Plant phenolic volatiles inhibit quorum sensing in pectobacteria and reduce their virulence by potential binding to ExpI and ExpR proteins.

Janak Raj Joshi,Iris Yedidia,Netaly Khazanov,Alexander Lipsky,Hanoch Senderowitz,Saul Burdman

doi:10.1038/srep38126

Abstract

Quorum sensing (QS) is a population density-dependent regulatory system in bacteria that couples gene expression to cell density through accumulation of diffusible signaling molecules. Pectobacteria are causal agents of soft rot disease in a range of economically important crops. They rely on QS to coordinate their main virulence factor, production of plant cell wall degrading enzymes (PCWDEs). Plants have evolved an array of antimicrobial compounds to anticipate and cope with pathogens, of which essential oils (EOs) are widely recognized. Here, volatile EOs, carvacrol and eugenol, were shown to specifically interfere with QS, the master regulator of virulence in pectobacteria, resulting in strong inhibition of QS genes, biofilm formation and PCWDEs, thereby leading to impaired infection. Accumulation of the signal molecule N-acylhomoserine lactone declined upon treatment with EOs, suggesting direct interaction of EOs with either homoserine lactone synthase (ExpI) or with the regulatory protein (ExpR). Homology models of both proteins were constructed and docking simulations were performed to test the above hypotheses. The resulting binding modes and docking scores of carvacrol and eugenol support potential binding to ExpI/ExpR, with stronger interactions than previously known inhibitors of both proteins. The results demonstrate the potential involvement of phytochemicals in the control of Pectobacterium.

Highlights

An appealing control strategy against bacteria is based on the use of plant essential oils (EOs) which play an important role in the protection of plants against bacterial and fungal pathogens[13]
We assessed the effect of the phenolic volatiles carvacrol and eugenol on the biofilm formation ability of two Pectobacterium strains, P. carotovorum subsp. brasiliense Pcb1692 and P. aroidearum PC1 (Table S1), using a microtiter dish assay with non-inhibitory concentrations of these compounds
Since carvacrol and eugenol dose-dependently inhibit the growth of Pectobacterium (Fig. S1), the concentrations of both volatiles for these assays were calibrated to a level that does not affect bacterial growth or membrane leakage (Fig. S2)

Summary

Introduction

An appealing control strategy against bacteria is based on the use of plant essential oils (EOs) which play an important role in the protection of plants against bacterial and fungal pathogens[13]. EOs comprise a broad range of antimicrobial phytochemicals that display potent activity against a variety of cellular targets in the bacterial membrane and cytoplasm[13,14] Some of these volatiles were previously designated as potential inhibitors of the QS machinery in bacteria[15,16]. The objectives of the current work were to elucidate the molecular mechanisms underpinning the antimicrobial activities of carvacrol and eugenol on these pathogens and to establish a potential mode of action Major virulence determinants such as biofilm formation, secretion of PCWDEs and QS-related gene expression were studied following exposure to both compounds in vitro, while infection capabilities and compensation assays with the QS signaling molecule AHL were studied in vivo using potato tubers, cabbage and calla lily leaves as hosts. The resulting binding modes highlight the importance of specific ligand-protein interactions, which could be used in the future for design of more active ExpI and ExpR blockers

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Methods

Results

Conclusion