruginosa viz. LasI-LasR, RhlI-RhlR, and PQS-MvfR. Recently, a new QS signal molecule i.e. 2-(2-hydroxyphenyl) thiazole-4-carbaldehyde (IQS) in P. aeruginosa has been identified that is associated with phosphate-stress response [7]. Moreover, cis-unsaturated fatty acids called as diffusible signal factors (DSFs) has also been recognized for their involvement in cell to cell signalling in production of virulence factor and formation of biofilms [8]. Serratia marcescens is another opportunistic pathogen. SwrI and SwrR are homologues proteins to I and R proteins of AHL-mediated QS systems [9]. The signal molecules bind to transcriptional regulator (SwrR) and control the expression of numerous proteins that are involved in pathogenicity and biofilm formation [9].

Camellia sinensis, commonly known as green tea, family Theaceae, whose leaves are used worldwide popularly as beverage. The plant is known for its broad-spectrum therapeutic efficacy and its prolonged consumption has been documented in improvement of antioxidant status in vivo [10]. Green tea contains bioactive components including, polyphenols, caffeine, catechins, volatile compounds, minerals, and many undefined compounds [11]. There are evidences that phytoconstituents of green tea may contribute in the prevention of cardiovascular diseases, cancer, and it has anti-inflammatory, neuroprotective, antibacterial, antiviral, anti-arthritic, anti-angiogenic and cholesterol-lowering effects [12]. A preliminary study found that green tea modulates the QS of C. violaceum and P. aeruginosa [13]. Tea polyphenols has also been reported to disrupt QS-regulated virulence factors of P. aeruginosa [14]. We hypothesize that activity guided fractionation may result in identification of most active fraction and its phytoconstituents as broad-spectrum QS inhibitors.

The primary objective of this study was to identify the most potent anti-QS fraction of green tea extract followed by its broad-spectrum efficacy against Gram-negative bacteria viz. C. violaceum 12472, P. aeruginosa PAO1, and S. marcescens MTCC 97 targeting QS regulated virulence and biofilms. To obtain closer insight into the possible role of phytoconstituents, molecular modelling was employed. In vivo efficacy of the most active fraction was also investigated in C. elegans.