Elsevier

Phytochemistry

Volume 117, September 2015, Pages 98-106
Phytochemistry

Flavonoids from Piper delineatum modulate quorum-sensing-regulated phenotypes in Vibrio harveyi

https://doi.org/10.1016/j.phytochem.2015.06.006Get rights and content

Highlights

  • A series of new flavonoids were isolated from Piper delineatum.

  • Their stereostructures were elucidated by NMR and biosynthetic considerations.

  • Four flavonoids strongly disrupted QS-mediated bioluminescence in Vibrio harveyi.

  • Phenotypic mutant analyses suggest a molecular target downstream LuxO.

  • These flavonoids also inhibited biofilm formation in V. harveyi WT.

Abstract

Quorum sensing (QS), or bacterial cell-to-cell communication, is a key process for bacterial colonization of substrata through biofilm formation, infections, and production of virulence factors. In an ongoing investigation of bioactive secondary metabolites from Piper species, four new flavonoids (14), along with five known ones (59) were isolated from the leaves of Piper delineatum. Their stereostructures were established by spectroscopic and spectrometric methods, including 1D and 2D NMR experiments, and comparison with data reported in the literature. The compounds were screened for their ability to interfere with QS signaling in the bacterial model Vibrio harveyi. Four compounds from this series (2, 3, 6, and 7) exhibited remarkable activity in the micromolar range, being compounds 3 and 7 particularly attractive since they did not affect bacterial growth. The results suggest that these flavonoids disrupt QS-mediated bioluminescence by interaction with elements downstream LuxO in the QS circuit of V. harveyi, and also, they exhibited a strong dose-dependent inhibition of biofilm formation. The present findings shed light on the QS inhibition mechanisms of flavonoids, underlining their potential applications.

Graphical abstract

A series of natural flavonoids from Piper delineatum exhibited the ability to disrupt QS-mediated bioluminescence downstream LuxO and showed a strong dose-dependent inhibition of biofilm formation, in the bacterial model Vibrio harveyi.

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Introduction

One of the most ground-breaking developments of microbiological research during the past 40 years has been the recognition of bacterial biofilms as the predominant bacterial lifestyle (Mielich-Suss and López, 2015). Biofilms play an important role in the fouling process (Yebra et al., 2004), and they are also postulated as being responsible for an overwhelming proportion of persistent antibiotic resistance (Bjarnsholt et al., 2013), one of the major obstacles in antimicrobial chemotherapy.

Quorum sensing (QS) is the term coined for bacterial cell-to-cell communication, a process that regulates coordinated behaviors in bacteria as a function of population density. It involves the production, excretion and detection of autoinducer (AI) molecules that trigger the expression of QS-regulated genes (Miller and Bassler, 2001). This process allows bacteria to optimize their energetic resources to carry out tasks that are more efficiently performed when a threshold population is achieved, for example, the production of virulence factors, biofilm formation and, in certain bacteria such as Vibrio harveyi, the generation of bioluminescence (Waters and Bassler, 2005). Since quorum sensing inhibitors (QSIs) do not target bacterial growth, they do not exert a selective pressure on bacterial populations and, consequently, they avoid the appearance of resistance towards chemical or antibiotic treatments (Kalia, 2013), although their prospects are still being debated (Defoirdt et al., 2010, García-Contreras et al., 2013). Furthermore, there is increasing evidence that QS signaling molecules interact directly with fouling colonizers (Joint et al., 2007), and conversely, QS disruption controls, directly or indirectly, biofilm formation and larval attachment onto underwater substrata (Dobretsov et al., 2007, Twigg et al., 2014). Renowned examples are the brominated furanones produced by the red alga Delisea pulchra (Givskov et al., 1996). Therefore, the search for effective and safe QSIs has become a priority for current antifouling research as well as for biomedicine, industry and other fields where bacterial biofilms are causes of sanitary or operational problems. In this context, flavonoids, which are among the richest members of plant-derived compounds in terms of structural diversity and biological activity (Andersen and Markham, 2006), have been reported as potential inhibitors of both biofilm formation and virulence factors in pathogenic bacteria by interfering with QS mechanisms (Cushnie and Lamb, 2011, Nazzaro et al., 2013).

The goal of this work is: (a) to report the isolation and structural elucidation of flavonoids from Piper delineatum, (b) to report the results from their screening as disruptors of QS-regulated phenotypes using V. harveyi as bacterial model, (c) to select the most active compounds and gain an insight into their possible mode of action through phenotypic analyses with three V. harveyi mutant strains, and (d) to check whether these compounds were also able to interfere with bacterial adhesion to material substrates (biofilm formation).

Thus, efforts have been focused on the characterization of new flavonoids as potential QSIs from P. delineatum Trel. (Piperaceae), a shrub native to tropical regions of the Americas, and whose chemical investigation has not been previously reported. In this work, the isolation, structure elucidation and bioactivity of nine flavonoids, including two new chalcones and two new flavanones (14) from the leaves of P. delineatum, are reported. Their sterostructures were elucidated by means of 1H and 13C NMR spectroscopic studies, including homonuclear (COSY and ROESY) and heteronuclear (HSQC and HMBC) correlation experiments. These flavonoids are able to interfere with bacterial QS-controlled processes using V. harveyi as bacterial model.

V. harveyi is a marine luminescent bacterium with a complex QS system (Henke and Bassler, 2004) (Fig. 1). V. harveyi employs three kinds of autoinducers for intra-species (HAI-1 signal, a homoserine lactone), intra-genera (CAI-1 signal, an α-amino ketone) and inter-species (AI-2 signal, a furanosyl borate diester) communication in three parallel circuits that converge in the σ54-dependent response regulator LuxO, which is phosphorylated (low cell densities, or more precisely, low AI concentrations) or dephosphorylated (high AI concentrations) via the phosphotransferase protein LuxU. At low AI concentrations, the phosphorylated LuxO activates the translation of five small quorum-regulatory RNAs (Qrr sRNAs) that, together with the chaperone Hfq destabilize the mRNA encoding the QS master regulator LuxR. Once a threshold concentration of AIs is achieved, the QS receptors LuxN, LuxPQ and CqsS switch from kinases to phosphatases, dephosphorylating LuxO, thus allowing the expression of LuxR and the subsequent translation of the genes in the QS regulon (Pompeani et al., 2008, Anetzberger et al., 2012, Nackerdien et al., 2008).

In order to characterize the bioactivities of the isolated flavonoids and gain an insight into their molecular targets, four compounds exhibiting a promising profile (2, 3, 6 and 7) were evaluated with V. harveyi QS mutants. Whereas compounds 2 and 6 exerted an activity with toxic side effects, compounds 3 and 7 disrupted QS-regulated bioluminescence in a non-toxic fashion, probably by interaction with elements downstream LuxO in the QS transduction pathway. These flavonoids also imposed strong, dose-dependently inhibitions on biofilm formation, a process controlled by QS in V. harveyi.

Section snippets

Isolation and structure identification

The EtOH extract of leaves of P. delineatum were partitioned into a CH2Cl2/H2O (1:1, v/v) solution. The CH2Cl2 fraction was subjected to multiple chromatographic steps, involving vacuum-liquid and medium-pressure liquid chromatography, centrifugal preparative TLC, and preparative TLC chromatography on Si gel, and Sephadex LH-20 to yield flavonoids 19 (Fig. 2). The structures of the new compounds were deduced as described below.

Compound 1 was obtained as a yellow amorphous solid and showed the

Conclusion

A series of nine flavonoids, including four new ones were isolated from the leaves of P. delineatum. Two compounds from this series, 3 and 7, displayed a potent QS inhibitory activity in V. harveyi without inhibition of bacterial growth up to 500 μM. Experiments with V. harveyi mutant strains suggest a molecular target downstream LuxO for these compounds. In addition, both flavonoids exhibited a strong inhibitory effect on biofilm formation in V. harveyi. These results emphasize the potential of

General experimental procedures

Optical rotations were measured on a Perkin Elmer 241 automatic polarimeter in CHCl3 at 25 °C and the [α]D values are given in 10−1 deg cm2/g. UV spectra were obtained in absolute EtOH on a JASCO V-560 spectrophotometer. IR (film) spectra were measured on a Bruker IFS 55 spectrophotometer. 1H and 13C (400 and 100 MHz, respectively) NMR spectra were recorded in (CD3)2CO on a Bruker Avance 400 spectrometer; the chemical shifts are given in δ (ppm) with TMS as internal reference and coupling constants

Acknowledgements

We are indebted to the Spanish Ministry of Economy and Competitiveness (MINECO) SAF2011-28883-C03-01 and CTQ2014-55888-C03-01-R; EU, FP7-REGPOT-2012-CT2012-316137-IMBRAIN; and Campus de Excelencia Internacional CEI10/00018 projects for financial support. A.J.M.-R. thanks PLOCAN for a 2+2 contract. The authors are grateful to Dr. Tom Defoirdt (University of Ghent) for his valuable advice in the experimental set-up with Vibrio harveyi.

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